JP2006518590A - Kinases and GPCRs involved in apoptosis - Google Patents

Abstract

The present invention provides a method for identifying an agent that modulates the function of an apoptosis-related polypeptide having the sequence shown in Table 1B, comprising: (a) a sample containing the apoptosis-related polypeptide having the sequence shown in Table 1B; Preparing a candidate agent and incubating under conditions that allow the candidate agent to bind to the polypeptide; (b) in the sample, an apoptosis-related polypeptide having the sequence set forth in Table 1B Measuring the binding to the candidate agent; and (c) the binding of the apoptosis-related polypeptide having the sequence described in Table 1B to the candidate agent in the sample, the polypeptide having the sequence described in Table 1B Compared to binding to a control substance known not to bind to a polypeptide having that sequence shown in Table 1B. Wherein the apoptosis-related polypeptide having the sequence described in Table 1B in the sample is compared to the control agent by the apoptosis-related polypeptide having the sequence described in Table 1B. The method relates to the method, wherein increased binding is indicated that the candidate agent modulates the function of an apoptosis-related polypeptide having that sequence listed in Table 1B.

Description

The present invention relates to the identification of a number of human genes that function in the course of apoptosis and the proteins they encode. The present invention relates to the use of these “apoptosis-related” genes and proteins in the modulation of apoptosis in cells, and methods for identifying modulators of these genes or proteins, and thus modulators of apoptosis. .

BACKGROUND OF THE INVENTION Programmed cell death or apoptosis is a genetically programmed process that causes cells to die both under physiological and various disease conditions (Kerr et al., Br. J. Cancer, 26th). 239-257, 1972). It functions as an anti-equilibrium force for mitotic fission in adulthood and is a major contributor that contributes to carving out the physiological structure in many developmental processes (Wyllie et al., Int. Rev. Cytol, 68th). Volume, 251-305, 1980). Apoptosis has been characterized by a number of distinct biochemical features, including DNA fragmentation caused by the activation of endogenous endonuclease enzymes (Wyllie, Nature, 284, 555). 556, 1980; Enari et al., Nature, 391, 43-50, 1998). The result can be easily visualized as a DNA ladder pattern in an agarose gel. With DNA fragmentation, cell shrinkage occurs (Wesselbory et al., Cell Immunol, 148, 234-41, 1993), where water is actively pushed out of the cell. Apoptotic cells are then fragmented into apoptotic bodies that are engulfed by adjacent cells or cells of the reticuloendothelial system.

  A second distinct feature is that when cells undergo apoptosis, phospholipid phosphatidylserine is exposed to the outer surface of the cell plasma membrane (Fadok et al., J Immunol, 148, 2207-16). Page, 1992). Usually, this lipid is localized inside the membrane lipid bilayer. The mechanism responsible for such lipid reversal is currently largely unknown, but its appearance serves as a signal for recognition and phagocytosis of apoptotic cells (Fadok et al., J Immunol, 148). Vol. 2207-16, 1992).

  Apoptosis is tightly regulated under normal physiological conditions. However, in some diseases, this process is unregulated and leads to specific pathologies. Examples of conditions where apoptosis is delayed or inhibited include tumor development and many inflammatory conditions such as acute respiratory distress syndrome (ARDS) and other related conditions (Matete-Bello et al., Am J Respir). Crit Care Med, 56, 1969-77, 1997). Inappropriate or excessive apoptosis may occur under ischemic conditions (stroke, myocardial infarction, etc.) (Linnik et al., Blood, 80, 1750-7, 1992; Gorman et al., J Neurol Sci, 139, 45 52, 1996), under a series of neurodegenerative conditions, under bone marrow suppression (Mori et al., Blood, 92, 101-7, 1998), after chemotherapy or radiation (Lotem et al., Blood, 80, 1750-7, 1992), and many other diseases where cell death is an important feature of the pathology.

Neutrophils, inflammation, and phosphatidylinositol signaling neutrophils are recognized as major cellular mediators in inflammation. They play an essential role in protecting the host, thereby being the first line of defense response mobilized against microbial invasion. Neutrophils are the predominant cell type in the cellular phase in an acute inflammatory response (Edwards S, “Biochemistry and Physiology of the Neutrophil”, Cambridge, UK, Cambridge University Press, 1994). The importance of neutrophils in host protection can be seen in patients who have severe neutropenia and consequently suffer from recurrent bacterial infections (Boxer L and Dale DC, “Neutropenia: causes and consequences ”, Semin Hematol, 2002, 39 (2), 75-81).

  In order to protect the host against pathogen invasion, neutrophils have a large reserve of germicidal weapons, including various reactive oxygen and reactive nitrogen molecular species, which follow the phagocytosis of the invading bacteria. Together with proteases and elastases, are released from the granules into the phagolysosomes. However, some of these damaging substances inevitably leak into the surrounding environment where they can cause tissue damage at the site of inflammation. Furthermore, neutrophils are interleukin-1α, interleukin 1β, interleukin 6, tumor necrosis factor α (TNFα), IL1 receptor antagonist α, and chemokine interleukin 8, macrophage inflammatory protein 1α (MIP− 1α), MIP-1β, and other immune cells because of their ability to synthesize both a number of pro- and anti-inflammatory cytokines, including growth related gene product-α (Cassatell MA, Cytokines Produced by Polymorphonuclear Neutrophils: Molecular and Biological Aspects, Austin, Tx, RG Landes, 1996).

  Inappropriate activation of these cells and the release of proteases and elastases are recognized to contribute to many diseases including chronic obstructive pulmonary disease (COPD) and acute respiratory distress syndrome (ARDS) (Nogueera) Thorax, 2001, 56, 432-437; Lamb et al., Crit Care Med, September 1999, 27 (9), 1738-44).

  One of the regulatory mechanisms that exist to limit the degree of harmful activation is priming. Through the action of priming, the activation level of the cell and the subsequent reaction can be adjusted so that a sequence of activation states can be achieved. One such priming factor found at that time at the site of inflammation is granulocyte macrophage colony stimulating factor (GM-CSF), which is a number of activated cell types including monocytes and T cells. Is a 22 kDa pro-inflammatory cytokine released in Preincubation of neutrophils with GM-CSF promotes their ability to respond (prime) to many stimuli, including chemotactic and phagocytic factors, thereby enhancing the inflammatory process ( Lopez et al., J Clin Invest, 1986, 78 (5), 1220-8). In addition, GM-CSF enhances superoxide production (Weisbart et al., Nature, March 28-April 3, 1985, 314 (6009), 361-3), calcium mobilization (Naccache et al., J Immunol). May 15, 1988, 140 (10), 3541-6, and arachidonic acid release and synthesis of leukotriene B4 (DiPersio et al., J Immunol, June 15, 1988, 140 (12). , 4315-22), etc., are known to enhance the biological activity of neutrophils.

  Granulocyte-macrophage colony-stimulating factor activation and neutrophil priming have previously been shown to coincide with stimulation of phosphatidylinositol 3 kinase (al-Shami et al., Blood, February 1, 1997, 89th). (3) Volume, pages 1035-44). More recent work has shown that regulation of phosphatidylinositol 3 kinase activity promotes the conversion of phosphatidylinositol 4,5 diphosphate to phosphatidylinositol 3,4,5-triphosphate, and increases and positives the superoxide anion reaction. (Cadwallader et al., J Immunol, September 15, 2002, 169 (6), 3336-44).

  In addition, neutrophils obtained from mice lacking the PI3K-p85α regulatory subunit produced superoxide anions at levels comparable to unprimed neutrophils, as did wild-type mice. , Production by them is significantly lower than the wild type after priming, which again confirms the role of PI3K in the priming phenomenon leading to superoxide production (Yasui et al., J Leukoc Biol, November 2002). 72 (5), 1020-6). In another knockout study, the PI3K-p110 catalytic subunit is now targeted, reducing the efficiency of neutrophil migration to various chemotactic agents, and knockout mice show reduced neutrophil oxidative burst. As a result, researchers have found that bacteria become persistently viable in the peritoneal cavity (Hirsch et al., Science, February 11, 2000, 287 (5455), 1049-53. ).

To limit the activated state, it is known that neutrophils can circulate between the unprimed state, the primed state, the deprimed state, and the reprimed state. This phenomenon is believed to be due to the control of the level of production and degradation of phosphoinositides, especially phosphatidylinositol 3,4,5-triphosphate. In fact, PtdIns (3,4,5) _{P 3} is an enzyme that hydrolyzes SHIP (SH2-inositol-5-phosphatase) Neutrophils obtained from mice lacking the priming factors such as GM-CSF It was found that the reactivity was increased. In these animals, both macrophages and neutrophils fatally invade the lungs, resulting in high mortality (Helgason et al., Genes Dev, June 1, 1998, Vol. 12 (11), 1610. 20)), indicating that failure to regulate phosphoinositide levels may cause excessive inflammation.

Neutrophils, in addition to the phosphatidylinositol signaling, and survival neutrophil priming, as another result of PtdIns (3,4,5) _{P 3} production, a cellular homologue of retroviral oncogene v-AKT AKT Phosphorylation and activation. The biological consequence of AKT activation in neutrophils is the inhibition of apoptosis and the resulting prolongation of survival, which has a major impact on the number of otherwise short-lived neutrophils It is a phenomenon that gives

  Studies have shown that the inhibition of neutrophil apoptosis that occurs after GM-CSF treatment is mediated by PI3K and AKT, which is inhibited by chemical inhibitors of either of its kinases (Klein et al., J Immunol, 2000, 164 (8), 4286-91). Similarly, in genetic knockout mice, failure to activate PI3K results in the failure of AKT activation by GM-CSF, and subsequent failure to delay neutrophil apoptosis. (Yasui et al., J Leukoc Biol, November 2002, 72 (5), 1020-6).

  The direct anti-apoptotic mechanism of AKT activation in neutrophils is currently unknown, but survival mediated by AKT activation is numerous in apoptosis, including caspase 9, Bad, and Forkhead transcription factors It is known that it appears to be due to AKT's ability to phosphorylate and inactivate facilitating proteins. In addition, NFκB is a transcription factor that promotes survival in response to several apoptotic stimuli, whereas AKT phosphorylates IκB kinase (IKK), a kinase that induces degradation of the NFκB inhibitor IκB. Can also have a positive effect on NFκB.

Phosphatidylinositol signaling and cancer phosphatidylinositol reveal that PI3K activity is physically and functionally related to the transforming activity of viral oncogenes such as SRC tyrosine kinase and polyomavirus middle T antigen Since then, it has been a major focus of cancer research for about 20 years. The same time, so far it is a lipid which has not been known to exist PtdIns (3,4,5) _{P 3} were detected in activated neutrophils (Traynor-Kaplan et al, 1988, Nature, 334, 353-356). Since then, the literature on the role of PI3K, phosphatidylinositol, and cancer has increased logarithmically (for review, see Vivanco I and Sawyers CL (2002), Nature Reviews Cancer, Volume 2, pages 489-501). reference). PtdIns (3, 4, 5) that there is oncogenic potential to _{P 3} is that it is kept under strict control, therefore, regulation of PI3K and PIP3 phosphatase (PTEN, SHIP 1, SHIP2) By means of unstimulated growth, it means that the level is hardly detectable. The PIP3 phosphatase with the clearest involvement in tumorigenesis is PTEN, which is a 3-position lipid phosphatase that converts PIP3 to the original PIP2. This phosphatase mutation, originally identified in breast cancer and glioblastoma, is now recognized as one of the most common proteins mutated in connection with cancer. Like PTEN, mutations associated with phosphatidylinositol and also with cancer include constitutive activity of PI3 kinase and increased AKT activity.

Neutrophil Model International Publication WO02 / 04657 for Apoptosis and Therapeutic Target Search describes a neutrophil model of apoptosis. GM-CSF provides a signal that acts through a signaling cascade and is associated with significant changes, or patterns of changes, in cellular gene expression. Thus, inhibition of neutrophil cell death via apoptosis is accomplished by regulation of “effector genes” that control the process of apoptosis. To date, however, the identity of such “effector genes” and their role in signal transduction pathways leading to biochemical events of cell death have been determined incompletely. So far, many of the genes that have been discovered have some fundamental disadvantage. For example, they may act late in apoptosis after the cells have surely entered the death program, or they may be ubiquitous, ie not limited to a particular cell type. The ideal target for controlling apoptosis is one that acts early in this process and is limited to specific cell types.

  Control of apoptosis is an important therapeutic target because many diseases are due to defects in this process. Therefore, the identification of genes that regulate this process is urgently needed. In other words, once a gene that prevents apoptosis is identified, this gene / gene product, or its function, can be blocked with a drug to cause apoptosis. Similarly, if excessive apoptosis occurs, blocking this process is an important therapeutic goal.

  Based on the above, phosphatidylinositol signaling is GM-CSF for both priming cells to promote cell activity and both delaying the apoptotic process and prolonging cell survival. Clearly it is a fundamental part of the biology of stimulated neutrophils. Similarly, an association between phosphatidylinositol signaling and tumor survival has been demonstrated. The identification of targets that are modulated in relation to phosphatidylinositol signaling in activated neutrophils would clearly be an effective target for cancer therapy. Therefore, it can be concluded that neutrophils stimulated with GM-CSF are an excellent model for conducting studies to identify new therapeutic targets, including targets in inflammation and cancer.

SUMMARY OF THE INVENTION The present invention identifies a number of genes whose expression is associated with early stages in the regulation of apoptosis, ie “apoptosis-related” genes. Identification of these genes and validation of their role in apoptosis can be achieved by using the model assay described in WO 02/04657 as described herein, and also knocking down gene expression using RNAi, This was done by assessing phenotypes that resulted in altered apoptosis progression. These genes are therefore novel targets for therapeutic drugs.

  The present invention provides a method for identifying an agent that modulates the function of an apoptosis-related polypeptide described in Table 1, ie, having the identified sequence, comprising: (a) an apoptosis-related polypeptide having the sequence described in Table 1. Preparing a containing sample and a candidate drug and incubating under conditions that allow the candidate drug (or test drug) to bind to the polypeptide; (b) a sequence described in Table 1B above Measuring the binding of the apoptosis-related polypeptide having to the candidate agent in the sample; and (c) the apoptosis-related polypeptide having the sequence described in Table 1B above to the candidate agent in the sample It is known that the binding does not bind to the polypeptide having the sequence described in Table 1B above to the polypeptide having the sequence described in Table 1. Comparing to binding to a reference substance, wherein the apoptosis-related polypeptide having the sequence described in Table 1B above has an apoptosis having a sequence described in Table 1B as compared to binding to the control substance. Increased binding of the relevant polypeptide to the candidate agent in the sample indicates that the candidate agent modulates the function of an apoptosis-related polypeptide having the sequence set forth in Table 1B. Regarding the method.

  The present invention provides a method for detecting the presence of an apoptosis-related polypeptide having a sequence described in Table 1B in a sample, wherein (a) a biological sample containing DNA or RNA is displayed under hybridization conditions. Contacting with a probe comprising a fragment of at least 15 nucleotides of a nucleic acid having the sequence of 1B; and (b) detecting a double-stranded molecule formed between the probe and the nucleic acid in the sample. And by detecting a double-stranded molecule, there is also provided a method wherein the presence of an apoptosis-related polypeptide having the sequence set forth in Table 1B above is indicated in the sample.

  The level of gene expression may be measured by any other suitable method. Reduced gene expression can be detected by measuring the expression of apoptosis-related genes in the treated cells relative to untreated cells. Gene expression may be preferably measured by detecting a nucleic acid encoding an apoptosis-related polypeptide, such as an apoptosis-related mRNA transcript or fragment thereof. In one embodiment, the amplification techniques described herein can be used as a method of measuring mRNA transcripts. In another embodiment, apoptosis-related gene expression can be measured by detecting an apoptosis-related polypeptide gene product or fragment thereof, eg, with an agent that binds to the apoptosis-related polypeptide. Suitable drugs include antibodies.

  Accordingly, in another aspect, the invention provides a method for detecting the presence of an apoptosis-related polypeptide having a sequence set forth in Table 1B in a sample, comprising: (a) an apoptosis-related polyply having a sequence set forth in Table 1B. Providing an antibody capable of binding to a peptide; (b) incubating a biological sample with the antibody under conditions that allow formation of an antibody-antigen complex; and (c) an antibody-antigen complex comprising the antibody. And detecting an antibody-antigen complex, wherein the presence of an apoptosis-related polypeptide having the sequence described in Table 1B is indicated in the sample.

  In another aspect, a method for modulating apoptosis of a cell comprising increasing, decreasing or otherwise altering the functional activity of an apoptosis-related polypeptide or nucleic acid encoding it. A method comprising: In one embodiment, the modulation of apoptosis is inhibition. In another embodiment, the modulation of apoptosis results in cell survival.

  Preferably, the apoptosis-related polypeptide has a sequence specified in Table 1B.

  In the context of the present invention, within the scope of the term “change in the functional activity of an apoptosis-related polypeptide or the nucleic acid that encodes it”, the natural protein, under normal circumstances, ie in a single cell under natural conditions This includes an increase, decrease, or other change in the activity of an apoptosis-related polypeptide as compared to its function. This further includes increasing or decreasing the expression level of the nucleic acid encoding the apoptosis-related polypeptide and / or changing the intracellular distribution and / or changing the intracellular distribution of the apoptosis-related polypeptide.

  In one embodiment, the method includes reducing the expression of an apoptosis-related gene. In a preferred embodiment, apoptosis-related gene expression is greater than 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500% of normal levels in untreated cells. Reduced by ultra or more.

  It may be preferable to reduce the expression of apoptosis-related genes by antisense expression. Other methods of reducing the expression of apoptosis-related genes will be recognized by those skilled in the art. This includes introducing small molecules, including dominant negative peptides, or RNA molecules such as siRNA molecules that cause reduced gene expression by RNA interference. Suitable siRNA molecules are described in the Examples section herein.

  In another embodiment, the method comprises increasing apoptosis-related gene expression. In a preferred embodiment, apoptosis-related gene expression is greater than 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500% of normal levels in untreated cells. Increased beyond or greater than this.

  Preferably, the method allows for providing an expression vector comprising a nucleic acid sequence encoding an apoptosis-related polypeptide, introducing the expression vector into a cell, and expressing the encoded polypeptide in the cell. Maintaining the cell under conditions to achieve. As defined herein, an apoptosis-related polypeptide or a nucleic acid encoding an apoptosis-related polypeptide also encompasses fragments thereof.

  Accordingly, the present invention is a method for modulating apoptosis in a cell, comprising the steps of (a) introducing and transforming into a cell a double-stranded nucleic acid sequence having the sequence shown in Table 1B or a complementary sequence thereof. The nucleic acid sequence is operably linked to a regulatory sequence; and (b) culturing the cell under conditions under which the nucleic acid sequence is expressed, thereby causing apoptosis in the cell. Relates to a method for modulating the signal.

  The present invention relates to a method of modulating apoptosis in a cell, comprising the steps of (a) introducing and transforming into a cell a double-stranded nucleic acid sequence encoding a polypeptide having the sequence described in Table 1B. The nucleic acid sequence is operably linked to a regulatory sequence; and (b) culturing the cell under conditions under which the nucleic acid sequence is expressed, whereby the cell It also relates to a method of modulating apoptosis.

  In another aspect, the invention relates to a method of modulating apoptosis in a cell, which encodes a polypeptide having (a) at least 80% sequence identity to a polypeptide having the sequence set forth in Table 1B Introducing a double stranded nucleic acid sequence into the cell for transformation, wherein the nucleic acid sequence is operably linked to a regulatory sequence; and (b) the nucleic acid sequence is expressed. A method of modulating apoptosis in said cells, comprising culturing said cells under conditions.

  Another aspect of the invention is a method of modulating apoptosis in a cell, comprising: (a) an isolated nucleic acid molecule comprising a regulatory sequence operably linked to a nucleic acid sequence encoding a ribonucleic acid (RNA) precursor. A sequence of 15-40 nucleotides in length, wherein the precursor is (i) identical to consecutive 15-40 nucleotides in the sequence set forth in Table 1B (Ii) a second stem portion comprising a 15-40 nucleotide long sequence complementary to the contiguous 15-40 nucleotides in the sequence set forth in Table 1B; and (iii) A loop portion connecting two stem portions, and the first stem portion and the second stem portion can hybridize with each other to form a double-stranded stem. That, steps; and, under the conditions (b) the nucleic acid sequence is expressed, comprising the step of culturing the cells, thereby to modulate apoptosis of the cells, relating to the method.

  The cells may be suitable therapeutic targets for the treatment of disease. For example, such cells can be cancer cells, cells involved in inflammatory diseases, cells involved in autoimmune diseases or neurodegenerative disorders.

  In any of the methods described herein, the nucleic acid sequence is the nucleic acid sequence listed in Table 1B, or a complementary sequence thereof. The nucleic acid sequence can encode a polypeptide having the sequence set forth in Table 1B. The nucleic acid sequence may encode a polypeptide having 80% sequence identity to a polypeptide having the sequence set forth in Table 1B.

  The methods described herein can be used to increase or decrease apoptosis.

  Another feature of the invention is an RNA precursor encoded by the nucleic acid sequence described or specified in Table 1B. Such RNA precursors can be included in the composition as a bioactive component. Such RNA precursors or compositions can be used by contacting the tissue with an RNA precursor or composition to treat a disease or condition characterized by abnormal apoptosis in mammalian tissue. The disease may suitably be a cancer or inflammatory disease.

  Another feature of the present invention is a host cell transformed with the methods described herein. Such host cells can contain all or part of the nucleic acid sequences set forth in Table 1B. Such host cells can express all or part of a polypeptide having the sequence set forth in Table 1B.

  In another aspect of the invention, the apoptosis-related genes identified in Table 1B can be used to identify downstream targets. For example, proteins that interact with or are regulated by apoptosis-related genes can be identified.

  The methods described herein can be used to provide an anti-apoptotic protein to a mammal, where the method introduces mammalian cells transformed by the methods described herein into the mammal. Including doing.

  Modulation of apoptosis can be done for therapeutic purposes. Accordingly, the present invention also relates to a pharmaceutical composition comprising an apoptosis-related nucleic acid sequence having the sequence shown in Table 1B as an active ingredient, and further comprising a pharmaceutically acceptable carrier.

  The present invention further relates to a pharmaceutical composition comprising an apoptosis-related polypeptide having the sequence shown in Table 1B as an active ingredient, and further comprising a pharmaceutically acceptable carrier.

  Accordingly, another aspect of the present invention provides a method for treating a disease characterized by abnormal apoptosis, comprising administering to an individual a modulator of apoptosis-related gene expression or functional activity.

  Thus, in one embodiment, the present invention further relates to a pharmaceutical composition comprising as an active ingredient an antibody against an apoptosis-related nucleic acid having the sequence described in Table 1B, and further comprising a pharmaceutically acceptable carrier. Such an antibody is a method of diagnosing a disease or pathological condition characterized by abnormal apoptosis in mammalian tissue, comprising contacting the tissue with the antibody and detecting an antibody / antigen complex. And can be used in methods where the detection indicates the presence of the disease or condition.

  In another aspect, the present invention relates to a pharmaceutical composition comprising as an active ingredient an antibody against an apoptosis-related polypeptide having the sequence shown in Table 1B, and further comprising a pharmaceutically acceptable carrier. Such an antibody is a method of diagnosing a disease or pathological condition characterized by abnormal apoptosis in mammalian tissue, comprising contacting the tissue with the antibody and detecting an antibody / antigen complex. And the detection can be used in a way that indicates the presence of the disease or condition.

  Yet another aspect of the invention is a method of treating a disease or pathological condition characterized by abnormal apoptosis in a mammalian tissue, wherein the tissue is an antagonist of an apoptosis-related polypeptide having the sequence set forth in Table 1B. And a method comprising contacting with.

  Another aspect of the invention is a method of treating a disease or pathological condition characterized by abnormal apoptosis in a mammalian tissue, wherein the tissue is treated with an agonist of an apoptosis-related polypeptide having the sequence set forth in Table 1B. It relates to a method comprising contacting.

  The present invention provides a kit for treating a disease or pathological condition characterized by abnormal apoptosis in mammalian tissue, wherein: (a) a polypeptide encoded by a nucleic acid having the sequence set forth in Table 1B; It also relates to a kit comprising a nucleic acid having the nucleotide sequence set forth in Table 1B; or (c) an antibody that recognizes an epitope of the polypeptide set forth in (a).

  Cells useful in the methods of the invention may be obtained from any source, such as those obtained from primary cultures or established cell lines, or those obtained from organ cultures or in vivo. Cell lines useful in the present invention include cells and cell lines derived from the hematopoietic system. Suitable cells include HeLa, U937 (monocytes), TF1, HEK293 (T), neutrophils or primary cultures of cells with neutrophil characteristics such as HL60 cells, mouse FDCP-1, FDCPmix, 3T3 Primary or human stem cells are included. Other suitable cells include cancer cell lines such as U251, SKOV3, OVCAR3, MCF7PC3, HCT15, 786-0, HT29, M-14, Molt4, CEM, K562, Daudi, DU145, NCI-H460 and LnCap. It is.

  In the case of methods for treatment, the cells can be disease-related cells such as cancer, inflammation, autoimmunity, or neurodegenerative related cells.

  In another aspect, there is provided the use of a modulator for apoptosis-related gene expression or activity in the manufacture of a medicament for use in the treatment of disease.

  The modulator is preferably an antisense molecule, or an RNA molecule that mediates RNA interference, and thus reduces the expression of apoptosis-related genes.

  Suitable diseases include cancer, inflammation, autoimmune diseases, and neurodegenerative disorders.

  A number of inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), rheumatoid arthritis (RA), and inflammatory bowel disease (IBD) are a) cytokines and primarily bone marrow cells. It is characterized by increased levels and expression of growth factors that act on, b) prolonged survival of bone marrow cells, and c) long-term activation of bone marrow cells.

  Thus, the increased number of activated bone marrow cells such as neutrophils is associated with the pathology of these many chronic and acute inflammatory diseases and has been strongly implicated (Williams TJ and Jose). PJ, Novartis Found Symp, 2001, 234, 136-41; Discussion 141-8; Barnes PJ, Chest, February 2000, 117 (2), 10S-4S; Nadel JA, Chest, February 2000, 117 (2), 10S-4S; Ward I et al., Trends Pharmacol Sci, December 1999, 20 (12), 503-9; Bradbury J and Lakatos L , Drug Discover Today, May 1, 2001, No. 6 (9 , Pp. 441-442).

  Accordingly, in one embodiment, there is provided the use of an apoptosis-related gene or an agent that alters cellular apoptosis-related gene expression in the treatment of inflammatory diseases through modulation of bone marrow cell apoptosis.

  In the context of the present invention, the term “bone marrow cells” refers to terminally differentiated, non-dividing cells of the myeloid lineage. These cells include neutrophils, eosinophils, and monocytes / macrophages. In one embodiment of aspects of the invention, the bone marrow cells are neutrophils, eosinophils, or monocytes / macrophages.

  Inflammatory diseases include sepsis, acute respiratory distress syndrome, preeclampsia, myocardial ischemia, reperfusion injury, psoriasis, asthma, COPD, bronchiolitis, cystic fibrosis, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease And diseases such as ulcerative colitis, but are not limited thereto.

  In another aspect of the invention, RNA molecules are provided that can interfere with the expression of apoptosis-related genes having the sequences set forth in Table 1B. The RNAi molecule preferably has the sequence described in the Examples section of this specification.

  Another aspect of the invention is an array comprising at least two apoptosis-related genes having the nucleic acid sequences set forth in Table 1B. The nucleic acid sequence can be a DNA sequence. The nucleic acid sequence can also be an RNA sequence.

  Another aspect of the invention is an array comprising at least two apoptosis-related proteins having a polypeptide having the sequence set forth in Table 1B.

  Apoptosis-related genes identified in Table 1B of the present specification include: 1) “effector” genes (anti-apoptotic genes) involved in cell defense mechanisms aimed at inhibiting apoptosis, and thus genes corresponding to therapeutic targets And 2) genes that constitute aspects of the apoptosis and / or GM-CSF signal cascade, and therefore correspond to therapeutic targets, and 3) genes that initiate the apoptotic process (proapoptotic genes), and thus therapeutic targets And 4) genes related to the process of apoptosis and defense, which will help in understanding the main pathways, processes and mechanisms and thus lead to the identification of therapeutic targets , Is included.

  Specifically, the genes identified in the present invention are protein kinases and G protein coupled receptors (GPCRs).

Protein kinase kinases regulate a number of different cell growth, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been linked to various disease states including inflammation, cancer, arteriosclerosis, and psoriasis. Protein reversible phosphorylation is a major strategy for controlling the activity of eukaryotic cells. Of the 10,000 proteins active in typical mammalian cells, it is estimated that over 1000 proteins are phosphorylated. Phosphorylation occurs in response to extracellular signals (such as hormones, neurotransmitters, growth factors, and differentiation factors), cell cycle checkpoints, and environmental or nutrient stress, which is roughly a molecular switch. Is similar to turning on. When the switch is turned on, the appropriate protein kinase activates metabolic enzymes, regulatory proteins, receptors, cytoskeletal proteins, ion channels, pumps, or transcription factors.

  Kinases are the largest of the known protein groups and constitute a superfamily of enzymes with a wide range of functions and specificities (eg, Hardie, G. and Hanks, S. (1995), The Protein Kinase Facts Books, Vol I: 7-20, Academic Press, San Diego, Calif.).

G protein-coupled receptors G protein-coupled receptors (GPCRs) constitute a major class of proteins that signal within cells. A GPCR has three structural domains. That is, the amino-terminal extracellular domain, the transmembrane domain, and the carboxy-terminal intracellular domain, which contains seven transmembrane segments, three extracellular loops, and three intracellular loops. Binding of a ligand to the extracellular portion of a GPCR transmits a signal into the cell, resulting in a change in the biological or physiological properties of the cell. GPCRs, together with G proteins and effectors (intracellular enzymes and channels modulated by G proteins), are components of a modular signaling system that connects the state of second messengers in cells to extracellular inputs.

  GPCR genes and gene products are potential causative agents of disease (Spiegel et al., J. Clin. Invest, 92, 1119-1125 (1993); McKusick et al., J. Med. Genet, 30 1-26 (1993)). Various forms of retinitis pigmentosa (Nathans et al., Annu. Rev. Genet, 26, 403-424 (1992)), depending on the specific deficiency in the rhodopsin gene and in the V2 vasopressin receptor gene, and It has been shown that nephrogenic diabetes insipidus (Holtzman et al., Hum. Mol. Genet, Vol. 2, 1201-1204 (1993)) is caused. These receptors are crucial for both the central nervous system and peripheral physiological processes. Evolutionary analysis suggests that the ancestors of these proteins originated in concert with complex body plans and nervous systems.

  The GPCR protein superfamily is divided into five families. That is, receptors represented by more than 200 unique members, typically Family I, rhodopsin and β2 adrenergic receptors (Dohlman et al., Annu. Rev. Biochem, 60, 653-688 (1991) )); Family II, parathyroid hormone / calcitonin / secretin receptor family (Juppner et al., Science, 254, 1024-1026 (1991); Lin et al., Science, 254, 1022-1024 (1991) Family III, a metabotropic glutamate receptor family (Nakanishi, Science, 258, 597-603 (1992)); Family IV, important for chemotaxis and development of D. discoideum cAMP receptor family (Klein et al., Science, 241, pp. 1467-1472 (1988)); and fungal conjugation pheromone receptors such as family V, STE2 (Kurjan, Annu. Rev. Biochem, vol. 61, 1097 to 1129, 1992).

  There are also seven putative hydrophobic segments, but there are also a few other proteins that are not associated with GPCRs, which do not show association with G proteins. Drosophila expresses a seven-transmembrane segment protein called the bride of sevenless (boss), a photoreceptor-specific protein, which has been studied in detail but evidence that it is still a GPCR (Hart et al., Proc. Natl. Acad. Sci. USA, 90, 5047-5051 (1993)). The Drosophila gene frizzled (fz) is also thought to be a protein with seven transmembrane segments. Like boss, fz does not show association with G protein (Vinson et al., Nature, 338, 263-264 (1989)).

  G proteins are a heterotrimeric protein family composed of α, β, and γ subunits that bind to guanine nucleotides. Usually these proteins are linked to cell surface receptors, for example receptors containing seven transmembrane segments. After the ligand binds to the GPCR, a conformational change is transmitted to the G protein, thereby causing the α subunit to exchange from the bound GDP molecule to the GTP molecule and the separation from the βγ subunit. GTP-linked α subunits usually function as effector modulating moieties, leading to the production of second messengers such as cAMP (eg, by activation of adenyl cyclase), diacylglycerol, or inositol phosphates. More than 20 different types of alpha subunit are known in humans. The pool of β and γ subunits to which these subunits bind is smaller. Examples of mammalian G proteins include Gi, Go, Gq, Gs, and Gt. The G protein is described in detail in Rodish et al., Molecular Cell Biology (Scientific American Books Inc., New York, NY, 1995), the contents of which are incorporated herein by reference. GPCRs, G proteins, and G protein-linked effectors and second messenger systems are reviewed in The G-Protein Linked Receptor Fact Book, edited by Watson et al., Academic Press (1994).

  Thus, kinases and GPCRs are major subjects of drug action and drug development. Therefore, identifying and characterizing previously unknown kinases and GPCRs is valuable for the field of pharmaceutical development. The present invention advances the state of the art by identifying the role in apoptosis of specific kinases and GPCRs whose roles have not been previously identified.

  Thus, GPCR and kinase polypeptide modulators identified herein as apoptosis-related genes can be identified using standard assays for GPCR activity and kinase activity. Suitable assays are well known to those skilled in the art.

  In a preferred embodiment of aspects of the invention, the apoptosis-related gene or protein, or the gene or protein whose sequence is specified in Table 1B, is MAK, GPR86, PCTAIRE protein kinase 3, BAI2, pp125, STK6, ULK1, serine / Threonine kinase 16, ribosomal S6 protein kinase, TLK2, ethanolamine kinase, MNK1 (MAP kinase-interacting serine / threonine kinase 1), FLJ20559, FLJ13351, NTKL, CDC42 binding protein kinase β (DMPK-like), RBSK6 ribokinase , DAGK, G protein-coupled receptor 12, PRK, mitogen-activated protein kinase kinase Selected from zase 5, phosphatidylinositol 4-kinase catalytic β polypeptide, fms-related tyrosine kinase 4, PSKH1 protein serine kinase H1, and inositol 1,4,5-triphosphate 3-kinase C.

  The present invention provides a method for identifying an inhibitor of apoptosis-related protein encoded by a gene selected from Table 1B, comprising providing a preparation containing the encoded protein; Incubating the preparation with the test agent under conditions that allow the agent to bind to the protein; and detecting the presence or absence of a signal generated from the interaction of the agent with the protein. By providing whether the test agent interacts with the protein, thereby determining whether the test agent inhibits an apoptosis-related protein.

  In the context of the present invention, a preparation containing the encoded protein comprises a cell expressing the protein, a purified protein including a recombinantly produced protein, or a membrane fraction containing the protein. May be included. Further, when using the methods of the invention to identify inhibitor compounds, the protein preferably comprises a human protein described herein, but in another embodiment, other mammalian homologs. Functional variants of this structure, including (eg, dogs, pigs, rats, mice), fusion proteins (eg, sequences added to aid purification, eg, GST, glutathione S-transferase sequences) Protein), functional domains or fragments of the protein (eg, the active catalytic domain of a protein kinase) can also be used.

  The present invention provides a method for identifying an inhibitor of apoptosis-related protein kinase encoded by a protein kinase gene selected from Table 1B, comprising the step of preparing a sample containing the encoded protein kinase; Incubating the sample with the test agent under conditions that allow the test agent to bind to the protein kinase; by detecting a change in the phosphotransferase activity of the protein kinase, Further provided is a method comprising determining whether to interact with a protein kinase, thereby determining whether the test agent inhibits the apoptosis-related protein kinase.

The present invention is a method for identifying an inhibitor of an apoptosis-related cell surface receptor protein encoded by a gene selected from Table 1B, encoded by a cell surface receptor gene selected from Table 1B Providing a cell expressing the protein on its surface linked to a second component capable of providing a detectable signal in response to binding of the agent to the protein; allowing binding to the protein Contact with a test agent to be screened under conditions; and whether the agent binds to and inhibits the protein, the presence or absence of a signal generated from the interaction of the compound with the protein. Determining by detecting, and thereby selected from Table 1B above The activity of apoptosis-associated cell surface receptor protein that is encoded by a gene, further comprising the step, determining whether the test agent inhibits. In the context of this method, the second component that can provide a detectable signal can be a G protein, eg, a G protein of Gi, Go, Gs, G ₁₆ , G ₁₅ , Gq or G _12-13 . In another embodiment, the second component can be βarrestin.

  The present invention is a method for determining whether a chemical compound specifically binds to and inhibits an apoptosis-related protein encoded by a gene selected from Table 1B, which produces a second messenger response. And contacting a cell expressing a protein encoded by a gene selected from Table 1B with a chemical compound under conditions suitable for protein inhibition, and in the presence and absence of the chemical compound. And measuring the second messenger response in such cells, such cells normally do not express the protein, and the compound is selected from Table 1B by the change in the second messenger response in the presence of the chemical compound. Apoptosis-related tongue encoded by the expressed gene Further provides a method that is shown to inhibit the click quality.

  In the context of the present invention, the second messenger reaction comprises activation of chloride channels, changes in intracellular calcium ion levels, release of inositol phosphate, release of arachidonic acid, GTPγS binding, activation of MAP kinase, accumulation of cAMP, It is selected from, but not limited to, a change in intracellular potassium ion level or a change in intracellular sodium ion level. Second messenger response can be measured by various methods well known in the art or by changes in reporter gene activity. Such reporter genes include, but are not limited to, secreted alkaline phosphatase, luciferase, and β-galactosidase.

  The present invention is a method for identifying an apoptotic activator comprising the steps of providing a preparation containing a protein encoded by a gene selected from Table 1B; a test agent to be screened binds to the protein Incubating the preparation with the test agent under conditions that allow the test agent to generate; whether the test agent binds to and inhibits the protein from interaction with the protein by the agent Further provided is a method comprising the steps of determining by detecting the presence or absence of a signal to be performed and determining whether any test agent that inhibits the protein is an active agent for apoptosis.

  The present invention is a method for identifying a drug having pro-apoptotic activity, wherein a test drug interacts with a protein encoded by a gene selected from Table 1B and a preparation containing the encoded protein. Further provided is a method comprising the steps of determining whether to act and determining whether any agent that interacts with the encoded protein is an active agent of apoptosis.

  The present invention is a method for identifying an inhibitor of tumor cell growth, comprising preparing a preparation containing a protein encoded by a gene selected from Table 1B; Incubating the preparation with the test agent under conditions that permit binding to the test; by detecting the presence or absence of a signal generated from the interaction of the agent with the protein by the agent. Further comprising: determining whether an agent binds to and inhibits the protein; and determining whether any test agent that inhibits the protein is an inhibitor of tumor cell growth. provide.

  The present invention relates to a method of identifying a drug that inhibits the growth of tumor cells, wherein the test drug is a protein containing a protein encoded by a gene selected from Table 1B and the protein encoded above. Further provided is a method comprising determining whether to interact and determining whether any agent that interacts with the encoded protein is an inhibitor of tumor cell growth.

  In the method of the present invention, the determination of whether an agent inhibits the growth of tumor cells may be performed by any method known to those skilled in the art, such as BrdU or radioisotope-labeled thymidine, DNA synthesis. This may be done by labeling possible cells.

  The present invention is a method for identifying an agent that inhibits the growth of tumor cells, wherein the test agent comprises a protein encoded by a gene selected from Table 1B and a preparation containing the encoded protein. There is further provided a method comprising the steps of: determining whether or not any agent that interacts with the encoded protein is an inhibitor of tumor cell growth.

  The present invention is a method for identifying an agent that inhibits tumor growth, wherein a test agent interacts with a protein encoded by a gene selected from Table 1B in a sample containing the encoded protein. Further provided is a method comprising determining whether to act and determining whether any agent that interacts with the encoded protein is an inhibitor of tumor growth.

  The present invention provides a method for identifying an agent having pro-apoptotic activity, comprising determining whether a test agent modulates the activity or expression of a protein encoded by a gene selected from Table 1B, and Further provided is a method comprising determining whether any agent that modulates activity or expression is an active agent of apoptosis.

  The present invention is a method for identifying an agent that inhibits tumor cell growth, comprising determining whether a test agent modulates the activity or expression of a protein encoded by a gene selected from Table 1B; and Further provided is a method comprising determining whether any agent that modulates the activity or expression is an inhibitor of tumor cell growth.

  The present invention is a method for identifying an agent that inhibits tumor cell growth, comprising determining whether a test agent modulates the activity or expression of a protein encoded by a gene selected from Table 1B; and Further provided is a method comprising determining whether any agent that modulates the activity or expression is an inhibitor of tumor cell growth.

  The present invention is a method for identifying an agent that inhibits tumor growth, comprising determining whether a test agent modulates the activity or expression of a protein encoded by a gene selected from Table 1B; and Further provided is a method comprising determining whether any agent that modulates the activity or expression is an inhibitor of tumor growth.

  In the above method, the test agent may be selected from, but not limited to, a low molecular weight (less than 5000 dalton) organic molecule, antibody, antibody fragment, antisense oligonucleotide, small molecule inhibitory dsRNA, or ribozyme. .

  The present invention also provides an inhibitor of tumor cell proliferation or tumor growth identified by the above method. Such inhibitors of tumor cell proliferation or tumor growth are low molecular weight organic molecules (less than about 5000 Daltons), antibodies, antibody fragments, antisense oligonucleotides, small molecule inhibitory dsRNA (eg, as used for RNA interference) ), Or a ribozyme, but is not limited thereto.

  The present invention is a method for inhibiting tumor growth in a mammal in need of such treatment, wherein a gene selected from Table 1B is used in the mammal in need of such treatment. Further comprising the step of administering an inhibitor to the activity or expression of the apoptosis-related protein encoded by said method, wherein said administration is performed in an amount effective to reduce tumor growth in said mammal. In the context of this method, the inhibitor may be selected from, but not limited to, low molecular weight (<5000 Dalton) organic molecules, antibodies, antibody fragments, antisense oligonucleotides, small molecule inhibitory dsRNAs, and ribozymes. Absent. The small molecule inhibitory dsRNAs described in the Methods section herein for performing RNA interference experiments that reduce the expression level of a protein encoded by a gene selected from Table 1B are used to perform this method. A specific example of an inhibitor that can be used.

  The present invention is a method for inhibiting the growth of tumor cells in a mammal in need of such treatment, and is selected from Table 1B for mammals in need of such treatment. Further provided is a method comprising administering an inhibitor to the activity or expression of a protein encoded by the gene, wherein the administration is performed in an amount effective to attenuate growth of tumor cells in the mammal. In the context of this method, the inhibitor may be selected from, but not limited to, low molecular weight (<5000 Dalton) organic molecules, antibodies, antibody fragments, antisense oligonucleotides, small molecule inhibitory dsRNAs, and ribozymes. Absent. The small molecule inhibitory dsRNAs described in the Methods section herein for performing RNA interference experiments that reduce the expression level of a protein encoded by a gene selected from Table 1B are used to perform this method. A specific example of an inhibitor that can be used.

  The present invention is a method of stimulating tumor cell apoptosis in a mammal in need of such treatment, selected from Table 1B for mammals in need of such treatment. Further provided is a method comprising administering an inhibitor to the activity or expression of a protein encoded by the gene, wherein said administration is performed in an amount effective to stimulate apoptosis of tumor cells in said mammal. In the context of this method, the inhibitor may be selected from, but is not limited to, low molecular weight organic molecules, antibodies, antibody fragments, antisense oligonucleotides, small molecule inhibitory dsRNAs, and ribozymes. The small molecule inhibitory dsRNAs described in the Methods section herein for performing RNA interference experiments that reduce the expression level of a protein encoded by a gene selected from Table 1B are used to perform this method. A specific example of an inhibitor that can be used.

  The present invention is a method for preparing a composition comprising a chemical compound that specifically binds to and inhibits an apoptosis-related protein encoded by a gene selected from Table 1B, wherein the composition is selected from Table 1B. Contacting an apoptosis-related protein encoded by the gene with a test chemical compound under conditions suitable for such compound to bind to the protein, against the encoded protein by the test chemical compound Detecting specific binding and inhibition, and the test chemical so identified, or a functional analog or homolog of the test chemical, mixed with a carrier, thereby preparing the composition Such cells are usually tampered with by the above encoded tamper Do not express the quality, further provides the above method.

  The present invention is a method of preparing a composition comprising a chemical compound that specifically inhibits an apoptosis-related protein encoded by a gene selected from Table 1B, wherein the composition is encoded by a gene selected from Table 1B. Contacting an apoptosis-related protein with a test chemical compound under conditions suitable for such compound to bind to the protein, detecting specific inhibition of the protein by the test chemical compound, and Further provided is a method comprising the step of mixing the test chemical so identified, or a functional analog or homologue of the test chemical with a carrier, thereby preparing the composition.

  The present invention relates to a method for identifying a neoplasm responsive to treatment with a compound that selectively inhibits neoplasia, which is directed against an apoptosis-related protein encoded by a gene selected from Table 1B. A method is provided that includes exposing a sample of a neoplasm to a compound having inhibitory activity and determining whether the compound inhibits the neoplasm.

  The present invention provides a method for identifying a neoplasm responsive to treatment with a compound that selectively inhibits a neoplasm, comprising: (a) removing a neoplastic tissue sample from a patient; (b) from the sample (C) contacting the sample of the cells with a compound having an inhibitory activity against an apoptosis-related protein encoded by a gene selected from Table 1B. (D) comparing cell growth in the presence of the compound to cell growth in the absence of the compound; and (e) neoplastic growth is responsive to inhibition by the compound. There is further provided a method comprising determining whether there is.

  The present invention provides a method for identifying a neoplasm responsive to treatment with a compound that selectively inhibits a neoplasm, comprising: (a) removing a neoplastic tissue sample from a patient; (b) from the sample (C) contacting the sample of the cells with a compound having an inhibitory activity against an apoptosis-related protein encoded by a gene selected from Table 1B. (D) comparing the number of apoptotic cells in the presence of the compound with the number of apoptotic cells in the absence of the compound; and (e) whether the compound promotes increased apoptosis in the tumor. Further provided is a method comprising the step of determining whether or not.

  The present invention is a method of identifying a neoplasm responsive to treatment with an inhibitor of an apoptosis-related protein encoded by a gene selected from Table 1B, comprising: Measuring the level of protein encoded by the selected gene, wherein the level of apoptosis-related protein encoded by the gene selected from Table 1B in the neoplastic tissue is compared to normal tissue. By raising, the neoplasm further provides a method that is shown to be potentially treated by an inhibitor to an apoptosis-related protein encoded by a gene selected from Table 1B.

  In one embodiment of this method, the measurement of the level of protein encoded by the gene selected from Table 1B in the neoplastic tissue is encoded by the gene selected from Table 1B in the neoplastic tissue sample. Measuring the amount of protein to be produced.

  In another embodiment of this method, the measurement of the level of protein encoded by a gene selected from Table 1B in a neoplastic tissue sample is determined by the gene selected from Table 1B in the neoplastic tissue sample. Measuring the amount of mRNA encoding the encoded protein.

  In yet another embodiment of this method, the determination of the level of protein encoded by a gene selected from Table 1B in a neoplastic tissue sample is performed by measuring the level of a gene selected from Table 1B in the neoplastic tissue sample. Measuring the biochemical activity of the protein encoded by. For example, when the apoptosis-related protein is a protein kinase, autophosphorylation or phosphorylation of a protein substrate or peptide substrate can be measured. In the case of apoptosis-related GPCR proteins, the measurement result of the activation level of downstream signal transduction pathway components can be evaluated.

  The present invention is a method for identifying a neoplasm in a patient responsive to treatment with an inhibitor of an apoptosis-related protein encoded by a gene selected from Table 1B, comprising (a) suspected neoplasm Obtaining a tissue sample from the patient; and (b) immunizing against an apoptosis-related protein encoded by a gene selected from Table 1B under conditions effective to allow formation of an immune complex. Contacting with a reactive antibody; and (c) detecting the complex so formed and encoded by a gene selected from Table 1B in said neoplastic tissue Due to the increased amount of apoptosis-related protein compared to normal tissue, the neoplasia contains a gene selected from Table 1B Therefore further provides methods indicates that there is likely to be treated with inhibitors to an apoptosis-related protein that is encoded.

  The present invention describes human genes involved in apoptosis. Such genes can be used in the assays described herein to determine whether a candidate substance is an inhibitor of apoptosis. These same assays can also be used to determine whether the substance is a pro-apoptotic substance.

  Such assays include binding assays, in which the extent of binding of the substance to the polypeptides described herein is measured. Such assays also include determining whether the substance prevents binding between the polypeptide described herein and another substance known to bind to the polypeptide. It is.

  In these assays, the presence or absence of a polypeptide described herein in a sample is added to the sample with a substance known to bind to the polypeptide, and whether or not binding occurs is determined. Also included are assays determined by Alternatively, a nucleic acid probe whose sequence is based on a nucleic acid encoding the polypeptide is used to determine whether the nucleic acid encoding the polypeptide is present or absent in the sample You can also By adding a probe to the sample and incubating under hybridization conditions, binding to the nucleic acid, if present in the sample, can be caused. Such probes can be labeled so that it can be more easily determined whether binding has occurred. Alternatively, instead of nucleic acid probes, antibodies specific for these nucleic acids or polypeptides can be used.

  In situations where it is desirable to know whether a mutation has occurred in a nucleic acid encoding a polypeptide described herein, a sample containing this nucleic acid is obtained and the mutation is considered to have occurred. A region specific nucleic acid probe can be used. A lack of binding compared to a sample containing an equivalent nucleic acid that does not contain a mutation indicates that the nucleic acid contains a mutation at that position. If it is not known where the mutation is occurring in the nucleic acid, a sequential series of probes covering the full length of the nucleic acid can be used.

  These assays should be used to determine whether the polypeptides described herein are overexpressed in certain tissues, such as tumors or tissues suspected of containing hyperproliferating cells. Can do. A fixed amount of a known ligand can be added to a sample obtained from the tissue, whereby the ligand binds to the polypeptide. If the amount of binding ligand-polypeptide complex in the sample is greater than the normal amount, the polypeptide is overexpressed. “Normal” means the amount of polypeptide detected in another tissue from the same organism, or a sample of equivalent tissue from another organism, or a baseline level of expression measured in advance. sell.

  Any of the assays described above can be incorporated into kits that measure the presence / absence of the nucleic acids or polypeptides described herein, or the level of expression of the polypeptides described herein.

Unless defined otherwise, all technical and scientific terms used herein are generally defined by persons of ordinary skill in the art (eg, cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Has the same meaning as understood. Standard techniques of molecular biological, genetic, and biochemical techniques are used. See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. And Ausubel et al., Short Protocols in Molecular Biology (1999), 4th edition, John Wiley & Sons, Inc. See also Guthrie et al., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press Inc. (1991), PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, Calif.), McPherson et al., PCR, Volume 1, Oxford University Press (1991), Culture of Animal Cells: A Manual of Basic Technique, 2nd edition (RI Freshney, 1987, Liss, Inc., New York, NY) and Gene Transfer and Expression Protocols, pages 109-128 (E See also J. Murray, The Humana Press Inc., Clifton, NJ). These documents are incorporated herein by reference.
Definition "apoptosis", or programmed cell death, is a controlled intracellular process characterized by condensation of the cell nucleus followed by fragmentation while the plasma membrane remains intact It is. Apoptosis is an active and tightly regulated process that is distinguished by cell shrinkage and packaging of cell contents into apoptotic bodies. Apoptotic bodies are later swallowed by macrophages, thereby avoiding activation of the inflammatory response (for review see Wyllie, Br. Med. Bull., 53, 451-465, 1997). ) Apoptotic death is different from other cellular processes including necrotic cell death and replicative aging.

  As used herein, the term “nucleic acid” refers to a single-stranded or double-stranded nucleic acid, including naturally occurring nucleic acids that exist in nature and / or artificially modified nucleic acids having a modified backbone or base known in the art. Refers to double-stranded DNA and RNA molecules. The “nucleic acid” of the present invention is a nucleic acid encoding the aforementioned apoptosis-related gene or protein. The term further includes polynucleotides that are capable of hybridizing under stringent hybridization conditions to the naturally occurring nucleic acids identified above or their complementary strands.

  As used herein, an “isolated” polypeptide or nucleic acid refers to a material that has been removed from its original environment (eg, the natural environment in which the material naturally exists), and as such, It has already been altered from its natural state by human hands. For example, an isolated polynucleotide may be part of a vector or composition, or contained within a cell, but the vector, composition, or particular cell This polynucleotide may still be in an “isolated” state since it is not in its original environment. The term “isolated” refers to genomic library or cDNA library, whole cell total mRNA sample or mRNA sample, genomic DNA sample (including those separated by electrophoresis and transcribed on a blot), shear Preferably, it does not refer to a prepared whole cell genomic DNA sample, or other composition in which the unambiguous characteristics of the nucleic acids of the invention have not been demonstrated by the art.

  “Vector” refers to a host such as a plasmid, cosmid, virus, autonomously replicating sequence, phage, linear single stranded, circular single stranded, linear double stranded, or circular double stranded DNA or RNA nucleotide sequence By any substance that holds exogenous DNA introduced into a cell or host organism is meant. Recombinant vectors can be obtained from any source and can be integrated into the genome or autonomously replicated.

  A ligand that “specifically binds” or “specifically recognizes” an apoptosis-related polypeptide of the present invention is higher than that bound to such a polypeptide to other unrelated polypeptides. A ligand that binds with affinity.

  “Modulating apoptosis” is to change, for a given cell, the normal tendency of that cell to undergo apoptosis by at least 10% compared to an untreated cell under certain environmental conditions. Means. For example, blood neutrophils produce apoptosis with a certain tendency. That is, more than 80% of the cell population will undergo apoptosis within the first 24 hours. Modulating the apoptosis of blood neutrophils means changing this normal apoptotic tendency, thereby enhancing or reducing apoptosis compared to normal rate. Similarly, blood neutrophils in the presence of GM-CSF have a reduced tendency to undergo apoptosis. Thus, modulating blood neutrophil apoptosis in the presence of GM-CSF means enhancing or reducing apoptosis under those conditions compared to their normal tendency to be reduced. . The reduced tendency to cause apoptosis may be a measurable increase in cell survival and may be the result of inhibiting apoptosis by inhibiting one or more components of the apoptotic pathway.

  The term “expression” refers to the transcription of a DNA template of a gene to produce the corresponding mRNA, and the translation of the mRNA into the corresponding gene product (ie, peptide, polypeptide, or protein). It means to generate. The term “activates gene expression” means to induce or enhance transcription of a gene in response to a treatment, such induction or enhancement being related to the amount of gene expression in the absence of said treatment. This is due to the comparison. Similarly, the terms “decrease gene expression” or “down-regulate gene expression” mean to suppress or prevent transcription of a gene by response to certain treatments, such suppression or blocking Is by comparison with the amount of gene expression in the absence of the treatment.

  In the context of the present invention, the “functional activity” of a protein describes the function that this protein performs in a natural environment for this protein. Changing the functional activity of a protein includes enhancing, reducing, or otherwise changing the original activity of the protein itself. It further includes increasing or decreasing the expression level and / or changing the intracellular distribution of the nucleic acid encoding the protein and / or changing the intracellular distribution of the protein itself.

  A “reporter gene” is a gene that is incorporated into an expression vector, placed under the same control as the gene of interest, and that expresses an easily measurable phenotype.

  The term “bone marrow cells” includes terminally differentiated non-dividing (ie, non-proliferating) cells derived from bone marrow cell lines, including neutrophils or polymorphonuclear neutrophils (PMN). , Eosinophils, and mononuclear phagocytes. The latter cells are known as mononuclear cells when in the blood and are known as macrophages when they migrate into the tissue. Terminal differentiation is the normal endpoint of cell differentiation and is usually irreversible.

  An “inflammatory disorder” or “inflammatory disease” is a disorder characterized by chronic or acute inflammation. This is then characterized by increased levels of cytokines and / or bone marrow cell survival factors. These disorders are characterized by prolonged survival of bone marrow cells, including neutrophils, eosinophils, and mononuclear cells / macrophages, which are a mixture of one or more of these cell types Can exist as a group. Thus, when referring to the treatment of an inflammatory disorder or disease, it includes treatment for individual cell types or treatment for a mixed population of different cell types. Consequently, if the number of these inflammatory cells increases, it is related to the pathology of the disease. In chronic inflammation, a persistent inflammatory response causes harmful effects such as tissue damage. Chronic inflammatory diseases include cystic fibrosis, adult respiratory distress syndrome, chronic obstructive pulmonary disease, inflammatory bowel disease, and rheumatoid arthritis. Other inflammatory diseases are known to those skilled in the art and include sepsis, pre-eclampsia, myocardial ischemia, reperfusion injury, psoriasis, asthma, bronchiolitis, Crohn's disease, and ulcerative colitis .

  “Polynucleotide” or “polypeptide” means the DNA and protein sequences disclosed herein. These terms also include variants that approximate their sequence, in which case these variants have the same biological activity as the reference sequence. Such mutant sequences include “alleles” (variant sequences found at the same locus of the same species or related species), and “homologs” (relative relationships with a common ancestral DNA sequence, depending on the second relationship. Genes that have a relationship to the other genes, such as speciation (“ortholog”) or gene duplication (gene separated by “paralog”), such variants are also the same organism as the reference sequences disclosed herein It is included as long as it retains activity.

  The present invention also includes silent polymorphisms and conservative substitutions of the polynucleotides and polypeptides disclosed herein as long as such variants retain the same biological activity as the reference sequences disclosed herein. .

Polypeptides specified herein are not limited to polypeptides specified in Table 1B, or polypeptides having amino acid sequences encoded by nucleic acid sequences specified in Table 1B or fragments thereof, and any It should be understood that homologous sequences obtained from sources, such as closely related virus / bacterial proteins, cellular homologues, and synthetic peptides, and variants or derivatives thereof are also included.

  Thus, reference herein to “polypeptide” refers to homologs of other species, including mammals (eg, mice, rats, or rabbits), particularly animals such as primates. Sequences are also included. Particularly preferred polypeptides include homologous human sequences.

  The term also encompasses variants, homologs, or derivatives of the amino acid sequence encoded by the nucleic acids identified in Table 1B, as well as variants, homologs, or derivatives of the nucleotide sequence that encodes this amino acid sequence. .

  In the context of the present invention, a homologous sequence spans at least 50 or 100 amino acids, preferably 200, 300, 400 or 500 amino acids, to any one of the polypeptide sequences disclosed herein at least 15, It is intended to include amino acid sequences that are 20, 25, 30, 40, 50, 60, 70, 80, or 90% identical, preferably at least 95 or 98% identical. In particular, homology should normally be considered with respect to regions of the sequence known to be essential for protein function and not with respect to adjacent non-essential sequences. This is particularly important when considering homologous sequences of closely related organisms.

  While homology can also be considered in terms of functional similarity (ie, amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to calculate homology in terms of sequence identity.

  Homology comparisons can be made visually and, more generally, using readily available sequence comparison programs. These computer programs, both publicly and commercially available, can calculate percent homology between two or more sequences.

  The percent homology is calculated with respect to a contiguous sequence, that is, one sequence is aligned with the other, and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. May be. This is called “no gap” alignment. Usually, such ungapped alignments are performed only on a relatively small number of residues (eg, less than 50 consecutive amino acids).

  This method is very simple and consistent, but for example, if an insertion or deletion occurs in the same sequence pair once, the subsequent amino acid residues will be out of alignment, thereby It does not take into account that when performing an overall alignment, a significant reduction in percent homology may be caused. As a result, most sequence comparison methods are designed to generate optimal alignments that allow for the possibility of insertions and deletions without adding excessive deductions to the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximize local homology.

  However, these more complex methods reflect sequence alignments with as few gaps as possible for the same number of identical amino acids—more closely related between the two sequences compared— Assign a “gap penalty” to each gap that occurs in the alignment so that it has a higher score than one with a large number of gaps. Using “affine gap costs” usually imposes a relatively high cost on the existence of a gap and a smaller penalty on each subsequent residue in the gap. This is the most commonly used gap scoring system. Increasing the gap penalty will of course produce an optimized alignment with a smaller number of gaps. Most alignment programs can change the gap penalty. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package (see below), the default gap penalty for amino acid sequences is −12 per gap and −4 for each extension.

  Therefore, to calculate the maximum homology percentage, it is first necessary to create an optimal alignment that takes into account the gap penalty. One suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research, Vol. 12, page 387). Examples of other software that can perform sequence comparison include BLAST package (Ausubel et al., 1999, ibid-chapter 18), FASTA (Altschul et al., 1990, J. MoL Biol, Vol. 215, 403-410), and GENEWORKS comparison tool suite, but are not limited to these. Both BLAST and FASTA can be used for offline and online search (Ausubel et al., 1999, ibid., 7-58 to 7-60). However, it is preferred to use the GCG Bestfit program.

  Although the final percent homology is calculated in terms of identity, the alignment process itself is usually not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is typically used that assigns a score to each pair-wise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix, which is the default matrix for the BLAST program suite. The GCG Wisconsin program typically uses either the public default values or a custom symbol comparison table if supplied (see user manual for more details). It is preferable to use a public default value for the GCG package and a default matrix such as BLOSUM62 for other software.

  Once the optimal alignment has been generated by the software, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and gives a numerical result.

  With respect to an amino acid sequence, the term “mutant” or “derivative” refers to any substitution, mutation, modification, exchange, deletion, or addition of one (or more) amino acids from or to that sequence. And the resulting amino acid sequence possesses substantially the same activity as the unmodified sequence, and is preferably encoded by a polypeptide specified in Table 1B or a nucleic acid sequence specified in Table 1B Has the same activity as a polypeptide.

  As used herein, the phrase “substantially the same activity” means that a variant polypeptide catalyzes the same reaction as compared to a known polypeptide having the sequence described herein; Specifically binding to at least one molecule to which the known polypeptide specifically binds, or binding of the molecule to the mutant polypeptide; and a substrate for the same enzyme or It means to show at least one of functioning as a cofactor.

  Polypeptides having amino acid sequences encoded by the nucleic acid sequences specified in Table 1B, or fragments or homologues thereof, can be modified for use in the present invention. Usually, the introduced modifications are those that maintain the biological activity of this sequence. Amino acid substitutions can introduce, for example, 1, 2, or 3 to 10, 20, or 30 substitutions if the modified sequence retains the biological activity of the unmodified sequence. Alternatively, modifications may be introduced to inactivate one or more functional domains in the polypeptides of the invention. Amino acid substitutions can also include the use of non-naturally occurring analogs, for example, to increase the plasma half-life of a therapeutically administered polypeptide.

  Conservative substitutions can be introduced, for example, according to the table below. The amino acids shown in the same block in the second column and preferably in the same row in the third column can be substituted for each other.

  As described above, polypeptides for use in the present invention include full-length sequence fragments. The fragment preferably contains at least one epitope. Methods for identifying epitopes are well known in the art. A fragment will usually contain at least 6 amino acids, more preferably at least 10, 20, 30, 50, or 100 amino acids.

  The protein is produced by recombinant methods, for example as described below. However, the protein may be produced by synthetic methods using techniques well known to those skilled in the art, such as solid phase synthesis. The protein may be produced as a fusion protein, eg, to aid in extraction and purification. Examples of fusion protein partners include glutathione S-transferase (GST), 6 × His, GAL4 (DNA binding domain and / or transcriptional activation domain), and β-galactosidase. It may also be advantageous to allow excision of the fusion protein sequence by including a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest. The fusion protein is preferably one that will not interfere with the function of the protein sequence of interest. The protein may be obtained by purification of cell extracts of animal cells.

  Multimeric proteins including apoptotic proteins are also intended to be encompassed by the present invention. “Multimer” means a protein containing two or more copies of a subunit protein. This subunit protein may be one of the proteins of the invention, for example, an apoptotic protein as described herein that has been repeated more than once. Such multimers may be fusion proteins or chimeric proteins, for example, a repetitive apoptotic protein may be linked to a polylinker sequence and / or one or more apoptotic peptides. And this apoptotic peptide may be present in a single copy or repeated in series, for example, a protein may contain more than one multimer in its total protein.

  The protein may be in a substantially isolated form. It should be understood that the protein may be admixed with a carrier or diluent that will not interfere with the original purpose of the protein and still be considered substantially isolated. A protein for use in the present invention may be in a substantially purified form, in which case it usually contains the protein in a sample and is greater than 90%, eg, 95% of the protein in the sample. , 98%, or 99% are proteins as specified herein.

The polypeptide may be labeled with a revealing label. The overt label can be any suitable label that allows detection of the polypeptide. Suitable labels include radioisotopes such as ¹²⁵ I, enzymes, antibodies, polynucleotides, and linkers such as biotin. The labeled polypeptide of the present invention can be used in a diagnostic method such as an immunoassay to measure the amount of the polypeptide of the present invention in a sample. The polypeptides or labeled polypeptides of the invention can also be used in serological or cell-mediated immunoassays to detect immune responsiveness to the polypeptides in animals and humans using standard protocols.

  The polypeptide or labeled polypeptide, or fragment thereof, may be immobilized on a solid phase, eg, the surface of a microarray, immunoassay well or dipstick. Such labeled and / or immobilized polypeptide may be packaged in a kit in a suitable container along with suitable reagents, controls, instructions, and the like. Such polypeptides and kits can be used in methods of detecting the polypeptides, or antibodies to their allelic or species variants, in an immunoassay.

  Immunoassay methods are well known in the art and typically include: (a) providing a polypeptide comprising an epitope to which an antibody against the protein can bind; (b) conditions that allow formation of an antibody-antigen complex. Incubating a biological sample with the polypeptide under, and (c) determining whether an antibody-antigen complex comprising the polypeptide is formed.

  Immunoassays can be used to detect polypeptides, for example, to detect modulation of protein expression or function.

  The polypeptides identified herein can be used in in vitro or in vivo cell culture systems to study the role of their corresponding genes and their homologs in cellular functions, including their function in disease. it can. For example, a truncated or modified polypeptide can be introduced into the cell to disrupt normal function performed in the cell. The polypeptide of the present invention may be introduced into a cell by in situ expression of a polypeptide from a recombinant expression vector (see below). The expression vector optionally retains an inducible promoter for controlling the expression of the polypeptide.

  By using appropriate host cells such as insect cells or mammalian cells, post-translational modifications (eg, myristoylation, glycosylation, truncation, etc.) that may be necessary to confer optimal biological activity to the recombinant expression product of the invention. Lipid addition and provision of tyrosine, serine, or threonine phosphorylation) is expected. Such a cell culture system in which the expression of the polypeptide of the present invention is performed can be used in an assay system for identifying candidate substances that interfere with or promote the function of the polypeptide of the present invention in cells.

Polynucleotide The polynucleotide or nucleic acid of the present invention includes any one or more of the polynucleotide specified in Table 1B, or the nucleic acid encoding the polypeptide encoded by the nucleic acid specified in Table 1B, and fragments thereof Is included. Usually, a fragment will comprise at least 15 contiguous nucleotides in a full length polynucleotide, more preferably at least 20, 30, 50, or 100 contiguous nucleotides in a full length polynucleotide. Nucleic acid sequences encoding such polypeptides can be readily identified by reference to the genetic code. In addition, computer programs are available that perform translation from nucleic acid sequences to polypeptide sequences and / or vice versa. Disclosure of a nucleic acid and corresponding polypeptide sequence includes disclosure of all nucleic acids (and their sequences) that encode the polypeptide sequence.

  One skilled in the art will appreciate that the same polypeptide can be encoded by a number of different polynucleotides as a result of the genetic code degeneracy. In addition, one of skill in the art will affect the polypeptide sequence encoded by the polynucleotide of the present invention by considering its codon usage in any particular host organism in which expression of the polypeptide of the present invention occurs. Without any doubt, it should be understood that nucleotide substitutions can be introduced using routine techniques.

  In a preferred embodiment of the invention, the nucleic acids of the invention include those polynucleotides, such as cDNA, mRNA, and genomic DNA. Such polynucleotides are typically human (Homo sapiens) cDNA, mRNA, genomic DNA, and the like. In the examples, accession numbers for these nucleic acid sequences are provided, and the polypeptides they encode are obtained by using such accession numbers in related databases, such as human sequence databases, including the Human Genome Sequence database. You can get it. Accession numbers can be used to identify such human polynucleotide or polypeptide sequences. Related sequences can also be obtained by searching sequence databases such as BLAST using polypeptide sequences. Specifically, a search using TBLASTN can be used.

  Nucleic acids for use in the present invention can include DNA or RNA. They may be single stranded or double stranded. They may be polynucleotides that contain synthetic or modified nucleotides therein. A number of different types of modifications to oligonucleotides are known in the art. These include the methylphosphonate backbone, the phosphorothionate backbone, and the addition of acridine or polylysine chains to the 3 'and / or 5' ends of the molecule. In the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be made to promote the in vivo activity or lifetime of the polynucleotides of the invention.

  With respect to nucleotide sequences for use in the present invention, the term “mutant”, “homolog”, or “derivative” includes any of one (or more) nucleic acids from or to that sequence. Substitutions, mutations, modifications, exchanges, deletions or additions are also included. Preferably, the mutant, homolog or derivative encodes a polypeptide having biological activity.

  As mentioned above, with respect to sequence homology, it is preferred to have at least 50% or 75% homology, more preferably at least 85%, more preferably at least 90% homology. More preferably it has at least 95% homology, more preferably at least 98%. Nucleotide homology comparisons can be performed as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty per nucleotide is -3.

  The invention also encompasses the use of nucleotide sequences that can selectively hybridize to the sequences presented herein, or any variant, fragment, or derivative thereof, or the complementary strand to any of the above. The nucleotide sequence is preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40, or 50 nucleotides in length.

  The term “hybridization” is intended to include “a process in which a strand of nucleic acid is ligated to a complementary strand through base pairing” and an amplification process that is performed by the polymerase chain reaction technique.

  There are at least 20, preferably at least 25 or 30, such as at least 40, 60, or 100 or more polynucleotides that can selectively hybridize to the nucleotide sequences presented herein, or their complementary sequences. It is at least 70%, preferably at least 80% or 90%, and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a number of contiguous nucleotide regions.

The term “selectively hybridizable” means that a nucleic acid is known to hybridize to a nucleic acid having a sequence specified in Table 1B at a level significantly above background. By background is meant the level of signal produced by the interaction of the test nucleic acid with a non-specific DNA member in the sample, this signal being observed with a specific interaction with the target DNA. Less than 1/10, preferably less than 100%. The intensity of interaction can be measured, for example, by radiolabeling the probe, eg with ³² P.

  Hybridization conditions, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Technologies, Methods in Enzymology, vol. 152, Academic Press, San Diego CA). ) And give a specific “stringency” as explained below. Stringency conditions are described in US Pat. No. 5,976,838, the teachings of which are hereby incorporated by reference in their entirety. Specifically, examples of high stringency, stringent, reduced minimal stringency conditions are provided in the table on page 15 of US Pat. No. 5,976,838. Examples of stringency conditions for solutions during and after hybridization are shown. High stringency conditions are at least as stringent as, for example, conditions A to F in the table. Stringent conditions are at least as stringent as, for example, conditions G to L in the table; and reduced stringency conditions are at least as high as conditions M to R in the table; This is a stringent condition.

  Maximum stringency is usually around Tm-5 ° C (5 ° C below the Tm of the probe); high stringency is 5 ° C to 10 ° C below the Tm; intermediate stringency is 10 ° C to 20 ° C below the Tm And low stringency occurs at 20-25 ° C. below the Tm. As one skilled in the art will appreciate, hybridization at maximum stringency can be used to identify or detect the same polynucleotide sequence, while hybridization at intermediate (low) stringency is similar. It can be used to identify or detect polynucleotide sequences or closely related polynucleotide sequences.

  Hybridization stringency conditions refer to conditions of temperature and buffer composition that allow a first nucleic acid sequence to hybridize to a second nucleic acid sequence, which conditions hybridize to each other. To determine the degree of identity between those sequences. Thus, “high stringency conditions” are conditions in which only nucleic acid sequences that are very similar to each other will hybridize. When a plurality of sequences hybridize under moderate stringency conditions, the similarity between the sequences may not be so high. And the similarity required for two sequences to hybridize under low stringency conditions is even lower. The conditions under which a given sequence will hybridize to the most similar sequence by changing the hybridization conditions from a stringency level at which no hybridization occurs to the level at which hybridization is first observed. Can be determined. The exact conditions that determine the stringency in a particular hybridization include not only the ionic strength, temperature, and concentration of destabilizing agents such as formamide, but the length of the nucleic acid sequences, their base composition, Factors such as the percentage of base pairs mismatched between the two sequences and the frequency of occurrence of sequence subsets in other non-identical sequences (eg, short regions of repetitive sequences) are also included. Washing is a step in which conditions are set to determine the lowest level of similarity between sequences that hybridize to each other. Typically, at any selected SSC concentration, for every 1% mismatch between the two sequences, the melting temperature (Tm) decreases by 1 ° C. from the lowest temperature at which only homologous hybridization occurs. Usually, Tm increases by about 17 ° C. every time the concentration of SSC is doubled. Using these guidelines, the cleaning temperature can be empirically determined depending on the desired mismatch level. Hybridization and washing conditions are described in Current Protocols in Molecular Biology (edited by Ausubel, FM et al., John Wiley & Sons, Inc., 1995, and supplemental updates) 2.10.1 to 2.10.16. Page 6 and pages 6.3.1 to 6.3.6.

High stringency conditions were: (1) 1 × SSC (10 × SSC = 3M NaCl, 0.3M Na ₃ -citric acid · 2H ₂ O (88 g / liter), pH adjusted to 7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg / ml salmon sperm denatured DNA, 65 ° C., (2) 1 × SSC, 50% formamide, 1% SDS, 0.1-2 mg / ml salmon sperm denatured DNA , 42 ° C., (3) 1% bovine serum albumin (fraction V), 1 mM Na ₂ .EDTA, 0.5 M NaHPO ₄ (pH 7.2) (1 M NaHPO ₄ = 134 g Na ₂ HPO ₄ · 7H ₂ per liter _{O, 4ml 85% H 3 PO} 4), 7% SDS, 0.1~2mg / ml denatured salmon sperm DNA, 65 ℃, (4) 50% formamide, 5 × SSC, 0.02M ris-HCl (pH 7.6), 1 × Denhardt solution (100 × = 10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), adjusted to 500 ml with water), 10% dextran sulfate, 1% SDS 0.1 to 2 mg / ml salmon sperm denatured DNA, 42 ° C., (5) 5 × SSC, 5 × Denhardt solution, 1% SDS, 100 g / ml salmon sperm denatured DNA, 65 ° C., or (6) 5 × Hybridization under the conditions of SSC, 5 × Denhardt solution, 50% formamide, 1% SDS, 100 g / ml, salmon sperm denatured DNA, 42 ° C. can be used. (1) 0.3-0. 1 × SSC, 0.1% SDS, 65 ° C., or (2) 1 mM Na ₂ EDTA, 40 mM NaHPO ₄ (pH 7) .2) with high stringency wash at either 1% SDS, 65 ° C. The above conditions are intended for use with 50 base pairs or longer DNA-DNA hybrids. If the length of the hybrid is considered to be less than 18 base pairs, the hybridization and wash temperatures should be 5-10 ° C. below the calculated hybrid Tm. In this case, Tm = (2 × number of bases A and T) + (4 × number of bases G and C) ° C. Hybrids considered to be about 18 to about 49 base pairs in length are: Tm = (81.5 ° C. + 16.6 (log ₁₀ M) +0.41 (% G + C) −0.61 (% formamide) −500 / L) ° C., where “M” is the molar concentration of a monovalent cation (eg, Na ⁺ ) and “L” is the length of the hybrid in base pairs.

The moderate stringency conditions are (1) 4 × SSC (10 × SSC = 3M NaCl, 0.3M Na ₃ -citric acid · 2H ₂ O (88 g / liter), pH adjusted to 7.0 with 1M HCl) 1% SDS (sodium dodecyl sulfate), 0.1-2 mg / ml salmon sperm denatured DNA, 65 ° C., (2) 4 × SSC, 50% formamide, 1% SDS, 0.1-2 mg / ml salmon sperm denatured DNA, 42 ° C., (3) 1% bovine serum albumin (fraction V), 1 mM Na ₂ · EDTA, 0.5 M NaHPO ₄ (pH 7.2) (1 M NaHPO ₄ = 134 g Na ₂ HPO ₄ · 7H per liter ₂ O, 4 ml 85% H ₃ PO ₄ ), 7% SDS, 0.1-2 mg / ml salmon sperm denatured DNA, 65 ° C., (4) 50% formamide, 5 × SSC, 0.02M Tris-HCl (pH 7.6), 1 × Denhardt solution (100 × = 10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), adjusted to 500 ml with water), 10% dextran sulfate, 1% SDS 0.1 to 2 mg / ml salmon sperm denatured DNA, 42 ° C., (5) 5 × SSC, 5 × Denhardt solution, 1% SDS, 100 g / ml salmon sperm denatured DNA, 65 ° C., or (6) 5 × SSC Hybridization under any of 5 × Denhardt solution, 50% formamide, 1% SDS, 100 g / ml, salmon sperm denatured DNA, 42 ° C. can be used, 1 × SSC, 0.1% SDS, With moderate stringency wash at 65 ° C. The above conditions are intended for use with 50 base pairs or longer DNA-DNA hybrids. If the length of the hybrid is considered to be less than 18 base pairs, the hybridization and wash temperatures should be 5-10 ° C. below the calculated hybrid Tm. In this case, Tm = (2 × number of bases A and T) + (4 × number of bases G and C) ° C. Hybrids considered to be about 18 to about 49 base pairs in length are: Tm = (81.5 ° C. + 16.6 (log ₁₀ M) +0.41 (% G + C) −0.61 (% formamide) −500 / L) ° C., where “M” is the molar concentration of a monovalent cation (eg, Na ⁺ ), and “L” is the length of the hybrid in base pairs.

Low stringency conditions were (1) 4 × SSC (10 × SSC = 3M NaCl, 0.3M Na ₃ -citric acid · 2H ₂ O (88 g / liter), pH adjusted to 7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg / ml salmon sperm denatured DNA, 50 ° C., (2) 6 × SSC, 50% formamide, 1% SDS, 0.1-2 mg / ml salmon sperm denatured DNA , 40 ° C., (3) 1% bovine serum albumin (fraction V), 1 mM Na ₂ · EDTA, 0.5M NaHPO ₄ (pH 7.2) (1M NaHPO ₄ = 134 g Na ₂ HPO ₄ · 7H ₂ per liter _{O, 4ml 85% H 3 PO} 4), 7% SDS, 0.1~2mg / ml denatured salmon sperm DNA, 50 ℃, (4) 50% formamide, 5 × SSC, 0.02M ris-HCl (pH 7.6), 1 × Denhardt solution (100 × = 10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), adjusted to 500 ml with water), 10% dextran sulfate, 1% SDS 0.1 to 2 mg / ml salmon sperm denatured DNA, 40 ° C., (5) 5 × SSC, 5 × Denhardt solution, 1% SDS, 100 g / ml salmon sperm denatured DNA, 50 ° C., or (6) 5 × SSC Hybridization under any of 5 × Denhardt solution, 50% formamide, 1% SDS, 100 g / ml, salmon sperm denatured DNA, 40 ° C. can be used, 2 × SSC, 0.1% SDS, 50 ° C. or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na ₂ EDTA, 40 mM Na With low stringency wash with either HPO ₄ (pH 7.2), 5% SDS. The above conditions are intended for use with 50 base pairs or longer DNA-DNA hybrids. If the length of the hybrid is considered to be less than 18 base pairs, the hybridization and wash temperatures should be 5-10 ° C. lower than the calculated hybrid Tm, where Tm = Calculated as (2 × number of bases A and T) + (4 × number of bases G and C) ° C. Hybrids considered to be about 18 to about 49 base pairs in length are: Tm = (81.5 ° C. + 16.6 (log ₁₀ M) +0.41 (% G + C) −0.61 (% formamide) −500 / L) ° C., where “M” is the molar concentration of a monovalent cation (eg, Na ⁺ ), and “L” is the length of the hybrid in base pairs.

In a preferred embodiment, the present invention is performed under stringent conditions (eg, 65 ° C. and 0.1 × SSC (1 × SSC = 0.15M NaCl, 0.015M Na ₃ -citric acid, pH 7.0)). Includes the use of nucleotide sequences that can hybridize to the nucleotide sequences of the invention.

  Where the polynucleotide is double stranded, the present invention encompasses the use of both strands of the double stranded molecule, and their individual or combinations. If the polynucleotide is single stranded, it should be understood that the invention also encompasses the use of the complementary sequence of the polynucleotide.

  Polynucleotides that do not have 100% homology to the sequences in Table 1B, but whose use is encompassed by the present invention, can be obtained in a number of ways. Other variants of the sequences described herein can be obtained, for example, by probing DNA libraries made from various individuals, such as individuals from different populations. In addition, other viral / bacterial homologues, or cellular homologues, particularly cellular homologues found in mammalian cells (eg, rat, mouse, bovine, and primate cells) can be obtained. Such homologues and fragments thereof will then typically be able to selectively hybridize to the sequences specified in Table 1B. Such a sequence may be any one of the sequences in Table 1B, such as a library of cDNA or genomic DNA libraries generated from other animal species using probes containing all or part of that sequence. Can be obtained by probing under moderate to high stringency conditions. To identify other primate / mammalian homologues or allelic variants, the nucleotide sequence of Table 1B, or the nucleotide sequence encoding the human protein specified in Table 3B, column 3 may preferably be used.

  Variants and cell line / species homologues can also be obtained using degenerate PCR. In such degenerate PCR, primers designed to target variants and homologues of the sequences in Table 1B will be used. For example, conserved sequences can be predicted by aligning the amino acid sequences of several variants / homologs. Sequence alignment can be performed by using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.

  Primers used in degenerate PCR contain one or more degenerate positions and have lower stringency conditions than those used when cloning sequences with a single sequence primer relative to a known sequence Would be used in As will be appreciated by those skilled in the art, the overall nucleotide homology between closely related sequences of organisms tends to be very low, so in such situations, the labeled fragment Rather than screening a library, performing degenerate PCR can be the method of choice.

  In addition, homologous sequences can also be identified by searching nucleotide and / or protein databases using search algorithms such as the BLAST program suite.

  Alternatively, such polynucleotides can also be obtained by site-directed mutation of the sequence being characterized. This can be useful, for example, when silent codon changes are required for the sequence to optimize codon usage for the particular host cell in which expression of the polynucleotide sequence is to take place. Other sequence modifications may be desired to introduce restriction enzyme recognition sites, or to alter the properties or functions of the polypeptide encoded by the polynucleotide. For example, further modifications may be desired to achieve certain coding changes in the nucleic acid sequence that produces the mutant gene that has lost its regulatory function. Probes based on such modifications can be used as diagnostic probes for detecting such mutants.

  Polynucleotides can also be used to generate primers, eg, PCR primers or primers for other amplification reactions, probes, eg, probes labeled with overt labels by conventional methods using radioactive or non-radioactive labels. Also, the polynucleotide may be cloned into a vector. Such primers, probes, and other fragments are at least 8, 9, 10, or 15 nucleotides in length, preferably at least 20 nucleotides, such as at least 25, 30, or 40 nucleotides, These are also encompassed by the term polynucleotide.

  Polynucleotides such as DNA polynucleotides and probes for use in the present invention may be produced by recombination, synthesis, or any method available to those skilled in the art. They can also be cloned by standard techniques. The invention also includes compositions comprising one or more isolated polynucleotides encoding apoptotic proteins, for example, vectors containing polynucleotides encoding apoptotic proteins, and host cells containing such vectors. Include. “Host cell” means a cell that is or can be used as a recipient cell for a nucleic acid introduced by a vector. The host cell may be prokaryotic, eukaryotic, mammalian, plant or insect, and is present in a tissue or organism as a single cell or as a collection, eg as a culture, in tissue culture can do. Host cells can also be obtained from multicellular organisms, such as normal or diseased tissues of mammals. As used herein, a host cell is intended to include not only the original cell transformed with a nucleic acid, but also the progeny of such a cell that still contain the nucleic acid. The exogenous polynucleotide may be maintained as a non-integrated vector, such as a plasmid, or may be inserted into the host genome. The vector may also contain regulatory sequences, such as sequences that allow expression of the polynucleotide.

  Typically, the primer will be generated by a synthetic technique that steps the desired nucleic acid sequence one nucleotide at a time. Techniques for doing this using automated methods are readily available in the art.

  Longer polynucleotides will usually be produced using recombinant methods, for example using PCR (polymerase chain reaction) cloning techniques. This generates a pair of primers (eg, about 15 to 30 nucleotides) adjacent to the region of the lipid targeting sequence that is desired to be cloned, and this primer is combined with mRNA or cDNA obtained from animal or human cells. Contacting, performing a polymerase chain reaction under conditions that cause amplification of the desired region, isolating the amplified fragment (eg, by purifying the reaction mixture on an agarose gel), and amplified Recovering the recovered DNA. The primer may be designed to contain an appropriate restriction enzyme recognition site so that the amplified DNA can be cloned into an appropriate cloning vector.

A polynucleotide or primer subjected to use in the present invention may carry a revealing label. Suitable labels include radioisotopes such as ³² P or ³⁵ S, enzyme labels, or other protein labels such as biotin. Such labels may be added to the polynucleotides or primers of the invention and detected by using techniques well known to those skilled in the art.

  A polynucleotide or primer, or fragment thereof, for use in the present invention is labeled or unlabeled in a nucleic acid based test to detect or sequence a polynucleotide of the present invention in a human or animal body. Can be used by those skilled in the art.

  Typically, such detection tests involve contacting a biological sample containing DNA or RNA with a probe comprising a polynucleotide or primer of the invention under hybridization conditions, and the probe and nucleic acid in the sample. Detecting any double-stranded molecules formed between them. Such detection can be accomplished by using techniques such as PCR, or by immobilizing the probe to a solid support, removing nucleic acid that has not hybridized with the probe from the sample, and then detecting the nucleic acid that has hybridized to the probe. Can be realized. Alternatively, the sample nucleic acid may be immobilized on a solid support and the amount of probe bound to such a support can be detected. Suitable assay methods according to this format or other formats can be found, for example, in International Publications WO 89/03891 and WO 90/13667.

  Tests for sequencing nucleotides for use in the present invention include contacting a biological sample containing target DNA or RNA with a probe containing a polynucleotide or primer of the invention under hybridization conditions, and For example, determining the sequence by the Sanger dideoxy chain termination method (see Sambrook et al.).

  Usually, such methods extend the primer in the presence of a suitable reagent by synthesizing a strand complementary to the target DNA or RNA, and the extension reaction is performed with an A, C, G, or T / U residue. Selectively terminating at one or more of the groups; causing chain extension and termination reactions; separating extension products by size to determine the sequence of nucleotides at which the selective termination occurred. Including. Suitable reagents include DNA polymerase enzymes, deoxynucleotides dATP, dCTP, dGTP, and dTTP, buffers, and ATP. Dideoxynucleotides are used for selective termination.

  Tests for detecting or sequencing the nucleotides specified in Table 1B in a biological sample can be performed in cells of individuals having or suspected of having altered gene sequences, such as leukemia cells, and Can be used to determine the presence of specific sequences in cancer cells, including solid tumors such as breast, ovary, lung, colon, pancreas, testis, liver, brain, muscle, and bone tumors. Cells obtained from patients with inflammatory diseases can be similarly tested.

  In addition, the identification of the genes described in the examples will allow the role of these genes in genetic diseases to be investigated. In general, this will involve establishing the state of these genes (eg, using PCR sequence analysis), eg, in cells obtained from an animal or human having a neoplasm.

  Probes for use in the present invention may be conveniently packaged in a suitable container in the form of a test kit. In such a kit, the probe may be bound to a solid support if the probe is required to be bound to a solid support in the assay format for which the kit is designed. The kit may also contain appropriate reagents, control reagents, instructions, etc. for processing the sample to be probed and for hybridizing the probe to the nucleic acid in the sample.

Homology Search Sequence homology (or identity) may be determined using any suitable homology algorithm, eg, using default parameters.

It is advantageous to use the BLAST algorithm with parameters set to default values. The BLAST algorithm is available from the United States government (“.gov”), National Institutes of Health (“nih”), National Center for Biotechnology Information (“.ncbi”) website (“www”), “/ Blast /” Directory, “blast help. It is described in detail in the “html” file. The search parameters defined as follows are advantageously set to specific default parameters.

  As assessed by BLAST, “substantial homology” corresponds to sequences that match with EXPECT values of at least about 7, preferably at least about 9, and most preferably 10 or greater. The default threshold for EXPECT in a BLAST search is typically 10.

BLAST (Basic Local Alignment Search Tool) is a heuristic search algorithm used by the blastp, blastn, blastx, tblastn, and tblastx programs, which are described in Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA, 87 (6), 2264-8, using statistical methods with some improvements (as described above, “blast”). help. html)), and calculate the significance of those search results. The BLAST program is designed for sequence similarity searches, for example, to identify homologues of query sequences. This program is not always useful for exploring motif styles. See Altschul et al. (1994) for a discussion of basic problems in sequence database similarity searches.

  The sequence comparison is most preferably performed using the simple BLAST search algorithm provided in the “/ BLAST” directory on the NCBI World Wide Web site described above.

Preparation of Apoptosis-Related Polypeptides Apoptosis-related polypeptides according to the present invention can be produced by any desired technique, including chemical synthesis, isolation from biological samples, and expression of nucleic acids encoding such polypeptides. The nucleic acid can then be synthesized or isolated from a biological source of apoptosis-related protein.

  The invention therefore relates to a vector encoding a polypeptide according to the invention, or a fragment thereof. The vector can be, for example, a phage, plasmid, virus, or retroviral vector.

  The nucleic acid according to the present invention may be part of a vector containing a selectable marker for propagation in a host. Usually, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then introduced into the host cell.

  The nucleic acid insert is operably linked to a suitable promoter such as the phage lambda PL promoter, E. coli lac, trp, phoA, tac promoter, SV40 early and late promoters, and the retroviral LTR promoter. Other suitable promoters are known to those skilled in the art. The expression construct further contains a transcription initiation and termination site and a translational ribosome binding site in the transcribed region. The coding portion of the transcript expressed by the construct preferably includes a translation initiation codon at the beginning of the polypeptide to be translated and a termination codon (UAA, UGA, or UAG) appropriately positioned at the termination.

  As indicated, it is preferred that the expression vector includes at least one selective marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for E. coli and other bacterial cultures. . Representative examples of suitable hosts include bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells (eg, Saccharomyces cerevisiae, or Pichia pichia ); Insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, bow melanoma cells; and plant cells, but are not limited thereto.

  Suitable media and conditions for the host cells described above are known in the art and are commercially available.

  Preferred vectors for use in bacteria include pQE70, pQE60, and pQE-9 sold by QIAGEN; pBluescript vector, Pagescript vector, pNH8A, pNH16a, pNH18A, pNH46m sold by Stratagene Cloning Systems; Ptrc99a, pKK2233, pKK233-3, pDR540, and pRIT5. Preferred eukaryotic cell vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG sold by Stratagene; and pSVK3, pBPV, pMSG and pSVL sold by Pharmacia. Preferred expression vectors for use in the yeast system include pYES2, pYDl, pTEFl / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K. And PA0815 (all available from Invitrogen, Carlsbad, CA).

  Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran transfection, cationic lipid mediated transfection, electroporation, transduction, infection, and other methods. Such methods are described in many standard laboratory manuals, such as Sambrook et al.

  Polypeptides according to the present invention can be obtained from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography. And can be recovered and purified by well known methods including lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

  Polypeptides according to the present invention can also be recovered from biological sources, including body fluids, tissues, and cells, particularly cells derived from the subject tumor tissue or suspected tumor tissue.

  Furthermore, polypeptides according to the present invention can be chemically synthesized using techniques known in the art (eg, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., N.). Y. and Hunkapiller et al., Nature, 310, 105-111 (1984)). For example, a polypeptide corresponding to a fragment of an apoptosis-related protein can be synthesized using a peptide synthesizer.

Antibodies The present invention provides the use of monoclonal or polyclonal antibodies against the polypeptides encoded by the nucleic acids specified in Table 1B, or fragments thereof. Methods of antibody production are well known to those skilled in the art. If a polyclonal antibody is desired, a selected mammal (eg, mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide having an epitope of a polypeptide. Serum obtained from the immunized animal is collected and processed according to known methods. Various adjuvants known in the art can be used to promote antibody production (for more details, see, for example, USing Antibodies, A Laboratory Manual, Harlow, E. and Lane, D., 1999, Cold, Spring Harbor Laboratory Press (see, eg, ISBN 0-87969-544-7). If serum containing polyclonal antibodies to an epitope of a polypeptide also contains antibodies to other antigens, the polyclonal antibody can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. To generate a greater immunogenic response, the polypeptide or fragment thereof can be haptenized to another polypeptide used as an immunogen in animals or humans.

  Antibodies can be isolated by, for example, concentrating immunoglobulin in the culture supernatant or ascites by precipitation with ammonium sulfate, dialysis against hygroscopic materials such as polyethylene glycol, filtration through selective membranes, or similar methods. can do. As required and / or desired, conventional chromatographic methods such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno) affinity chromatography such as target antigen or protein A Purify the antibody by affinity chromatography.

  While antibodies useful in the practice of the invention may be polyclonal, preferred antibodies are monoclonal antibodies. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules in a continuous cell system in culture. Production and isolation techniques include the hybridoma method first described by Kohler and Milstein (Nature, 1975, 256, 495-497); the human B cell hybridoma method (Kosbor et al., 1983, Immunology Today). Cote et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 2026-2030); and the EBV-hybridoma method (Cole et al., 1985, Monoclonal Antibodies and). Cancer Therapy, Alan R. Liss Inc., pages 77-96), but is not limited thereto.

Alternatively, techniques described for the production of single chain antibodies (see, eg, US Pat. No. 4,946,778) can be adapted to produce single chain antibodies against the apoptosis-related protein of interest. it can. Antibody-based agents useful for practicing the present invention include, but are not limited to, F (ab ′) ₂ and F (ab ′) ₂ fragments that can be generated by pepsin digestion of complete antibody molecules. Antibody fragments are also included, including Fab fragments that can be generated by reducing the disulfide bridges of Alternatively, Fab and / or scFv expression libraries can be constructed (eg, Huse et al., 1989) to allow rapid identification of fragments with the desired specificity for the apoptosis-related protein of interest. Year, Science, 246, 1275-1281).

  Techniques for producing and isolating monoclonal antibodies and antibody fragments are well known in the art and in addition to other literature, among others, Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, and J . W. Goding, 1986, Monoclonal Antibodies: Principles and Practice, Academic Press, London. Humanized antibodies and antibody fragments are also described in Vaughn, T .; J. et al. Et al., 1998, Nature Biotech, Vol. 16, pp. 535-539, and references cited therein, such antibodies or fragments thereof can also be prepared according to the present invention. Is useful to implement.

  One alternative technique is to screen a phage display library, where, for example, phage express scFv fragments with complementarity determining regions (CDRs) with great diversity on their coat surface. To do. This technique is well known in the art.

  Antibodies raised against epitopes of polypeptides encoded by the nucleic acids specified in Table 1B, both monoclonal and polyclonal, are particularly useful in diagnosis, and neutralizing antibodies are useful for passive immunotherapy. Useful. In particular, monoclonal antibodies may be used to produce anti-idiotype antibodies. Anti-idiotypic antibodies are immunoglobulins that retain an “internal image” of the antigen of the drug for which protection is desired.

  Techniques for producing anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful in therapy.

In the present invention, the term “antibody” also includes fragments of whole antibodies that retain binding activity to the target antigen, unless specified to the contrary. Such fragments include Fv, F (ab ′), and F (ab ′) ₂ fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanized antibodies, for example as described in EP 239400.

  The antibody comprises (a) providing an antibody of the invention; (b) incubating a biological sample with the antibody under conditions that allow formation of an antibody-antigen complex; and (c) the antibody Can be used to detect an apoptosis-related polypeptide as specified herein in a biological sample by a method comprising determining whether an antibody-antigen complex comprising is formed.

  Suitable samples include extracts from tissues such as brain, mammary gland, ovary, lung, colon, pancreas, testis, liver, muscle, and bone tissue, or neoplastic growth derived from such tissue .

  Antibodies that specifically bind to apoptotic proteins can be used in diagnostic methods and diagnostic kits well known to those skilled in the art for detecting or quantifying apoptotic proteins in body fluids or tissues. Results from these tests can be used to diagnose or predict the onset or recurrence of cancer and other apoptosis-mediated diseases.

  The present invention includes the use of apoptotic proteins, antibodies against these proteins, and compositions comprising these proteins and / or these antibodies in the diagnosis or prognosis of diseases characterized by proliferative activity. As used herein, the term “prognostic method” means a method that allows a prediction regarding the progression of disease in a human or animal diagnosed with a disease, particularly an apoptosis-dependent disease. As used herein, the term “diagnostic method” means a method that allows the determination of the presence or type of an apoptosis-dependent disease in or on a human or animal.

  Apoptotic proteins can be used in diagnostic methods and diagnostic kits to detect and quantify antibodies that can bind to these proteins. These kits will allow detection of circulating antibodies against apoptotic proteins. This indicates, for example, that apoptosis of diseased cells is promoted.

  The antibody to be used in the present invention may be bound to a solid carrier and / or packaged in a kit in an appropriate container together with appropriate reagents, controls and instructions.

  When used in a diagnostic composition, the antibody preferably provides a means for detecting the antibody, such means by an enzyme, by fluorescence, by a radioisotope, or other means. It can be. The antibody and its detection means can be provided for use in diagnosis kits intended for diagnosis, for simultaneous use, separate simultaneous use, or sequential use.

  Immunospecific binding of the antibodies of the invention can be assayed by any method known in the art. Available immunoassays include Western blot, radioimmunoassay, ELISA, sandwich immunoassay, immunoprecipitation assay, precipitation reaction, in-gel precipitation reaction, immunodiffusion assay, aggregation assay, complement binding assay, immunoradiometric assay, fluorescence Competitive and non-competitive assay systems using techniques such as immunization and protein A immunoassays include, but are not limited to. These assays are routine in the art (see, eg, Ausubel et al., 1994, Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons, Inc., New York, This document is incorporated herein by reference in its entirety). In the following, an exemplary immunoassay is briefly described.

  Usually, the immunoprecipitation protocol consists of RIPA buffer (1% NP-40 or Triton X-100, 1) supplemented with protein phosphatases and / or protease inhibitors (eg EDTA, PMSF, aprotinin, sodium vanadate). Lysing the cell population with a lysis buffer such as sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.01M sodium phosphate pH 7.2, 1% tradiolol) Adding to cell lysate, incubating at 4 ° C. for a fixed time (eg, 1 to 4 hours), adding protein A and / or protein G sepharose beads to cell lysate, about 1 hour or more at 4 ° C. Incubating, washing the beads with lysis buffer, and S Including resuspending the beads in DS / sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed, for example, by Western blot analysis.

Western blot analysis usually involves preparing a protein sample, performing electrophoretic movement of the protein sample in a polyacrylamide gel (eg, 8% to 20% SDS-PAGE, depending on the molecular weight of the antigen), protein Transfer sample from polyacrylamide gel to membrane such as nitrocellulose, PVDF, nylon, etc., blocking membrane with blocking solution (eg PBS containing 3% BSA or non-fat milk), wash buffer (For example, washing the membrane with PBS-Tween 20), blocking the membrane with a primary antibody (target antibody) diluted with a blocking buffer, washing the membrane with a washing buffer, blocking buffer Enzyme substrate (e.g. horseradish peroxidase or Cali phosphatase) or radioactive molecule (e.g., ^{32 P} or ^{125 I)} in the coupled secondary antibody (antibody recognizing the primary antibody, e.g., to perform the blocking of the membrane with anti-human antibody), membrane in washing buffer Washing and detecting the presence of the antigen.

  ELISA prepares the antigen, coats the wells of a 96-well microtiter plate with the antigen, wells the antibody of interest conjugated to a detectable compound such as an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase). Incubating for a while and detecting the presence of the antigen. In ELISA, it is not necessary to bind the antibody of interest to a detectable compound; instead, a secondary antibody bound to the detectable compound (an antibody that recognizes the antibody of interest) can be added to the well. Further, instead of coating the well with the antigen, the well can be coated with an antibody. In this case, a secondary antibody conjugated to a detectable compound can be added after the antigen of interest is added to the coated well.

The binding affinity of the antibody for the antigen and the dissociation rate of the antibody-antigen interaction can be measured in a competitive binding assay. An example of a competitive binding assay involves incubating an antibody of interest with a labeled antigen (eg, ³ H or ¹²⁵ I) in the presence of unlabeled antigen whose amount is gradually increased and bound to the labeled antigen. A radioimmunoassay comprising detecting an antibody. The affinity and binding dissociation rate of the antibody of interest for a particular antigen can be determined from the data by Scatchard plot analysis. The competition with the secondary antibody can also be measured using a radioimmunoassay. In this case, the antigen is incubated with the antibody of interest conjugated to a labeled compound (eg, ³ H or ¹²⁵ I) in the presence of an unlabeled secondary antibody that is gradually increased in amount.

Methods for detecting proteins that interact with apoptosis-related proteins One technique that is useful for identifying interacting proteins is the immunoprecipitation reaction as described above.

  Another technique useful for identifying interacting proteins is the yeast two-hybrid described in, for example, Bartel et al. (Edit), The Yeast Two Hybrid System, Oxford University Press (1997) (ISBN: 0195109384). There is a system. This disclosure is incorporated herein by reference.

  Protein interactions can also be analyzed using protein arrays. Protein arrays can be made by a variety of different techniques, which allow proteins to be deposited on a flat plane at different densities. High density protein arrays can be generated using automated methods similar to those described for DNA arrays (see below). Proteins that interact with apoptosis-related proteins can be identified, for example, by using apoptosis-related proteins for probing expression arrays. A positive interaction may then be detected, for example, by the presence of a labeled antibody or by a tag placed on the apoptosis-related protein. The identity of interacting proteins can be determined by techniques such as mass spectrometry.

Measuring Gene Expression The level of gene expression can be measured using a number of different techniques.

a) Measuring gene expression at the RNA level Gene expression can be detected at the RNA level. RNA can be extracted from cells using RNA extraction techniques including, for example, acidic phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), or use of RNeasy RNA preparation kits (Qiagen). Typical assay formats using ribonucleic acid hybridization include nuclear run-on assay, RT-PCR, and RNase protection assay (Melton et al., Nuc. Acids Res., Vol. 12, p. 7035). Available detection methods include radioactive labels, enzyme labels, chemiluminescent labels, fluorescent labels, and other suitable labels.

  Usually, RT-PCR is used to amplify RNA targets. In this process, reverse transcriptase can be used to convert RNA to complementary DNA (cDNA), which is then amplified to facilitate detection.

  Many DNA amplification methods are known, most of which rely on enzyme chain reactions (such as polymerase chain reaction, ligase chain reaction, or self-sustained sequence replication), or if the DNA is cloned This is due to replication of the entire vector or a part thereof.

  A number of target amplification and signal amplification methods have been described in the literature; for example, a general review of these methods includes Landegren, US; Science, 242, 229-237 (1988), and Lewis, R., et al. , Genetic Engineering News, 10 (1), 54-55 (1990).

  PCR is a nucleic acid amplification method described in US Pat. Nos. 4,683,195 and 4,683,202, among others. PCR can be used to amplify any known nucleic acid in the diagnostic context (Mok et al. (1994), Gynecological Oncology 52: 247-252). Self-sustained sequence replication (3SR) is a variant of TAS, a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase, and nuclease activity mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990), Proc. Natl. Acad. Sci. USA, 87, 1874). The ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described in Wu and D.W. Y. And Wallace, R .; B. (1989), Genomics, vol. 4, page 560. The Qβ replicase technology uses bacteriophage Qβ RNA replicase to replicate target DNA, as described in Lizardi et al. (1988), Bio / Technology, Vol. 6, page 1197. Amplify.

  Other amplification techniques can also be used in the present invention. For example, rolling circle amplification (Lizardi et al. (1998), Nat Genet, 19, 225) is a commercial amplification technique (RCAT ™) driven by DNA polymerase, but under isothermal conditions, Circular oligonucleotide probes can be replicated with a linear or geometric rate equation. Yet another technique, strand displacement amplification (SDA; Walker et al. (1992), PNAS (USA), 80, 392), starts with a specially specified sequence unique to a particular target.

  Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.

  Once the nucleic acid is amplified, a number of techniques are available for quantifying DNA and thus for quantifying RNA transcripts present. Available detection methods include radioactive labels, enzyme labels, chemiluminescent labels, fluorescent labels, and other suitable labels.

  In the context of the present invention, detection of nucleic acids encoding apoptosis-related proteins can be used to identify early apoptosis of cells.

b) Measuring gene expression at the polypeptide level may be detected by measuring an apoptosis-related polypeptide. This can be achieved by using a molecule that binds to an apoptosis-related polypeptide. Suitable molecules / agents that bind directly or indirectly to this protein to detect the presence of apoptosis-related proteins include naturally occurring molecules such as peptides and proteins, eg antibodies, or those May be a synthetic molecule.

  Using standard laboratory techniques such as immunoblotting as described above, it can be detected whether the level of apoptosis-related protein is altered by comparison with untreated cells in the same cell population.

  Gene expression can also be measured by detecting a post-translational process of the polypeptide or a change in post-transcriptional modification of the nucleic acid. For example, differential phosphorylation of polypeptides, cleavage of polypeptides, or alternative splicing of RNA, and the like can be measured. The level of expression of gene products such as polypeptides, as well as their post-translational modifications, can be detected using specialized protein assays or techniques such as two-dimensional polyacrylamide gel electrophoresis.

Monitoring the development of apoptosis A number of methods are known in the art for monitoring the occurrence of apoptosis. These include morphological analysis, DNA ladder formation, cell cycle analysis, membrane phospholipid phosphatidylserine externalization, and caspase activation analysis. Cell survival can be monitored by a number of techniques, including cell cycle analysis and cell viability measurements. Cell proliferation measurements can be made using a number of techniques including plaque assays. In the plaque assay, adherent cells are plated on tissue culture plates, grown, fixed and stained. The number of colonies formed reflects the amount of cell proliferation.

Modification of the functional activity of an apoptotic protein The functional activity of an apoptotic protein can be modified by an appropriate molecule / agent that binds directly or indirectly to the apoptotic protein or the nucleic acid encoding it. Agents can be naturally occurring molecules such as peptides and proteins, eg antibodies, and they can be synthetic molecules. Methods for modulating the expression level of apoptotic proteins include, for example, the use of antisense techniques.

  Thus, a test agent or candidate agent for use in the present invention can be based on an antisense oligonucleotide construct. Antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, directly block the translation of the mRNA for the apoptosis-related protein of interest by binding to it in the cell, thereby blocking protein translation Alternatively, it is believed to have the effect of enhancing the degradation of the mRNA, thereby reducing the level of the target apoptosis-related protein and thereby also reducing its activity. For example, antisense oligonucleotides of at least about 15 bases that are complementary to a unique region of an mRNA transcript sequence encoding an apoptosis-related protein of interest can be synthesized, for example, by the conventional phosphodiester method. Methods of using antisense techniques that specifically suppress gene expression of genes of known sequence are well known in the art (eg, US Pat. Nos. 6,566,135, 6,566,131, 6,365,354, 6,410,323, 6,107,091; 6,046,321 and 5,981,732).

  Suitable antisense molecules can be chemically modified variants based on these molecules. For example, antisense nucleic acids that are modified and often contain internucleotide linkages that are nuclease resistant can be useful. Hartmann et al. (Editing), Manual of Antisense Methodology (Perspectives in Antisense Science), Kluwer Law International (1999 years) (ISBN: 079238539X); Stein et al. (Editing), Applied Antisense Oligonucleotide Technology, Wiley Liss (1998 years) (ISBN: 0471172790); Chadwick et al. (Edit), Oligonucleotides as Therapeutic Agents Symposium No. 209, John Wiley & Son Ltd (1997) (ISBN: 0479772797).

  Other modified oligonucleotide backbones include, for example, methyl and other alkyl phosphonates, including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, 3 ′ alkylene phosphonates and chiral phosphonates. Phosphoramidates, including phosphinates, 3 'aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and normal 3' Boranophosphates with -5 'linkage, 2'-5' linkages of these analogs, and reversed polarity, where adjacent pairs of nucleoside units are 3'-5 'to 5' -3 ', 2'-5' It is connected to the '2'.

Modified oligonucleotide backbones that do not contain other phosphorus atoms for antisense use are short chain alkyl or cycloalkyl internucleotide linkages, heteroatoms and mixed internucleotide linkages of alkyl or cycloalkyl, or one or more short chains It has a backbone formed by heteroatoms or heterocyclic internucleotide linkages. These include morpholino linkages (partially formed from the sugar portion of the nucleoside); siloxane backbone; sulfide, sulfoxide, and sulfone backbone; formacetyl and thioformacetyl backbone; methyleneformacetyl and thioformacetyl Skeleton; alkene-containing skeleton; sulfamic acid skeleton; methyleneimine and methylenehydrazino skeleton; sulfonate and sulfanamide skeleton; amide skeleton; and other skeletons with mixed components of N, O, S, and CH ₂ Things are included.

  Other methods of modulating gene expression are known to those skilled in the art and include a dominant negative approach and the introduction of peptides or small molecules that suppress gene expression or functional activity.

  Small molecule inhibitory RNA (siRNA) can also function as a test agent for use in the present invention. Gene expression of the apoptosis-related protein of interest brings the tumor, subject, or cell into contact with a vector or construct that causes the production of small double-stranded RNA (dsRNA) or small double-stranded RNA, thereby causing the desired apoptosis-related protein It can be reduced by specifically suppressing protein expression (ie RNA interference or RNAi). Methods for selecting a dsRNA suitable for a gene of known sequence, or a vector encoding a dsRNA, are well known in the art (eg, Tuschi, T. et al. (1999) Genes Dev, 13 (24 ) 3191-3197; Elbashir, SM et al. (2001), Nature, 411, 494-498; Hannon, GJ (2002), Nature, 418, 244- 251; McManus, MT and Sharp, PA (2002), Nature Reviews Genetics, 3, 737-74; Bremmelkamp, TR, et al. (2002), Science, 296. Vol., 550-553; US Pat. No. 6,573,099; International Publication See International Publication WO01 / 68836); O01 / 36646; WO99 / 32619; U.S. Pat. No. 6,506,559.

  Ribozymes can also function as test agents for use in the present invention. Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of ribozyme molecules to complementary target RNA, followed by nucleotide strand breaks. Engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze nucleotide strand breaks in mRNA sequences encoding apoptosis-related proteins of interest are thereby useful within the scope of the present invention. The specific ribozyme cleavage site within any potential RNA target is first identified by scanning the target molecule for a ribozyme cleavage site. Ribozyme cleavage sites typically include the following sequences: GUA, GUU, and GUC. Once identified, predicted structures that can make a short RNA sequence of about 15 to 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site unsuitable for its oligonucleotide sequence, such as secondary structure It can be evaluated in terms of properties. The suitability of target candidates can also be assessed by testing their accessibility for hybridization with complementary oligonucleotides, for example in a ribonuclease protection assay.

  Antisense oligonucleotides and ribozymes useful as test agents can both be prepared by known methods. These include, for example, chemical synthesis methods by solid phase phosphoramadite chemical synthesis. Alternatively, antisense RNA molecules can be generated by in vitro or in vivo transcription of a DNA sequence encoding the RNA molecule. Such DNA sequences can be incorporated into a variety of vectors incorporating an appropriate RNA polymerase promoter, such as a T7 or SP6 polymerase promoter. As a method for increasing intracellular stability and half-life, various modifications can be introduced into the oligonucleotide of the present invention. Possible modifications include adding flanking sequences of ribonucleotides or deoxyribonucleotides at the 5 ′ and / or 3 ′ ends of the molecule, or 2′-O-methyl instead of phosphodiesterase linkage in the phosphorothioate or oligonucleotide backbone. Is included, but is not limited thereto.

  In addition, changes in events that occur immediately downstream of apoptosis-related proteins, such as the expression of genes whose transcription is regulated by the expression of apoptosis-related proteins, indicate that the molecule in question affects the functional activity of such proteins. It can be used as an indicator.

Expression of apoptosis-related proteins in cells Apoptosis-related proteins can be expressed in cells by introducing a vector encoding an apoptosis-related polypeptide.

  In the present invention, vectors that cause the expression of polypeptides from inserted heterologous nucleic acids (“expression vectors”) are particularly useful. These are often various other genetic elements operably linked to heterologous nucleic acid inserts that encode proteins, typically genetic elements that cause transcription, such as promoter and enhancer elements, transcription termination signals and / or polyadenylation signals. Elements that facilitate RNA processing, and elements that facilitate translation, such as ribosome consensus sequences. As will be appreciated by those skilled in the art, the conditions allowing expression of the polypeptide from these vectors will depend on the type of vector and the cell expression system selected.

  Vectors for expressing proteins are known to express in prokaryotic cells, yeast cells, usually S. cerevisiae, and mammalian cells, each of which is specific for expression in a specific cell type. Contains genetic elements.

  Protein expression performed by the vector may be constitutive or inducible. Inducible vectors include naturally inducible promoters such as the trc promoter regulated by the lac operon, the pL promoter regulated by tryptophan, and the MMTV-LTR promoter induced by dexamethasone, which are also synthetic. Additional elements that confer inducibility to the promoter and / or adjacent promoters may also be included.

  Methods for introducing vectors and nucleic acids into host cells are well known in the art, and the choice of technique will depend primarily on the particular vector being introduced and the host cell selected. Plasmid vectors will usually be introduced into bacterial chemically competent or electrocompetent bacterial cells. Introduction of a vector into yeast cells can be performed by spheroplast treatment with lithium salt, electroporation, or protoplast fusion. Mammalian and insect cells can be directly infected with packaged viral vectors or transfected by lipid, chemical, or electrical methods.

  Expression vectors can be designed such that the expressed polypeptide is fused to a small protein tag that facilitates purification and / or visualization.

  For example, the protein can be expressed with a tag that facilitates purification of the fusion protein. Suitable tags and methods for their purification are known and can be detected with poly-His / immobilized metal affinity chromatography, glutathione S-transferase / glutathione affinity resin, detectable with Xpress epitope / anti-Xpress antibody (Invitrogen, Carlsbad, CA, USA), myc tag / anti-myc tag antibody, V5 epitope / anti-V5 antibody (Invitrogen, Carlsbad, CA, USA), and FLAG® epitope / anti-FLAG® antibody (Stratagene, La Jolla, CA, USA).

  An appropriate sequence encoding a secretion signal, such as a leader peptide, can be included in the vector so that the expressed protein is secreted. Expression vectors can also be designed such that the protein encoded by the heterologous nucleic acid insert is fused to a polypeptide that is larger than the purification and / or identification tag. Useful protein fusions include those that allow the encoded protein to be displayed on the surface of a phage or cell, or those that have a green fluorescent protein (GFP) -like chromophore, to proteins that fluoresce intrinsically. Fusions to the IgG Fc region, and fusions for use in a two-hybrid system.

  Apoptosis-related proteins can be expressed and purified from a system, such as a system for use in a method for detecting a molecule that interacts with an apoptosis-related protein.

  Biochemical analysis tools and methodologies used in the above methods or processes for identifying inhibitors of target proteins are well known in the art. For example, in the case of a GPCR receptor protein, once a gene for a target protein such as the human apoptosis-related GPCR protein of the present invention is cloned, it is introduced into the recipient cell, and then the target protein is expressed on its surface. This cell expresses a single set of target human receptor subtypes, but this cell is expanded to establish a cell line. This cell line constitutes a drug discovery system and is used in two different types of assays: binding assays and functional assays. In a binding assay, the affinity of a compound for a receptor subtype that is the target of a particular drug discovery program and other receptor subtypes that may be associated with side effects is measured. These measurements make it possible to predict the potency of the compound and the degree of selectivity that the compound has over the target receptor subtype, separating other receptor subtypes. The data obtained from the binding assay is designed by the chemist to resemble or differ from one or more related subtypes as needed to obtain optimal therapeutic efficiency. It also makes it possible. In functional assays, the response characteristics of a receptor subtype to a compound are measured. The data obtained from the functional assay indicates whether the action of the compound inhibits or promotes the activity of the receptor subtype so that pharmacologists can determine their final human receptor subtype target With respect to, the compounds can be evaluated quickly, allowing chemists to rationally design drugs that are more effective and have fewer or substantially less severe side effects than existing drugs.

  Approaches to design and synthesize receptor-subselective compounds are well known and include traditional medicinal chemistry and newer combinatorial chemistry techniques, both of which are computer-based Assist with molecular modeling. Such an approach allows chemists and pharmacologists to structure the target receptor subtype and compounds that bind to this receptor subtype and / or activate or inhibit its activation. Is used to design and synthesize structures that will have activity against these receptor subtypes.

  Combinatorial chemistry is the automated synthesis of various new compounds by assembling them with different combinations of chemical components. The use of combinatorial chemistry has greatly accelerated the process of producing compounds. The resulting array of compounds is called a library and is used to screen to obtain compounds that exhibit a sufficient level of activity for the receptor of interest ("lead compound"). Using combinatorial chemistry, it is possible to synthesize “intensive” libraries of compounds that are expected to be highly biased towards the desired receptor target.

  Once the lead compound has been identified using combinatorial chemistry or traditional medicinal chemistry, or by other methods, various homologues and analogs can be prepared to determine the chemical structure and biological or functional activity. Promote understanding of relationships. Such studies reveal structure-activity relationships, which are then used to design drugs with improved potency, selectivity, and pharmacokinetic properties. Combinatorial chemistry is also used to generate various structures for lead optimization. Traditional medicinal chemistry involves the synthesis of one compound at a time, which is also used to achieve further refinement and to produce compounds that cannot be produced by automated methods. Once such drugs are defined, production is expanded using standard chemical manufacturing methods utilized throughout the pharmaceutical and chemical industries.

Thus, in one aspect of the invention, the activity of the apoptosis-related GPCR protein of the invention can be monitored by measuring the readout of G protein binding. For example, such readouts can be achieved by measuring the activity of Ca ²⁺ or K ⁺ channels regulated by G protein using electrophysiological methods, or by using fluorescent dyes to measure intracellular Ca ²⁺ levels. It can be monitored by measuring changes. Other methods that can typically be used to monitor receptor activity include measuring the level or activity of GTPγS or cAMP.

Detection of molecules that interact with apoptosis-related proteins To detect molecules that interact directly with apoptosis-related proteins, techniques such as analytical centrifugation, affinity binding analysis using chromatography or electrophoresis can be used. . Those skilled in the art will appreciate that this list is by no means exhaustive. More specifically, the apoptosis-related protein is labeled with a detectable label and used as a probe to detect the apoptosis product on an electrophoretic gel; the apoptosis-related protein target is labeled and By using to probe a library of genes and / or cDNA; a clone that synthesizes a protein that can bind to the target by labeling an apoptosis-related protein target and using it to probe a cDNA expression library By finding; performing UV cross-linking experiments to identify agents that can bind to the target; using apoptosis-related proteins in gel retardation assays to determine their ability to bind to the nucleic acid encoding the identified agent. By detecting; Performing footprint analysis, in the nucleic acid, by identifying a region where the target is bound, it can be used as an affinity ligand to identify agents that bind thereto. Those skilled in the art will recognize other suitable techniques and will appreciate that this list is not intended to be exhaustive.

  Other techniques that allow identification of protein-protein interactions include the yeast two-hybrid method and immunoprecipitation reactions as described above.

  Yeast assays can be used for screening to obtain agents that modulate the activity of the apoptosis-related GPCR proteins or mutant polypeptides of the invention. In a typical yeast assay, the apoptosis-related GPCR protein or mutant polypeptide of the present invention is heterologously expressed in a modified yeast strain containing multiple reporter genes. The multiple reporter genes are typically FUS1p-HIS-3 and FUS1p-lacZ, each linked to an endogenous MAPK cascade-based signaling pathway. This pathway is normally linked to pheromone receptors, but can be linked to foreign receptors by replacing the yeast G protein with a yeast / mammalian G protein chimera. Yeast strains may contain additional genetic defects, such as SST2 deletion and FAR1 deletion, to enhance the assay. Heterogeneous receptor ligand activation can be monitored, for example, as cell growth in the absence of histidine or with an appropriate substrate for β-galactosidase (lacZ). Such techniques are well known in the art. See, for example, International Publication Nos. WO99 / 14344, WO00 / 12704, or US Pat. No. 6,100,042.

  Alternatively, the melanocyte assay may be used for screening to obtain modulators of the apoptosis-related GPCR proteins of the invention. Apoptosis-related GPCR proteins can be expressed heterogeneously in Xenopus laevis melanocytes, and their activation or suppression can be measured by melanosome dispersal or aggregation. Basically, melanosome dispersal is promoted by activation of adenylate cyclase or phospholipase C, ie Gs-mediated and Gq-mediated signaling, respectively, while aggregation is a Gi protein that causes inhibition of adenylate cyclase. It happens as a result of activation. Thus, ligand activation of heterologous receptors can be measured simply by measuring changes in light transmission through the cell or by imaging cellular responses.

  In order to confirm whether the observed response is the result of modulation of apoptosis-related GPCR protein, it is preferred to perform a control experiment using cells that do not express the apoptosis-related GPCR protein of the present invention.

  Protein interactions can also be analyzed using protein arrays. Protein arrays can be made by a variety of different techniques, which allow proteins to be deposited on a flat plane at different densities. High density protein arrays can be generated using automated methods similar to those described for DNA arrays (see below). Proteins that interact with apoptosis-related proteins can be identified, for example, by using these proteins for probing expression arrays. A positive interaction may then be detected, for example, by the presence of a labeled antibody or by a tag placed on the apoptosis-related protein. The identity of interacting proteins can be determined by techniques such as mass spectrometry.

Cells Cells useful in the methods of the invention may be derived from any source, for example, primary cultures, established cell lines, organ cultures, or in vivo. Cell lines useful in the present invention include cells and cell lines derived from the hematopoietic system. Suitable cells include HeLa, U937 (mononuclear cells), TF1, HEK293 (T), primary cultures of cells with neutrophil or neutrophil characteristics, eg, HL60 cells, mouse FDCP-1, FDCPmix, 3T3, primary or human stem cells are included.

Measurement of global gene expression Expression of recombinant apoptosis-related proteins in cells can be induced using expression systems including any of those described herein.

  Measuring the expression level of genes associated with apoptosis-related proteins will allow the identification of other known or novel genes that play a role in apoptosis.

  Regulation of gene activity can occur at many levels. Most commonly, regulation is at the transcription level. Certain transcription factors modulate the expression of a subset of target genes. Post-transcriptional regulation (translational regulation) is determined by the rate and mechanism of intracellular RNA processing, ie, accumulation, translation, and degradation. Subsequent regulation of gene activity at the protein level occurs through post-translational modifications.

  In the present invention, screening can be performed for a number of individual gene product types whose expression or function is associated with apoptosis-related protein gene expression. These products include polypeptides and nucleic acids. The expression level evaluated can be an absolute level for the production of a particular polypeptide or nucleic acid, or it can be the production level of any polypeptide or nucleic acid derivative. For example, the present invention may be configured to measure the expression level of a particular mRNA splice variant, or the amount of phosphorylated derivative of a particular polypeptide present.

  If it is desirable to monitor the expression level of a known gene product, conventional assay techniques including nucleic acid hybridization analysis and activity-based protein assays may be used. Kits for quantifying nucleic acids and polypeptides are commercially available.

  However, if the gene product being monitored is unknown, methods are used that facilitate the identification of the gene product whose expression is being measured. For example, if the gene product is a nucleic acid, an array of oligonucleotide probes can be used as a basis for screening mRNA populations obtained from cells.

a) Gene arrays of oligonucleotides specific for gene sequences recorded in public databases such as the array GenBank are commercially available from a number of suppliers (Incyte Genomics, USA, etc.). Examples of such commercially available arrays include those in which nucleotides are spotted on the surface of either a membrane filter (such as nitrocellulose) or a solid support (such as glass). Commercially available gene arrays are used to obtain expression pattern profiles of genes associated with the apoptotic process of neutrophils and other cell types.

  Gene arrays can also be additionally constructed by spotting nucleotide sequences obtained from cDNA clones generated from new libraries or purchased cDNA clones. Such arrays allow the acquisition of expression profiles of proprietary and / or new nucleotide sequences.

  Gene arrays are additionally constructed by spotting nucleotide clones generated from new libraries or purchased cDNA clones, also from commercial sources (eg, Genescreen). Such arrays allow the acquisition of expression profiles of proprietary and / or new nucleotide sequences.

  Many of the cDNA or EST (expressed gene sequence fragment) sequences recorded in public domain databases are derived from a limited set of tissue types, such as liver, brain, and fetal tissue. Cloning homemade cDNA libraries focused on specific cellular events such as ROS-mediated apoptosis offers the possibility to identify, clone and characterize new genes associated with this process. Similarly, cloning a homemade cDNA library focused on a specific tissue type such as neutrophils has the potential to identify, clone and characterize new genes whose expression is restricted to this cell type. provide. Libraries (cDNA) constructed using physical subtraction, such as the ClonTech “Select” SSH (inhibition hybridization) method, and novel variants thereof, are described in the process or cell type studied. Allows selective cloning of genes whose expression is differentially regulated. Gene array technology is used in conjunction with an SSH cDNA library to identify false positives and further focuses on genes that are truly differentially expressed. Clones obtained from each of the constructed SSH libraries are picked, cultured and stored as glycerol stocks. CDNA inserts contained in individual plasmid clones are PCR amplified and spotted on a homemade array. Differential expression is confirmed using hybridization with radiolabeled probes generated from the mRNA used for each reciprocal subtraction.

  An array of nucleic acids may be prepared by direct chemical synthesis of nucleic acid molecules. In chemical synthesis, a nucleic acid array is synthesized on the surface in such a way that each of the identifiable nucleic acids (eg, unique nucleic acid sequences) is placed at a separate predetermined location in the array. The identity of each nucleic acid is determined by the spatial position of the array. These methods are described in US Pat. No. 5,143,854; International Publication Nos. WO 90/15070 and WO 92/10092; Fodor et al. (1991) Science 251: 767; Dower and Fodor (1991), Ann. Rep. Med. Chem. , Vol. 26, page 271 may be adapted.

  In a preferred embodiment of the invention, the nucleic acid array can be prepared by the gridding of nucleic acid molecules. Because robotic technology allows the most accurate and enriched gridding of nucleic acid molecules, it would be advantageous to align oligonucleotides by robotic picking. However, any technique suitable for finding the location of the molecule at a distinct location on the carrier, including manual techniques, can be utilized.

  The gridding may be regular so that each colony is a given distance from the next colony, or it may be random. If the distance between the molecules is determined randomly, the density of those molecules can be adjusted so that the probability of overlapping on the selected support is statistically reduced or eliminated.

  Devices for producing nucleic acid microarrays are commercially available, and can be purchased from, for example, Genetix and Genetic Microsystems. Furthermore, a nucleic acid molecule array prepared in advance is commercially available, and can be purchased from, for example, Incyte Genomics (Human LifeGrid ™). Such arrays will usually contain ESTs (expressed gene sequence fragments) that are representative of most or all of the genes expressed in a cell or organism. Thus, it provides a platform for screening mRNA collections obtained from multiple ROS treated cells.

  The sample for mRNA population analysis may be isolated and purified by any suitable mRNA production method. For example, RNA isolation kits can be purchased from Stratagene.

  Furthermore, when the gene product is a polypeptide, an antibody array may be used as a basis for screening a collection of polypeptides obtained from cells. Examples of protein arrays and antibody arrays are described in Proteomics: A Trends Guide, Elsevier Science Ltd., incorporated herein by reference. , In July 2000.

  In the context of the present invention, array technology can also be used, for example, in analyzing the expression of one or more of the apoptotic proteins specified herein. In one embodiment, array technology may be used to simultaneously assay the effects of candidate compounds on a number of apoptotic proteins identified herein. Accordingly, another aspect of the invention provides a microarray or protein array or antibody array comprising at least one, at least two, or at least some of the nucleic acids identified in Table 1, or fragments thereof.

b) 2D PAGE
Separation techniques such as two-dimensional gel electrophoresis are used to monitor unknown polypeptide gene products. Usually, in 2D PAGE, sample preparation, first dimension electrophoresis in an immobilized pH gradient, second dimension SDS-PAGE electrophoresis, and sample detection are performed. Protocols for 2D PAGE are extensive in the art, see, for example, http: // www. expasy. available at ch / ch2d / protocols /, the contents of which are as of November 30, 2001, incorporated herein by reference.

Samples for 2D PAGE may be prepared by conventional techniques. In the present case, HeLa cells transfected with apoptosis-related proteins are grown in a suitable medium such as RPMI 1640 containing 10% fetal calf serum (FCS). The suspension is transferred to a tube and the cells are centrifuged at 1000 g for 5 minutes. Discard the supernatant and wash the cells with RPMI 1640 without FCS. After centrifugation to remove RPMI 1640, 0.8 × 10 ⁶ cells were treated with urea (8M), CHAPS (4% w / v), Tris (40 mM), DTE (65 mM), and a small amount of bromophenol. Mix with 60 μl of the solution containing blue and dissolve. The entire final diluted HeLa sample is added to the first dimension separation.

  Advantageously, the method of the invention uses a step of determining the reference expression level of the gene product to be investigated. This can be done by using untransfected HeLa cells as a standard for one or more subsequent assays, or it can be an integral part of all assays. . For example, mRNA or polypeptide populations obtained from transfected and non-transfected HeLa cells can be simultaneously evaluated by nucleic acid array or 2D PAGE to identify changes in expression patterns by direct comparison. May be.

  Analysis of 2D PAGE results is performed using appropriate software as needed to reveal the presence of the polypeptide of interest. These polypeptides can be isolated, sequenced and further used to identify the genes that encode them.

Assays The present invention binds to the polypeptides of the invention and is characterized by, for example, apoptosis, characterized by caspase activation, DNA fragmentation, cell contraction and fragmentation, phospholipid exposure at the plasma membrane, etc. Provide an assay suitable for identifying substances that affect progression. In addition, GPCRs and assays suitable for identifying modulators of protein kinases including PI3 kinase have also been incorporated.

  Typically, a substance that inhibits one or more of these apoptotic features either completely inhibits its protein activity at a concentration of 500 mM or less, or is significant (ie, greater than 50%) compared to a control. Leading to a significant decline. This inhibition is preferably 75% inhibition compared to the control, more preferably 90%, and most preferably 95% or 100% inhibition. Agents that promote or increase one or more of these apoptotic characteristics are those that lead to a significant (ie, greater than 50%) increase in protein activity compared to controls at concentrations of 500 mM or less. This increase is preferably a 75% increase compared to the control, more preferably a 90% increase, and most preferably a 95% or 100% increase.

  Furthermore, the assay can also be used to identify substances that block the binding of the polypeptides of the invention. This binding, if appropriate, is binding to a component of the apoptotic mechanism. Usually such assays are performed in vitro. Also provided are assays that test the effects of candidate substances identified in preliminary in vitro assays on intact cells in whole cell assays. The assays described below, or any suitable assay known in the art, can be used to identify these agents.

  Thus, as described in detail below, according to one aspect of the present invention, we enable the assays described below: apoptosis assays, kinase assays, kinase inhibitor assays, GPCR assays, and whole cell assays. One or more substances identified by any of them are provided.

Modulator screening assays Methods for inducing apoptosis are well known in the art and include, without limitation, exposure to chemotherapeutic or radiotherapeutic agents and, where appropriate, absolute survival factors. (For example, exclusion of GM-CSF, NGF). Differences between treated and untreated cells indicate the effect due to the test compound.

  Bone marrow cells die spontaneously during culture, but the time course varies depending on the cell type. Neutrophils in culture undergo apoptosis within 24 hours, which can be delayed to over 48 hours in the presence of survival factors. Eosinophil apoptosis is observed over 48 hours and is delayed to several days in the presence of survival factors. Macrophages usually survive much longer. Thus, the potency of a compound that modulates myeloid cell apoptosis is determined in the presence or absence of the test compound and, where appropriate, the absolute survival factor (eg, GM-CSF, IL-8, IL-5, G- After the elimination of CSF, or BAL), it can be assessed by monitoring the rate of apoptosis. The difference between the treated and untreated cells shows the effect due to the test compound.

  Compounds having inhibitory, activating, or modulating activity, such as ligands, agonists, antagonists, and their homologues and mimetics are identified using in vitro and in vivo assays of apoptotic protein activity and / or expression can do. Modulator screening can be performed by adding a putative modulator test compound to a tissue or cell sample and monitoring the effect of the test compound on the function and / or expression of apoptotic proteins. Parallel samples to which no test compound is added are also monitored as controls. Treated and untreated cells are then not limited to microscopic analysis, viability testing, replication capacity, histological examination, levels of polypeptides associated with specific RNA or cells, Comparison is made by any suitable phenotypic criteria, including the level of enzyme activity expressed in the cell or cell lysate, and the ability of the cell to interact with other cells or compounds.

  A substance that inhibits apoptosis as a result of interaction with a polypeptide of the present invention may do so in several ways. For example, if the substance inhibits the PI3K or GPCR signaling pathway, it can be signaled by, for example, binding to this polypeptide to mask or modify the site of interaction with other components. May directly break the binding of the polypeptide of the invention to these components. Agents that inhibit the PI3K or GPCR signaling pathway may do so by inhibiting phosphorylation or may involve dephosphorylation of the protein. This type of candidate agent may be conveniently screened, for example, in an in vitro binding assay as described below and then tested in a whole cell assay, eg as described below. Examples of candidate substances include antibodies that recognize the polypeptides of the present invention.

  A substance capable of directly binding to the polypeptide of the present invention alters the intracellular localization of the polypeptide, thereby altering its ability to interact with normal substrates, thereby altering the polypeptide in apoptosis. Function can also be inhibited. Such substances can change their subcellular localization by binding directly to the polypeptide or by indirectly disrupting the interaction of another component with the polypeptide. .

  These materials can be tested using, for example, the whole cell assay described below. Non-functional homologues of the polypeptides of the present invention are unable to exercise the normal function of this protein, but compete with wild-type proteins for binding to components of the apoptotic machinery or Such homologues can also be tested for inhibition of apoptosis because they can interfere with the function of the bound protein. Such non-functional homologues can include naturally occurring variants and modified sequences or fragments thereof.

  As another possibility, this substance may not inhibit the assembly of the components directly, but may inhibit the bioavailable amount of the polypeptide of the invention. This can be done by repressing the expression of the component, for example, at the level of transcription, transcript stability, translation, or post-translational stability. Examples of such substances would include antisense RNA or double stranded interfering RNA sequences. These suppress the amount of mRNA biosynthesis.

  Suitable candidate substances are peptides, in particular peptides with a size of about 5 to 30 amino acids or about 10 to 25 amino acids, or one or more residues, based on the polypeptide sequences described in the examples. Variants of such peptides are included. Peptides from the peptide panel containing random sequences, i.e., sequences that are uniformly varied to provide a maximally diversified peptide panel may also be used.

  Suitable candidate substances also include antibody products specific for the polypeptides of the invention (eg, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, and CDR-grafted antibodies). In addition, combinatorial libraries, peptides and peptidomimetics, specific chemical entities, oligonucleotides, and natural product libraries can also be screened for activity as inhibitors of binding of the polypeptides of the invention to the apoptotic mechanism. Candidate substances may be used in initial screening, for example, in batches of 10 substances per reaction, and batches of substances showing inhibition may be tested individually. Candidate substances that are active in in vitro screening, such as those described below, can then be tested in whole cell systems such as mammalian cells. In this case, mammalian cells will be exposed to the inhibitor and tested for inhibition at any stage of apoptosis.

  Other suitable test or candidate substances that can be tested in the above assay include combinatorial libraries, specific chemical entities, peptides and peptidomimetics, oligonucleotides, natural product libraries, displays (eg, phage display Library), and antibody products. The assay may be performed in the presence of a ligand or active substance for a G protein-coupled receptor. Test substances may be used in initial screening, eg, in batches of 10 substances per reaction, and those batches of substances showing inhibition or activation may be tested individually. The test substance can be used at a concentration of 1 nM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 10 μM.

  Cells expressing the apoptosis-related GPCR protein of the present invention can be used for screening to obtain a ligand using a functional assay. The same assay can be used to identify agonists or antagonists of apoptosis-related GPCR proteins that can be utilized for various therapeutic purposes in the presence or absence of ligand. It is well known to those skilled in the art that overexpression of GPCRs can cause constitutive activation of intracellular signaling pathways. Similarly, overexpression of apoptosis-related GPCR proteins in any of the cell lines described above can result in activation of the functional responses described below, and any assay described herein can be It can be used for screening to obtain both agonist and antagonist ligands of related GPCR proteins.

  A wide variety of functional assays are available for screening to obtain modulators of apoptosis-related GPCR proteins. These assays, for example, from traditional measurements of total inositol phosphate accumulation, cAMP concentration, intracellular calcium mobilization, and potassium or sodium current, for example; measure these same second messengers, but with higher throughput. To systems that are modified or adapted to be more versatile and more responsive; and more general, such as metabolic fluctuations, differentiation, cell division, or proliferation resulting from receptor activation It extends to cell-based assays that report cellular events.

  In the foregoing methods or processes of the invention for identifying inhibitors of apoptosis-related proteins, these proteins or types of detectable compounds that act on biochemical pathways that they modulate in cells include antagonists, inverse agonists , Partial inverse agonists, allosteric or allotopic antagonists, agonists, partial agonists, and allosteric or allotopic agonists. Assays to detect such compounds are in the presence of alterations by activating ligands or other activation mechanisms, such as binding of activated proteins or factors, or phosphorylation or another reversible covalent modification mechanism. Or it can be done in the absence.

  Any of the commonly used in the practice of the invention, including immunoprecipitation reactions, immunoblotting (Western blotting), and ELISA assays, to detect the interaction of the protein kinase of the invention with a test agent. These immunoassay techniques may be used to quantify the phosphotransferase activity of the protein kinase apoptosis-related protein of the invention. In one embodiment, an anti-kinase antibody is used to isolate a kinase protein, for example by immunoprecipitation, and a change in protein activity is used to assess protein autophosphorylation, using a labeled antiphosphoamino acid antibody. Or by the phosphotransferase activity of the protein against the peptide or protein substrate. In another embodiment, an ELISA assay is used to first capture the kinase with an anti-kinase antibody and then assess autophosphorylation with a labeled antiphosphoamino acid antibody in a second step.

  In an ELISA assay, the specific binding pair may be immune type or non-immune type. Immune specific binding pairs are exemplified by antigen-antibody systems or anti-hapten / hapten systems. Examples of such include fluorescein / anti-fluorescein, anti-dinitrophenyl / dinitrophenyl, biotin / anti-biotin, anti-peptide / peptide, and the like. Antibody members of a specific binding pair can be generated by routine methods familiar to those skilled in the art (eg, Using Antibodies, A Laboratory Manual, Harlow, E. and Lane, D., 1999, Cold Spring Harbor Laboratory). Press (see, eg, ISBN 0-87969-544-7). In such a method, an animal is immunized with an antigen member of a specific binding pair. If the antigen member of a specific binding pair is not immunogenic (eg, a hapten), it can be covalently bound to a carrier protein to confer it immunogenicity.

Further, with respect to the apoptosis-related protein kinases of the present invention, methods for detecting activity changes in such kinases are particularly suitable for purified proteins, or proteins or peptides, where assaying for proteins in membrane fractions is contemplated. a assay of phosphotransferase activity in this assay, the activity is monitored by radioactive phosphorus taken up by radioactive phosphorus is incorporated into the protein via autophosphorylation by P ³² phosphate, or alternatively a substrate protein or the peptide . Alternatively, many non-radioactive assay formats such as ELISA, scintillation proximity, electrophoresis, bead-based immunoprecipitation reactions, and Western blots can be used to assess kinase activity in purified enzyme samples. Detection methods include the use of radioactive tracers, colorimetric or chemiluminescent substrates, time-resolved fluorescence, resonance transfer (FRET), and fluorescence polarization. Many of these assay formats are readily available as commercial kits.

  If a substance is known to inhibit, reduce, enhance, promote or activate apoptotic activity, the substance is identified as a modulator of apoptotic activity. Typically, an agent that inhibits one or more of these apoptotic features completely inhibits its protein activity at a concentration of 500 mM or less, or a control (ie, one or more of the apoptotic features). Leads to a significant (ie, more than 50%) decrease compared to a substance known not to modulate. This inhibition is preferably 75% inhibition compared to the control, more preferably 90%, and most preferably 95% or 100% inhibition. This inhibition may block apoptosis or may simply delay or prolong apoptosis. Agents that promote or increase one or more of these apoptotic characteristics are those that lead to a significant (ie, greater than 50%) increase in protein activity compared to controls at concentrations of 500 mM or less. This increase is preferably a 75% increase compared to the control, more preferably a 90% increase, and most preferably a 95% or 100% increase.

Polypeptide Binding Assays One type of assay for identifying substances that bind to a polypeptide of the present invention involves contacting a polypeptide of the present invention immobilized on a solid support with a non-immobilized candidate substance, and Determining whether the polypeptide of the invention and the candidate substance bind to each other and / or determining to what extent it. Alternatively, the candidate substance may be immobilized and the polypeptide of the present invention may be unimmobilized.

  The binding of the substance to the apoptosis-related polypeptide can be transient, reversible, or permanent. Preferably, the substance binds to the polypeptide with a Kd value that is lower than the Kd value when binding to a control polypeptide (ie, a polypeptide known not to be an apoptosis-related polypeptide). The Kd value of this substance is preferably 2 times less than the Kd value when binding to the control polypeptide, more preferably the Kd value is 100 times less than the Kd value when binding to the control polypeptide, most preferably , Kd value is 1000 times smaller.

  In a preferred assay, the polypeptide of the invention is immobilized on beads such as agarose beads. Usually this is achieved by expressing the component as a GST fusion protein in a bacterial, yeast, or higher eukaryotic system and purifying the GST fusion protein from the crude cell extract using glutathione agarose beads ( Smith and Johnson, 1988, Gene 67 (10), 31-40). As a control, the binding of a candidate substance that is not a GST fusion protein to the immobilized polypeptide of the present invention is measured in the absence of the polypeptide of the present invention. Next, the binding of the candidate substance to the immobilized polypeptide of the present invention is measured. This type of assay is known in the art as a GST pull-down assay. Again, the candidate substance may be immobilized and the polypeptide of the present invention may be unimmobilized.

  This type of assay can also be performed using different affinity purification systems, such as Ni-NTA agarose and histidine tagged components, to immobilize one of the components.

  Binding of a polypeptide of the present invention to a candidate substance can be measured by various methods well known in the art. For example, non-immobilized components may be labeled (eg, with a radioactive label, epitope tag, or enzyme-antibody complex). Alternatively, binding may be measured by immunological detection methods. For example, a Western blot of the reaction mixture can be performed and the blot can be probed with antibodies that detect non-immobilized components. An ELISA method can also be used.

  Candidate substances are usually added to a final concentration of 1-1000 nmol / ml, more preferably 1-100 nmol / ml. In the case of antibodies, the final concentration used is usually 100 to 500 μg / ml, more preferably 200 to 300 μg / ml.

Other In Vitro Assays Other assays that identify substances that bind to a polypeptide of interest are also provided. The binding of the substance to the apoptosis-related polypeptide can be transient, reversible, or permanent. Preferably, the substance binds to the polypeptide with a Kd value that is lower than the Kd value when binding to a control polypeptide (ie, a polypeptide known not to be an apoptosis-related polypeptide). The Kd value of this substance is preferably 2 times less than the Kd value when binding to the control polypeptide, more preferably the Kd value is 100 times less than the Kd value when binding to the control polypeptide, most preferably , Kd value is 1000 times smaller.

  A substance that affects kinase activity is the substance of interest.

Substances that inhibit or affect kinase activity can be obtained by kinase assays known in the art, eg, ³² P incorporated into an appropriate peptide or other substrate in the presence of a candidate substance. It can be identified by measuring. Similarly, agents that inhibit or affect proteolytic activity can be assayed by detecting an increase or decrease in cleavage of the appropriate polypeptide substrate.

  These and other protein or polypeptide activity assays are known to those skilled in the art and are suitable for use in the identification of substances that bind to and affect the polypeptide of interest.

Whole Cell Assay The effect of candidate substances on apoptosis can also be tested on whole cells. The candidate substance is preferably identified by the in vitro method described above. Alternatively, as a preliminary screen, use a rapid throughput screen to obtain a substance that can inhibit or promote apoptosis, and then use it in the in vitro assay described above to determine the effect of the specific polypeptide of interest. Confirm that it is for.

  Candidate substances, ie, test compounds, can be administered to cells in several ways. For example, it may be added directly to the cell culture medium or injected into the cells. Alternatively, in the case of a polypeptide candidate substance, the cell may be transfected with a nucleic acid construct that directs the expression of the polypeptide in the cell. The expression of the polypeptide is preferably under the control of a regulatable promoter.

  In general, an assay that measures the effect of a candidate substance identified by the method of the present invention on apoptosis involves administering the candidate substance to a cell and measuring whether the substance inhibits apoptosis. Techniques for measuring cell population apoptosis are well known in the art. If there is inhibition, the degree of apoptosis of the treated cells is determined as compared to the degree of apoptosis of the untreated control cell population. For example, inhibitors of apoptosis can be assayed by measuring the percentage of cells in the sub-G1 population and comparing this to the percentage of cells in the untreated population.

  The concentration of the candidate substance used will usually be such that the final concentration in the cell is similar to that described above for the in vitro assay.

  A candidate substance is usually apoptotic when the percentage of cells undergoing apoptosis is reduced to less than 50%, preferably less than 40, 30, 20, or 10% of the percentage seen in an untreated control cell population. It is considered an inhibitor.

  In the context of the above assay, the polypeptide of interest is a polypeptide encoded by any of the nucleic acid sequences specified in Table 1B.

  Methods for determining whether a compound that interacts with an apoptosis-related protein is a tumor growth, tumor cell growth, or an inhibitor of tumor cell proliferation, or an active substance of tumor cell apoptosis are well known in the art.

  The effect of a compound on tumor growth can be monitored by any of a number of methods known to those skilled in the art or variations thereof. For example, the effectiveness of a compound can be tested in an animal model. The efficacy of the compound alone or in combination with conventional anti-tumor agents such as cytotoxins / anti-tumor agents and anti-angiogenic agents can be compared to that of conventional agents alone. Usually, tumors of a given size are present in rats or mice. Mice are treated with drugs and tumor size is measured over time. The average survival time of animals can also be measured. The compound that modulates the activity of the apoptosis-related protein used must bind to the receptor of the animal being tested. Xenografts can be transplanted into their animals to test the species-specific inhibitory ability of the compounds. Suitable test animals include, but are not limited to, inbred rats such as Fischer 344 and Lewis rats, and athymic NCR-NU mice.

  The effect of a compound on tumor cell growth or tumor cell proliferation can be monitored by any of a number of methods known to those skilled in the art or variations thereof (see, eg, methods herein).

  The effect of a compound on apoptosis can be monitored by any of a number of methods known to those skilled in the art or variations thereof (see, eg, the methods section herein). Specific examples of apoptosis assays are also illustrated in the following references. The assay for lymphocyte apoptosis is disclosed by: Li et al. (1995), Science 268: 429-431; Gibellini et al. (1995), Br J. et al. Haematol, 89, 24-33; Martin et al. (1994), J. MoI. Immunol, 152, 330-42; Terai et al. (1991), J. Am. Clin Invest. 87, 1710-5; Dhein et al. (1995), Nature, 373, 438-441; Katsikis et al. (1995), J. MoI. Exp. Med. 1815, 2029-2036; Westendorp et al. (1995), Nature, 375, 497; and DeRossi et al. (1994), Virology, 198, 234-44. An assay for fibroblast apoptosis is disclosed by: Vossbeck et al. (1995), Int. J. et al. Cancer, 61, 92-97; Goruppi et al. (1994), Oncogene, 9, 1537-44; Fernandez et al. (1994), Oncogene, 9, 2009-17; Harrington et al. ( 1994), EMBO J. et al. 13, 3286-3295; and Itoh et al. (1993), J. MoI. Biol. Chem. 268, 10932-7. An assay for neuronal apoptosis is disclosed by: Melino et al. (1994), Mol. Cell Biol. 14, 6584-6596; Rosenblum et al. (1994), Ann. Neurol. 36, 864-870; Sato et al. (1994), J. MoI. Neurobiol, 25, 1227-1234; Ferrari et al. (1995), J. MoI. Neurosci, 1516, 2857-2866; Talley et al. (1995), Mol. Cell Biol. 1585, 2359-2366; Talley et al. (1995), Mol. Cell. Biol. 15, 2359-2366; and Wakinshaw et al. (1995), J. MoI. Clin. Invest. 95, 2458-2464. Insect cell apoptosis assays are disclosed by: Clem et al. (1991), Science, 254, 1388-90; Crook et al. (1993), J. MoI. Virol, 67, 2168-74; Rabizadeh et al. (1993), J. MoI. Neurochem, 61, 2318-21; Birnbaum et al. (1994), J. MoI. Virol, 68, 2521-8, 1994; and Clem et al. (1994), Mol. Cell. Biol. 14: 5212-5222.

Therapeutic applications Many disease states are associated with defects in abnormal apoptosis. Such conditions include cancer, inflammation, autoimmune diseases, and neurodegenerative disorders. Therefore, modulating apoptosis can be one of therapeutic approaches for treating such diseases. Such an approach can generally be used to treat any proliferative disease. Thus, the polypeptides of the present invention have been found to be necessary for normal apoptosis and are therefore targets to modulate their function, particularly in disease cells, including tumor cells and inflammatory cells.

  Cancers or proliferative diseases / disorders include malignant tumors and preneoplastic disorders. The present invention is particularly useful for the treatment or diagnosis of adenocarcinomas such as small cell lung cancer, kidney cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, and breast cancer. For example, malignant tumors that can be treated according to the present invention include acute and chronic leukemia, lymphoma, myeloma, fibrosarcoma, myxosarcoma, liposarcoma, lymphatic endothelial sarcoma, angiosarcoma, endothelial sarcoma, chondrosarcoma, osteogenic Sarcoma, chordoma, lymphangiosarcoma, synovial sarcoma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma and other sarcomas, colon carcinoma, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, squamous cell carcinoma, basal cell Cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchogenic carcinoma, choriocarcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma seminoma, fetus Sex cancer, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, marrow Blastoma, craniopharyngioma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and membrane germplasm Included tumor is.

  One possible approach is to selectively express an antisense construct directed against the polynucleotide of the invention, preferably in diseased cells, to inhibit gene function or otherwise modulate it. Inducing or inhibiting apoptosis. Another approach is to use non-functional variants of the polypeptides of the invention that compete with endogenous gene products for cellular components of the apoptotic machinery and result in functional inhibition. Alternatively, a compound identified by the above-described assay as binding to a polypeptide of the invention can help disease cells block or promote the function of the polypeptide. This can be done, for example, by gene therapy or by direct administration of the compound. Suitable antibodies of the invention can also be used as therapeutic agents.

  Alternatively, double stranded (ds) RNA is a powerful method of interfering with gene expression in various organisms and has recently been shown to be successful in mammals (Wianny and Zernica-Goetz, 2000). Year, Nat Cell Biol, 2000, Volume 2, pages 70-75). Double-stranded RNA corresponding to the sequence of the polynucleotide according to the present invention can be introduced into and expressed in a diseased cell of an individual in order to prevent normal apoptosis.

  Usually, a disease is defined as being “treated” if abnormal apoptosis associated with the disease or condition is significantly (ie, greater than 50%) inhibited compared to a control. This inhibition is preferably 75% inhibition compared to the control, more preferably 90%, and most preferably 95% or 100% inhibition. This inhibition may simply prevent apoptosis, or may be delayed or prolonged.

Dosing Substances identified or identifiable by the assay of the present invention are preferably combined with various components to produce the compositions of the present invention. This composition is preferably synthesized with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for use in humans or animals). Suitable carriers and diluents include isotonic saline, such as phosphate buffered saline. The compositions of the invention may be administered by direct injection. The composition may be formulated for parenteral administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration, or transdermal administration. Usually, each protein can be administered at a dose of 0.01-30 mg / kg body weight, preferably 0.1-10 mg / kg, more preferably 0.1-1 mg / kg body weight.

  A polynucleotide / vector encoding a polypeptide component (or antisense construct) for use in inhibiting apoptosis may be administered directly as a naked nucleic acid construct. They may contain flanking sequences that are homologous to the host cell genome. When administering a polynucleotide / vector as a naked nucleic acid, the amount of nucleic acid administered will normally be in the range of 1 μg to 10 mg, preferably 100 μg to 1 mg. It is particularly preferred to use polynucleotides / vectors that specifically target tumors or proliferating cells, for example, by appropriate regulatory constructs or by the use of targeted viral vectors.

  The uptake of naked nucleic acid constructs by mammalian cells is facilitated by several known transfection techniques, including, for example, the use of transfection agents. Examples of these agents include cationic agents (eg, calcium phosphate and DEAE-dextran), and lipofectants (eg, lipofectam ™ and transfectam ™). Usually, the nucleic acid construct is mixed with a transfection agent to produce a composition.

  Preferably, a polynucleotide, polypeptide, compound or vector described herein is linked, associated, linked, fused or otherwise associated with a membrane translocation sequence.

  The polynucleotides, polypeptides, compounds, vectors, and the like described herein can be combined, associated, linked, fused, or another method with a protein (ie, a membrane translocation sequence) that can cross the plasma membrane and / or the nuclear membrane. It is preferably delivered into the cell by being linked in The substance of interest is preferably fused or bound to a domain or sequence derived from a protein responsible for metastatic activity. Translocation domains and sequences include, for example, domains and sequences derived from HIV-1 transactivating protein (Tat), Drosophila Antennapedia homeodomain protein, and herpes simplex-1 virus VP22 protein. In a highly preferred embodiment, the substance of interest is bound to penetratin protein or a fragment thereof. Penetratin contains the sequence RQKIKIWFQNRRMKWKK and is described in Derossi et al. Biol. Chem. 1994, 269, 10444-50; the use of penetratin-drug conjugates for intracellular delivery is described in International Publication No. WO 00/01417. Truncated or modified penetratin can also be used, as described in International Publication WO 00/2927.

  In the context of the present invention, the carrier used in preparing the composition is preferably a pharmaceutically acceptable carrier. For therapeutic applications, the composition comprises a pharmaceutically acceptable carrier and a non-toxic therapeutically effective amount of a compound that inhibits apoptosis-related proteins (or pharmaceutically acceptable salts of such compounds). Including.

  Furthermore, in this preferred embodiment, the present invention provides a pharmaceutical composition for the treatment of a disease wherein the use thereof results in inhibition of tumor cell, benign or malignant tumor growth, or metastasis, comprising pharmaceutical And a non-toxic therapeutically effective amount of a compound that inhibits apoptosis-related proteins. Methods for preparing pharmaceutical compositions for therapeutic application of compounds that inhibit apoptosis-related proteins are well known in the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. , 18th edition (1990).

  The pharmaceutical composition of the present invention may comprise an inhibitor compound which is an active ingredient, a pharmaceutically acceptable carrier, and optionally other medicinal ingredients or adjuvants. Other therapeutic agents can include cytotoxins, chemotherapeutic agents, anticancer agents, or agents that promote the effects of such agents. This composition includes compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous administration), but is most appropriate in any case The route will depend on the particular host and the nature and severity of the condition for which the active ingredient is administered. The pharmaceutical composition is conveniently provided in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art.

  Indeed, the inhibitor compounds of the invention can be synthesized as an active ingredient into a complete mixture with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, eg, oral or parenteral (including intravenous). Accordingly, the pharmaceutical compositions of the invention can be provided as discrete units suitable for oral administration such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient. Further, the composition may be provided as a powder drug, a granule, a solution, an aqueous liquid suspension, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. In addition to the general dosage forms described above, inhibitor compound compositions can also be administered by controlled release means and / or delivery devices. The composition can be prepared by any of the pharmaceutical methods. Such methods typically include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. Ordinarily, the composition is prepared by uniformly and intimately mixing the active ingredient with a liquid carrier, a finely divided solid carrier, or both. The product can then conveniently be formed into the desired presentation.

  The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, gypsum, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gas carriers include carbon dioxide and nitrogen.

  Any convenient pharmaceutical vehicle may be utilized in preparing a composition for oral administration. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, colorants, and the like can be used to form oral liquid formulations such as suspensions, elixirs, solutions; Carriers such as sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrants can be used to form oral solid preparations such as powders, capsules, and tablets. Tablets and capsules are the preferred oral dosage units because of their ease of administration, and solid pharmaceutical carriers are employed for them. Optionally, the tablets can be coated by standard aqueous or non-aqueous techniques.

  A tablet containing the composition of this invention may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets consist of a free-flowing form of the active ingredient, such as powders or granules, optionally mixed with a binder, lubricant, inert diluent, surfactant, or dispersing agent in a suitable machine. It can be prepared by compression. A ground tablet may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Each tablet preferably contains from about 0.05 mg to about 5 g of the active ingredient, and each cachet or capsule preferably contains from about 0.05 mg to about 5 g of the active ingredient.

  For example, formulations intended for oral administration to humans are synthesized from about 0.5 mg to about 5 g synthesized with a suitable and convenient amount of carrier material that can vary from about 5 to about 95 percent of the total composition. Of active agents. Dosage unit forms will usually contain between about 1 mg to about 2 g of the active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg. Let's go.

  The pharmaceutical compositions of the invention suitable for parenteral administration can be prepared as aqueous solutions or suspensions of the active compounds. For example, a suitable surfactant such as hydroxypropylcellulose may be included. Dispersions can also be prepared with glycerol, liquid polyethylene glycols and mixtures thereof in oil. In addition, preservatives can be included to prevent harmful growth of microorganisms.

  Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. In addition, the compositions can take the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid to facilitate syringe operation. The pharmaceutical composition must be stable under the conditions of manufacture and storage. Therefore, it should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

  The pharmaceutical composition of the present invention can take a form suitable for topical administration, such as aerosol, cream, ointment, lotion, and garment. Further, the composition can take a form suitable for use in a transdermal device. These formulations can be prepared by conventional processing methods using the inhibitor compounds of the present invention. As an example, a cream or ointment is prepared by mixing a hydrophilic substance and water with about 5 wt% to about 10 wt% of a compound to produce a cream or ointment having the desired hardness.

  The pharmaceutical composition of the invention may take a form suitable for rectal administration wherein the carrier is a solid. Desirably, the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories are conveniently formed by first mixing the composition with a softened or melted carrier and then cooling and molding in a mold.

  In addition to the carrier components described above, the above-described formulations optionally contain diluents, buffers, flavors, binders, surfactants, thickeners, lubricants, and preservatives (including antioxidants). One or more additional carrier components such as In addition, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing inhibitor compounds can also be prepared in powder or concentrated liquid form.

  The dosage level of the compound in the combination of the invention will be approximately as described herein. However, specific dosage levels for any particular patient include age, weight, general health, sex, diet, time of administration, route of administration, elimination rate, combination drug, and severity of the particular disease being treated It is understood that it will depend on various factors.

  To determine whether a patient has a type of neoplasm that is likely to respond to treatment with an inhibitor of an apoptosis-related protein encoded by a gene selected from Table 1B, the neoplasm obtained from the patient Tissue samples are exposed to such inhibitors (eg, compounds identified in the screening methods of the present invention; small molecule inhibitory dsRNA, eg, those described in the methods section), tested, and neoplastic tissue samples Is responsive to treatment with an inhibitor.

  For example, in a patient with a solid tumor, a suspected tumor tissue sample is obtained, processed and cultured in the presence and absence of an inhibitor with appropriate tissue culture medium and conditions, and the tumor tissue sample is displayed. It is determined whether apoptosis-related proteins encoded by genes selected from 1B are responsive to treatment with such inhibitors. Responsiveness to inhibitors can be characterized by growth inhibition or increased apoptosis of tumor cells treated with inhibitors compared to untreated tissue samples.

  In one embodiment, the diagnostic methods of the invention determine whether a neoplastic tissue sample is responsive to treatment with an inhibitor of an apoptosis-related protein encoded by a gene selected from Table 1B. A neoplastic tissue sample is exposed to such an inhibitor and determined by determining whether such treatment reduces in vitro growth of tumor cells. Briefly, a suspected neoplastic tissue sample is removed from a patient and grown in vitro as an explant. This tissue sample is grown in the presence and absence of inhibitors of apoptosis-related proteins encoded by genes selected from Table 1B. After growth in culture, cells can be fixed by adding chilled trichloroacetic acid, and protein levels are determined according to Skehan, P. et al. Et al., New Colorimetric Assay For Anticancer-Drug Screening, J. Org. Natl. Cancer Inst. 82, 1107-1112, 1990, as described previously, using a sulforhodamine B (SRB) colorimetric protein staining assay. Alternatively, many other methods are available for measuring growth inhibition and can be used in place of the SRB assay. These methods include counting viable cells after trypan staining, labeling cells capable of DNA synthesis with BrdU, or radioisotope-labeled thymidine, neutral red staining of viable cells, or MTT or WST- A one cell viability assay is included. Cell growth inhibition indicates that the tumor in question is responsive to inhibitors of apoptosis-related proteins encoded by genes selected from Table 1B. Cell growth inhibition indicates that the patient will be a good candidate for treatment with such inhibitors.

  In another embodiment of the diagnostic method of the present invention, the responsiveness of neoplastic tissue to treatment with inhibitors of apoptosis-related proteins encoded by genes selected from Table 1B is tested in an apoptosis assay. For example, a suspected neoplastic tissue sample is processed and exposed to such inhibitors. Responsiveness to the inhibitor is characterized by increased apoptosis of neoplastic tissue samples treated with the inhibitor compared to untreated tissue samples. Inappropriate regulation of apoptosis is thought to play an important role in many pathological conditions, including cancer, AIDS, Alzheimer's disease, and the like. Patients with neoplasms that exhibit increased cell death via apoptosis after treatment with inhibitors of apoptosis-related proteins encoded by genes selected from Table 1B are candidates for treatment with such inhibitors. It is.

  In one type of apoptosis assay, suspected neoplastic cells are removed from the patient. The cells are then cultured in the presence or absence of an inhibitor of apoptosis-related protein encoded by a gene selected from Table 1B. Apoptotic cells are measured by combining the adherent compartment of the culture with the “floating” compartment. Appropriate and detailed protocols for assessing apoptosis in tumor cell cultures treated with pro-apoptotic compounds have been described in the literature (eg, Piazza, GA, et al., Cancer Research, Vol. 55, 3110-16). Page, 1995). Alternatively, other apoptosis assays described herein or in the references cited herein, such as morphological assays or DNA fragmentation assays may be used.

  As an alternative to measuring cell proliferation inhibition or increased apoptosis in response to exposure to inhibitor compounds in the above methods of the invention, promotes apoptosis or inhibits cell proliferation and thereby inhibits tumor growth. Various surrogate markers that may indicate the efficacy of the compound can be evaluated. For example, if the apoptosis-related protein is a protein kinase, autophosphorylation or inhibition of phosphorylation of cellular substrate proteins can be measured, which would predict an effect on apoptosis or cell proliferation. For apoptosis-related GPCR proteins, measurement of inhibition of downstream signaling pathways would predict effects on apoptosis or cell proliferation.

  For purposes of illustration only, the invention will be further described in the following examples.

Example 1 Neutrophil Model of Apoptosis A model system for identifying early regulatory genes in apoptosis of human primary neutrophils is described in International Publication WO 02/04657. International Publication WO 02/04657 describes the isolation of human primary neutrophils, the dose-responsive effect of GM-CSF on neutrophil survival, and the effect of gliotoxin, a fungal metabolite on GM-CSF. Therefore, from this model, parameters of neutrophil apoptosis are established, which makes it possible to identify apoptosis-related genes.

  Furthermore, neutrophil apoptosis can be measured by DNA fragmentation as follows.

Neutrophils are isolated from blood and resuspended at a concentration of 2 × 10 ⁶ / ml as described in International Publication WO 02/04657. Pipette 500 ml into a well of a 24-well plate and incubate in the presence or absence of survival factor.

  Following this incubation, the neutrophils are carefully resuspended by gentle agitation and the entire contents of the wells are placed in an Eppendorf tube and centrifuged in a minifuge at 2800 rpm for 2 minutes at room temperature. The cell pellet is carefully resuspended in 300 μl ice cold hypotonic fluorochrome solution (50 μg / ml propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100) and placed in a 4 ° C. refrigerator for 24 hours. Put the time.

  Prepared samples are analyzed on a FacsCalibre flow cytometer (Becton Dickinson) using double discrimination with Log Forward and Side Scatter parameter acquisition to ensure a single cell suspension.

  As shown in FIG. 1, normal cells (neutrophils cultured overnight in the presence of GM-CSF) give rise to a single G1 phase peak (since neutrophils have a cell cycle arrest) Only a few cells are present at the Sub-G1 phase peak. In contrast, neutrophils cultured in the absence of GM-CSF have enhanced apoptosis, which has a decreased number of cells in the G1 phase peak and an increased number of Sub-G1 phase peaks ( Suggesting DNA fragmentation).

  Furthermore, the present inventors confirmed that the PI3 kinase / AKT pathway plays a role in GM-CSF mediated survival signals as follows.

Neutrophils are seeded at 2 × 10 ⁶ / ml in complete medium (1 ml / well) in a 24-well plate. PI3K inhibitor (Ly294002) is added to neutrophils at a final concentration of 20 μM, followed by GM-CSF (50 units / ml). In control wells, no GM-CSF is added or F GM-CSF (50 units / ml) is added in the absence of inhibitor. Neutrophils are cultured at 37 ° C. for 18 hours and then harvested and prepared for Sub-G1 phase analysis as previously described.

  Inhibition of PI3 kinase by Ly294002 is shown in FIG. As is apparent from the figure, neutrophils cultured for 18 hours in the absence of GM-CSF resulted in apoptosis as measured by Sub-G1 phase analysis compared to those cultured in the presence of GM-CSF. The number of cells increases. In contrast, GM-CSF mediates survival through the PI3K pathway because neutrophils are cultured in the presence of both GM-CSF and PI3K inhibitors, since survival is abolished. To suggest.

  The inventors also demonstrate the role of AKT in GM-CSF mediated survival as follows.

Neutrophils are seeded at 2 × 10 ⁶ / ml in complete medium (1 ml / well) in a 24-well plate. AKT inhibitor ([1L-6-hydroxymethyl-cairo-inositol 2-O-methyl-3-O-octadecyl carbonate] (Calbiochem)) was added to neutrophils at a final concentration of 20 μM and incubated for 2 hours. GM-CSF (50 units / ml) is then added. In control wells, no GM-CSF is added or GM-CSF (50 units / ml) is added in the absence of inhibitor. Neutrophils are cultured for 18 hours at 37 ° C. as previously described, then harvested and prepared for Sub-G1 phase analysis.

  Inhibition of AKT by [1L-6-hydroxymethyl-cairo-inositol 2-O-methyl-3-O-octadecyl carbonate] (Calbiochem) is shown in FIG. As is apparent from the figure, neutrophils cultured for 18 hours in the absence of GM-CSF resulted in apoptosis as measured by Sub-G1 phase analysis compared to those cultured in the presence of GM-CSF. The number of cells increases. In contrast, GM-CSF mediates survival through the PI3K pathway because neutrophils are cultured in the presence of both GM-CSF and AKT inhibitors, since survival is abolished. Is suggested.

Example 2: Gene expression analysis in a neutrophil model of apoptosis was performed using commercially available microarrays to identify regulators of apoptosis, global gene expression associated with neutrophil apoptosis, GM-CSF inhibition of neutrophil apoptosis, And to measure the inhibition of this effect by the use of the fungal metabolite gliotoxin. Control experiments use methylgliotoxin, an inactive analog of gliotoxin. Analysis of such microarray results identifies genes whose expression pattern is altered (up-regulated or down-regulated) in association with a measurable apoptotic phenotype.

  This model exploration assay is configured to target “early” regulatory events that occur in apoptosis and in particular in the inhibition of apoptosis by GM-CSF. When apoptosis by GM-CSF is itself inhibited by drugs such as gliotoxin, these changes or by clustering changes that are common and both increase and / or decrease in response to treatment. The pattern of change can be targeted. For example, a change that “decreases” after induction of apoptosis is a candidate target gene, but in addition, a change that “increases” after inhibition of apoptosis by GM-CSF is more likely to be a target gene. This is because the regulation of this gene is highly correlated with the process. Similarly, further “decreasing” changes after inhibition of the inhibitory effect by GM-CSF are even more likely to be target genes since gene regulation is highly correlated with the process.

  This example describes the use of an Affymetrix Human U95 microarray chipset obtained from Affymetrix (USA). This chip set contains oligonucleotide probes corresponding to about 50,000 human mRNAs.

Isolation and purification of neutrophil RNA for Affymetrix gene expression profiling Primary human neutrophils were isolated and purified from peripheral blood of 15 normal healthy individuals using standard techniques. Each sample was divided into the following treatment groups.
1) T0 untreated control 2) Spontaneous apoptosis T4 (incubate in serum at 37 ° C. for 4 hours)
3) Treatment with GM-CSF (50 U) and gliotoxin (10 μM) (incubate at 37 ° C. for 4 hours)
4) Treat with GM-CSF (50 U) and methylgliotoxin (10 μM) (incubate at 37 ° C. for 4 hours).

  Total RNA was then prepared according to the manufacturer's instructions (RNAzol B; Biogenesis) using two sequential acidic phenol / guanidine isothiocyanate extractions, followed by the RNeasy RNA preparation kit ( Qiagen). Samples were pooled according to treatment.

Using reverse transcription of neutrophil RNA and second strand synthesis Superscript Choice system (Gibco) for Affymetrix profiling, using T7- (dT) oligomer provided by Molecular Diagnostics Laboratory (Aarhus), According to the protocol of the Affymetrix GeneChip expression manual, reverse transcription reaction and second strand synthesis reaction for neutrophil RNA samples were performed.

  PCR with primers designed to target the 5 ', middle and 3' ends of GAPDH cDNA was performed to confirm the quality of the second strand cDNA. This allows comparison of the relative product quantities and gives an indication of the percentage of full-length cDNA. Thereafter, to obtain profiles for Affymetrix Hu95A, B, C, D, and E chips, cDNA was obtained from Molecular Diagnostic Laboratory, Dept. Sent to Clinical Biochemistry, Aarhus University Hospital, Skejby Symehus, DK 8200 Aarhus N Denmark.

Bioinformatics analysis of differential gene expression for the entire treatment group is provided with various measurements by Affymetrix Genechip software analysis that identifies regulators of apoptosis . These are used to calculate the significance of differential gene expression. The following parameters are determined:

Positive : The number of probe pairs determined to be positive. A probe pair is considered positive if the following two criteria are met:

1) Perfect match strength (PM) Minus mismatch strength (MM)> Statistical discrimination threshold (SDT, also called discrimination threshold) ^*
PM-MM> SDT
2) After background subtraction, PM / MM> statistic ratio threshold (SRT, called ratio threshold) ^*
PM / MM> SRT
^* SDT and SRT are set by the user.

Negative : Number of probe pairs determined to be negative. A probe pair is considered negative when the following two criteria are met:

1) MM minus PM> statistical discrimination threshold MM-PM> SDT
2) After background subtraction, MM / PM> statistic ratio threshold MM / PM> SRT
This removes probe pairs from the analysis that have little PM and MM differences. Some probe pairs may not meet the requirements of either set, so in that case, neither Positive nor Negative will be determined.

Pairs : total number of probe pairs for this gene on the probe array.

Pairs Used : This is the number of probe pairs per gene used in the analysis. This may be the total number of probes per gene or per chip, or a preselected subset of probe pairs that can be specified in a probe mask file.

Pairs in average : This is the number of probe pairs used to calculate Log Avg and Avg Diff. There are three modes for measuring Pairs in average.

All-inclusive : All probe pairs for a gene are used in the calculation. This is the default if the number of probe pairs for the gene is less than 8.

Olympic scoring : Olympic scoring excludes probe pairs that have maximum and minimum intensity differences (PM-MM). This method is used when the number of probe pairs is in the range from 9 to the probe threshold number (#) (NPT ^* , default = 15).

Super Olympic scoring : Only data with a standard deviation of the mean intensity difference (STP ^* ) calculated by excluding both maximum and minimum values within 3 is used. Exclusion of maximum and minimum values is enforced. That is, they are excluded even if they are within the specified range. This method is used when the number of probe pairs for the gene is greater than the probe threshold number (#).

Positive fraction : This is the ratio of the number of probe pairs determined to be positive divided by the number of probe pairs used. Pos Fraction is a good indicator of whether a transcript is present. If the majority of the probe pair is positive, the probe utilization is good and probably the transcript is present.

Log Avg (Log Average Ratio) : The intensity ratio (PM / MM) of each probe pair used is calculated by dividing the exact match intensity by the mismatch intensity. Calculate the average intensity ratio from Pairs in Avg for each gene and multiply this by 10.

Log Avg = 10 × [S log (PM / MM)] / (# Probe Pairs In Avg)
Note: Log Avg = 0 indicates random cross-hybridization. The higher the Log Avg, the more certain the gene transcript is.

PM Excess (Perfect match excess) : The number of probe pairs whose intensity ratio exceeds a given value (ratio limit value).

MM Excess (Mismatch excess) : The number of probe pairs whose reciprocal intensity ratio exceeds a given value (ratio limit value).

  The numbers shown in PMexcess and MMexcess indicate how much of the PM / MM ratio or MM / PM ratio exceeded a specific value. For example, when it is 5, it means that 5 of all PM / MM or MM / PM (ratio) exceeded 10 (default value).

Pos / Neg (Positive / Negative) : This is the ratio of a probe pair determined to be positive and a probe pair determined to be negative.

Avg Diff (Average Difference) : The intensity difference (PM / MM) of each probe pair used is calculated by subtracting the intensity for mismatch from the intensity for perfect match. The average intensity difference is calculated for each gene from the probe Pairs in Avg.

S (PM-MM) / (# Probe Pairs In Avg)
Abs Call (Absolute Call) : There are three possible outcomes for Abs Call: “P” (present), “A” (absent), and “M” (boundary). This determination is based on the values of Pos Fraction, Log Avg, and Pos / Neg.

Inc (Increase) : For each gene, the number of probe pairs for which PM minus MM in the experimental data is significantly greater than the value in the baseline data. To be considered significant, the following two conditions must be met:

1) (PM-MM) experimental value-(PM-MM) baseline> change threshold (CT) ^*
2) [(PM-MM) experimental value− (PM-MM) baseline] / (PM-MM) baseline> percent change threshold (PCT) ^* / 100
Dec (Decrease) : For each gene, the number of probe pairs for which PM minus MM in the baseline data is significantly greater than the value in the experimental data. To be considered significant, the following two conditions must be met:

1) (PM-MM) Baseline-(PM-MM) Experimental value> CT
2) [(PM-MM) Baseline-(PM-MM) Experimental Value] / (PM-MM) Baseline> PCT / 100
Pos Fraction (Positive fraction) : This is the ratio of the number of probe pairs used divided by the number of probe pairs determined to be Positive. Pos Fraction is a good indicator of whether a transcript is present. If the majority of the probe pair is positive, the probe utilization is good and probably the transcript is present.

Log Avg (Log Average Ratio) : The intensity ratio (PM / MM) for each probe pair used is calculated by dividing the exact match intensity by the mismatch intensity. Calculate the average intensity ratio for each gene from Pairs in Avg and multiply this by 10.
Log Avg = 10 × [S log (PM / MM)] / (# Probe Pairs In Avg)
Note: Log Avg = 0 indicates random cross-hybridization. The higher the Log Avg, the more confident that the gene transcript is present.

Avg Diff (Average Difference) : The intensity difference (PM / MM) for each probe pair used is calculated by subtracting the intensity for mismatch from the intensity of perfect match. The average intensity difference for each gene is calculated from the probe Pairs in Avg.
S (PM-MM) / (# Probe Pairs In Avg).

Avg Diff Change (Average Difference Change) : The difference between the experimental mean difference and the baseline mean difference (Avg Diff).
Avg Diff (experimental value)-Avg Diff (baseline)
Inc Ratio (Increase Ratio) : The ratio of the number of probe pairs identified as increased (Inc) to the number of probe pairs used in the analysis for each gene.

Dec Ratio (Decrease Ratio) : The ratio of the number of probe pairs identified as reduced (Dec) to the number of probe pairs used in the analysis for each gene.

Max Inc & Dec Ratio (Maximum Increment Ratio and Decrease Ratio) : The larger of Inc Ratio or Dec Ratio.

Pos Change (Positive Change) : The difference between the number of probe pairs that were determined to be positive in each of the experimental and baseline files for each gene.

Neg Change (Negative Change) : The difference between the number of probe pairs that were determined to be negative in each of the experimental and baseline files for each gene.

Inc / Dec (Increase / Decrease) : Ratio of increased number of probe pairs to reduced number of probe pairs, see Inc and Dec.

Diff Call (Difference Call) : There are five possible results for the Difference Call. The expression level of RNA is increased (I), decreased (D), slightly increased (MI), slightly decreased (MD), or no detectable change in expression level (NC). The difference between all determinations is based on the values of Inc / Dec, Inc Ratio (or Dec Ratio), Dpos-Dneg Ratio, and Log Avg Ratio Change.

Fold Change : Fold Change is the ratio of the average difference (Avg Diff) between the experimental file and the baseline file. This number reflects the magnitude of change, not the direction of change (see Diff Call). For example, if the Avg Diff experimental value is 3000 and the Avg Diff baseline value is 300, the Fold Change is 10. The Fold Change is 10 when the Avg Diff experimental value is 300 and the Avg Diff baseline value is 3000. If either the Avg Diff experimental value or the Avg Diff baseline value is <20, this value is set to 20 for convenience of calculation.

Sort Score : Sort Score is based on both Fold Change and Avg Diff Change. This score can be used to assess differences in gene expression between the experimental file and the baseline file. The higher the Sort Score value, the more “significant” the measurement difference in gene expression between the experimental file and the baseline file, ie, the higher the reliability. For example, when Fold Change is 5, the significance value when the Avg Diff Change value changes from 200 to 1000 is larger than the significance value when the Avg Diff Change value changes from 20 to 100.

We identified the regulators of apoptosis using the following differential gene expression patterns across treatment groups according to the principles of the model screening method already outlined in this specification and in International Publication WO 02/04657. Went.
1. 1. Down-regulation (T4: T0) in the untreated group (T = 4 hours) compared to the untreated group (T = 0 hour), and 2. down-regulation (G: T0) in the GM-CSF + gliotoxin group (T = 4 hours) compared to the untreated group (T = 0 hours); 3. Up-regulation or no change (M: T4) in the GM-CSF + methylgliotoxin group (T = 4 hours) compared to the untreated group (T = 4 hours), and Up-regulation or no variation (M: G) in the GM-CSF + methyl gliotoxin group (T = 4 hours) compared to the GM-CSF + gliotoxin group (T = 4 hours)
Or 1. 1. Up-regulation (T4: T0) in the untreated group (T = 4 hours) compared to the untreated group (T = 0 hour), and Up-regulation (G: T0) in the GM-CSF + gliotoxin group (T = 4 hours) compared to the untreated group (T = 0 hour); and
3. Down-regulation or no variation (M: T4) in the GM-CSF + methylgliotoxin group (T = 4 hours) compared to the untreated group (T = 4 hours), and
4). Down-regulation or no variation (M: G) in the GM-CSF + methyl gliotoxin group (T = 4 hours) compared to the GM-CSF + gliotoxin group (T = 4 hours).

  We used these parameters to identify a number of differentially regulated genes, including those shown in Tables 1A and 1B.

  Table 2 shows the fold change in gene expression for the entire treatment group compared to untreated neutrophils following the treatment pattern listed above.

  Specific genes that are differentially regulated are further characterized using bioinformatics gene function analysis using the Gene Ontology database (www.geneonology.org). Such genes identified in Tables 1A and 1B have been found to have homology with kinases and G protein coupled receptors.

Confirmation of array data using QPCR Quantitative PCR (QPCR) provides highly accurate detection among gene expression profiling techniques. Furthermore, real-time analysis can complement microarray analysis by providing high sensitivity and accurate quantification. In addition, since QPCR requires gene-specific primers, the specificity of the target gene is increased. In this example, a number of genes identified by microarray analysis as being regulated in apoptosis are further profiled using QPCR for various treatment groups and compared to untreated neutrophils.

  Neutrophil RNA was isolated and purified from the various treatments listed above. Similarly, reverse transcription and second strand synthesis were performed as described above. Quantitative PCR was performed under the conditions detailed in Example 3.

  The results of QPCR analysis for a number of target genes are shown in FIG. For the seven genes that were compared, there was a good correlation between the microarray analysis results and the QPCR results across the various treatment groups.

  Therefore, this result confirms the regulation of the target gene by measurement with a microarray.

Description for several apoptosis modulators Table 1A shows differentially regulated genes encoding exemplary kinases and GPCRs that have been characterized in detail for apoptosis regulators. They have become therapeutic targets for cancer and inflammatory diseases. These include known genes whose function has already been described, such as:

1) Protein kinase C zeta (PKCζ)
Protein kinase C is a serine threonine kinase that plays a central role in signal transduction pathways involved in cell regulation, cancer, and apoptosis. The 11 isoforms that have been identified are structurally related but exhibit different cofactor requirements. They constitute three families: (i) classical isoforms (cPKC-α, βI, βII, and γ) are activated by DAG and are Ca2 + -dependent; (ii) novel isoforms Forms (n PKC-δ, ε, η, θ, and μ) are activated by DAG but are Ca2 + independent; (iii) atypical isoforms (PKC-ζ and ι / λ) Is DAG non-responsive and Ca 2+ independent. These isoforms migrate from the cytosol and membrane fraction upon activation.

  Protein kinases are implicated in several signaling pathways that regulate mammalian cell differentiation, malignant transformation, and apoptosis. Two of the main pathways directly related to PKCζ are the sphingomyelinase / ceramide pathway and the PI3 / AKT pathway, but signal transduction via either of these is not mutually exclusive ( Calcerrada et al., FEBS Lett, 2002 13, 514 (2-3), pages 361-5.

  The fact that PKCζ can be activated by phosphatidylinositol 3,4,5-triphosphate (Nakanishi et al., J Biol Chem, 1993, 268 (1), 13-6) indicates that it is PI3K / Suggests that it functions in the AKT survival pathway. Full activation of the AKT enzyme requires phosphorylation of threonine and serine amino acids. Threonine phosphorylation is due to PDK1, but little is known about the mechanism leading to serine phosphorylation by PDK2. Recent evidence suggests that PDKζ binds to PDK2 and AKT, thereby functioning as an adapter, thereby promoting serine phosphorylation of AKT and leading to its full activation (Hodgkinson CP et al., 2002). Biochemistry, Vol. 412 (32), pages 10351-9.

  In addition, mouse erythroleukemia cells (friends), in response to high dose ionizing radiation, cause activation and nuclear translocation of the phosphatidylinositol-3-kinase (PI-3-kinase) p85α subunit, resulting in: It leads to downstream activation of PKCζ and nuclear translocation. Treatment with wortmannin, a relatively specific inhibitor of PI-3-kinase, subsequently detects both an increase in the number of apoptotic cells and inhibition of protein kinase C zeta translocation. This result suggests that the PI-3-kinase / PKC zeta pathway plays a potential role in protecting Friend cells from ionizing radiation-induced apoptosis, so that PKC zeta is (Cataldi et al., Int J Oncol, January 2003, 22 (1), 129-35).

  The product of the par-4 gene has its expression correlated with apoptosis and specifically interacts with the regulatory domain of PKCζ to inhibit its activity. Overexpression of par-4 induces apoptosis in NIH-3T3 cells, but this apoptosis can be abrogated if PKCζ (but not a kinase-inactive mutant) is overexpressed, Suggests that can control survival (Ddiaz-Meco et al., Cell, 1996, 86 (5), 777-86). Another example of how PKCζ protects cells has been demonstrated by de Thonel et al. (Blood, 2001, 98 (13), 3770-7), which shows that Fas in acute myeloid leukemia (AML) It was shown that the mechanism of resistance is due to PKCζ expression. Chemical inhibition or overexpression of par-4 restored Fas-induced apoptosis.

  Similarly, overexpression of dominant negative PKCζ without kinase activity in the U937 human leukemia cell line resulted in a decrease in BCL2 expression, accompanied by an increase in Bax expression. Furthermore, inhibition of PKCζ accelerated the development of apoptosis in leukemic cells exposed to etoposide and TNFα, suggesting that this inhibition increased the sensitivity of the cells to these reagents. Inhibition of PKCζ also enhanced the sensitivity of tumor cells grown in nude mice to etoposide. As a result, the authors conclude that inhibition of PKCζ is an attractive target for chemical sensitization of tumor cells (Filomenko et al., Cancer Res, 62 (6), 1815-21. ).

  One consequence of PKCζ activation is NFκB activation by indirectly inducing phosphorylation of IκK, which allows NFκB to translocate to the nucleus and transcribe anti-apoptotic genes. (Diaz-Meco et al., EMBO J, 1994, 13 (12), 2842-8). Mice in which PKCζ was specifically inactivated by homologous recombination appeared normal in appearance, but were found to have phenotypic changes in their secondary lymphoid organs. Furthermore, this mouse is defective in the activation of the NFκB pathway, and in particular in the lung, IκK activation was inhibited (Leitges et al., Mol Cell, October 2001, Vol. 8 (4), 771- 80).

  Protein kinase Cζ has also been implicated in the production of reactive oxidants in endothelial cells stimulated with TNFα. Treatment of endothelial cells with TNFα resulted in phosphorylation of p47 and gp91 (NADPH oxidase subunit), events necessary for oxidant production. Inhibition of PKCζ blocked TNFα-induced phosphorylation of p47 (phox) and translocation to the membrane, followed by inhibition of oxidant production (Frey et al., Circ Res, May 17, 2002, 90 ( 9) Volume, pages 1012-9).

  Contrary to the anti-apoptotic example described above, PKCζ has also been suggested to be pro-apoptotic in neutrophils treated with TNFα. Exposure to TNFα enhanced PKCζ expression in a dose-dependent manner, which correlated with death (Das et al., Mol Cell Biochem, 1999, 197 (1-2), 97-108. page).

2) 3 ′ phosphoinositide-dependent kinase 1 (PDK1)
PDK1 plays a central role in the activation of AGC protein kinases Akt, S6K, RSK, MSK, and SGK. Specifically, PDK1 plays an important role in the regulation of cell survival by its ability to phosphorylate and activate Akt. For full activation, Akt requires phosphorylation of threonine-308 and serine-473 residues, but for partial activation, phosphorylation of threonine alone is sufficient. Following activation, Akt survival is mediated by a number of events including phosphorylation and inhibition of Bad, Caspase 9, and Forkhead transcription factors.

  Overexpression studies have demonstrated that PDK1 plays a role in mammary tumorigenesis. COMMA-1D mouse mammary epithelial cells transduced with PDK1 using retrovirus are highly transformed (measured by anchorage-independent growth in soft agar) with activation of Akt and protein kinase C showed that. The authors conclude that PDK1 activation can lead to mammary tumorigenesis, suggesting that PDK1 expression may be an important target in human breast cancer (Zeng et al., Cancer Res, 2002, 62nd). (12) Volume, pages 3538-43).

  Consistent with this hypothesis, Arico et al. (J Biol Chem, 2002, 277 (31), 27613-21) show that the antitumor effect of celecoxib, a COX-2 specific inhibitor, is due to inhibition of PDK1. It was demonstrated that it can be mediated. Celecoxib has previously been shown to reduce the number of adenomatous colorectal polyps. Celecoxib induced apoptosis of the colon cancer cell line HT-29 by inhibiting PDK-1, resulting in inhibition of Akt phosphorylation. Expression of Akt in a constitutively active form had a low protective effect against celecoxib-induced cell death, whereas expression of a constitutively active mutant of PDK1 strongly inhibited apoptosis. This confirms that the antitumor activity of celecoxib is due to PDK-1 inhibition.

  Similarly, UCN-01 (7-hydroxystaurosporine) is a drug currently in clinical trials and has a unique fingerprint pattern, which induces Akt dephosphorylation and inactivation, which Leads to loss of survival signal and induction of apoptosis. By further analysis, UCN01-mediated Akt inactivation resulted in PDK1 functioning as an upstream Akt kinase in vitro and not by inhibition of Akt itself or phosphatidylinostide 3-OH kinase Showed that it was caused by inhibition (IC50 = 33 nM). UCN-01-induced PDK1 inhibition was also observed in vivo in mouse and human tumor xenografts. Overexpression of active Akt attenuated UCN-01's cytotoxic effect, which is due to UCN-01 exerting its cytotoxicity in part by inhibiting the PDK1-Akt survival pathway Suggests that. Since UCN-01 has already been demonstrated to have potent antitumor activity in vivo, the PDK1-Akt survival pathway is a novel and attractive target for cancer chemotherapy (Sato et al., Oncogene, 2002). March 7, Volume 21 (11), pages 1727-38).

  Yet another example demonstrating the pivotal role played by PDK1 in cancer is Ballif et al. (J Biol Chem, 2001, 276 (15), 12466-75), which shows anti-tumorigenic and anti-tumor properties. The proliferative drug n-α-tosyl-1-phenylalanyl-1-chloromethyl ketone (TPCK) was demonstrated to block phosphorylation of the PDK1 regulatory site of S6K1 and Akt. This, in turn, resulted in a reduction in the phosphorylation state of BAD, a proapoptotic protein, resulting in cell death of IL3-dependent 32D cells.

  In a knockout study, Lawlor et al. (EMBO J, 2002, 21 (14), 3728-38), PDK1, a mouse with a lower form allele of PDK1, which expresses only 10% of the normal level of PDK1, Mice deficient in were produced. PDK1 (− / −) embryos show multiple malformations and die at embryonic age 9.5 days. In contrast, low-order PDK1 mice are 40-50% smaller than control animals, but are viable. Similarly, organs and cell volumes are decreasing proportionally. The author concludes that PDK1 is essential for mouse embryonic development and regulates cell size.

3) Jak3
STATs constitute a family of DNA binding proteins that are localized in the cytoplasm until activated by tyrosine phosphorylation. STAT phosphorylation is catalyzed by members of the Janus family of tyrosine kinases, including JAK3. However, it has been found that the Jak family has not only activation of gene transcription through the STAT family but also a more general role. In at least one example of cell activation, Jak3 has been shown to associate with the regulatory subunit of PI3K and modulate IL-7-induced P13 kinase activation by mediating tyrosine phosphorylation of the p85 subunit. Yes. Specific inhibition of IL-7-induced Jak kinase activity eliminates p85 tyrosine phosphorylation, followed by activation of PI3 kinase, and ultimately proliferation (Sharfe et al., Blood, 1995, 86 (6 ), 2077-85).

  The importance of Jak3 in apoptosis and survival is recognized in inhibitor studies. WHI-P131, a specific inhibitor of Jak3, causes clonogenic growth of JAK3-positive leukemia cell lines DAUDI, RAMOS, LC1; 19, NALM-6, MOLT-3, and HL-60 in a concentration-dependent manner. Inhibits (but does not inhibit JAK3-negative BT-20 breast cancer, M24-MET melanoma, or SQ20B squamous cell carcinoma cell lines). Based on these findings, the authors found that potent and specific inhibitors of JAK3, such as WHI-P131, are the basis for the design of new therapeutic strategies for acute lymphoblastic leukemia, the most common form of childhood cancer. (Sudbeck et al., Clin Cancer Res, 1999, 5 (6), 1569-820). Furthermore, inhibitors of Jak3, WHI-P131 and WHI-p159, are potent inhibitors of glioblastoma cell adhesion and migration (Narla et al., Clin Cancer Res 4 (10) 2463-71).

  Jak3-deficient mice showed a significant reduction in the number of lymphoid cells. Thymocytes and peripheral T cells obtained from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides a survival signal. Wen et al. (Mol Cell Biol, 2001, 21 (2), 678-89) showed that regulation of T lymphocyte neogenesis by Jak3 is at least partially mediated by selective regulation of Bax and BCL2. It is reported that it is. Jak3-deficient thymocytes have higher Bax expression levels and lower BCL2 expression levels than wild-type littermates. They show that Jak3 regulates cell survival by selective and cell content-dependent regulation of pro- and anti-apoptotic BCL2 family proteins, and that Bax and BCL2 play distinct roles in T cell development Suggested and concluded.

4) Phosphofructokinase, liver (PFKL)
Phosphofructokinase (PFK; ATP: D-fructose-6-phosphate-1-phosphotransferase) is a tetramer formed by the random association of products at two distinct loci, thereby Five possible tetramers are formed. Muscle and liver PFKs are homotetramers of M and L subunits, respectively. Red blood cells have all five isozymes: M4, M3L, M2L2, ML3, and L4. Liver phosphofructokinase functions in glycolysis and phosphorylates fructose-6-phosphate to fructose-1,6-diphosphate.

  6-phosphofructokinase (PFK) plays a central role in the regulation of glycolysis in both normal and tumor cells. Since PFK also mediates the Pasteur effect, it coordinates two methods of energy production, glycolysis and respiration, in most cellular systems. Energy production in cancer cells is characterized by the predominance of aerobic glycolysis (Warburg effect) and reduced or absent Pasteur effect.

  Staal et al. (Cancer Res, 1987, 47 (19), 5047-51) show that PFKL levels in glioma tissue are increased compared to normal brain tissue and that this is normal. It was demonstrated that it corresponds to a decrease in the level of PFK. Other researchers have reported a significant increase in PFK activity in carcinomas compared to phosphofructokinase in normal thyroid tissue. The specific activity of vesicular adenoma is fairly heterogeneous. When these tumors are divided into three groups according to increased proliferative activity as judged by histopathological criteria, the highest specific activity of phosphofructokinase is found in the group with the highest proliferative activity. It was. The latter group of enzyme activities is similar to that seen in carcinomas. There were all three isozyme M (muscle), L (liver), and P (platelet) types of PFK (van der Heijden et al., Tumor Biol, 1986, 7 (1), 7- 9).

  PFK isozymes from human leukemia, lymphoma, viral transformed cell lines, and malignant cells from established malignant cell lines derived from lymphoid, bone marrow, erythroid, and fibroblasts, and their normal counterparts Further studies of the profile revealed that PFKL levels were increased in all malignant tissues (Vora, Cancer Res, 1985, 45 (7), 2993-30001).

  Thus, among the genes identified using the screening approaches described herein, there are a significant number of well-characterized apoptosis regulators that are recognized as therapeutic targets for cancer and inflammatory diseases. is there. Therefore, the gene identified as an apoptosis regulator for the first time in the present invention is also a therapeutic target candidate.

Example 3: Differential Expression of Identified Gene Targets Compared with Normal and Transformed Tissue Samples, and Their Expression in Cancer Cell Lines This example shows normal tissue for target gene expression levels And describes the screening of transformed tissues. In addition, the expression of these genes was examined across a wide range of cancer cell lines isolated from various tissues. Expression was measured using real-time QPCR.

A) Differential expression in normal and transformed tissues and cancer cell lines
Sources of Normal Tissue RNA and Tumor Tissue RNA For the tissues shown below, a group of tumors and corresponding adjacent normal tissue (NAT) RNA were obtained from Ambion. Each sample was obtained from an unpooled individual human tumor and adjacent normal tissue. Tumor classification is as follows.

Cancer Cell Lines The cancer cell lines shown in Table 3 were obtained from the National Cancer Institute and maintained with RPMI containing 10% FCS. Cells are cultured at 37 ° C. in a humidified atmosphere containing 5% CO ₂ and then harvested by trypsinization when confluent to isolate total cellular RNA for QPCR.

Isolation of total cellular RNA The following cell lines are grown logarithmically. RNA is prepared using the RNeasy kit (Qiagen) according to the manufacturer's instructions. For each cell line, 5 × 10 ⁶ cells are harvested and the cell pellet is lysed in 600 μl RLT buffer containing β-mercaptoethanol. Before adding the sample to the RNeasy column, the DNA is sheared by passing the lysate through a KIAshredder column (Qiagen). DNA was removed by digestion on the column with RNase-free DNase, followed by washing with buffer and elution in RNase-free H ₂ O.

Preparation of cDNA for QPCR analysis RNA (1-2 μg) primed with oligo (dT) primers in a reaction containing Superscript II (Life Sciences), RT buffer, and dNTPs, according to the manufacturer's instructions Reverse transcribe into first strand cDNA. The reaction is incubated at 42 ° C. for 2 hours and stored at −20 ° C. until use.

Performing Quantitative RT PCR The Qiagen QuantiTect SYBR Green PCR kit provides a master mix containing hot start Taq polymerase and an optimized concentration of SYBR Green with a dedicated buffer. The buffer contains a balanced combination of KCl and (NH ₄ ) ₂ SO ₄ so that the ratio of specific binding to non-specific primer binding in the annealing step of each PCR cycle is high. This eliminates the need to optimize the MgCl ₂ concentration. This kit was used according to the manufacturer's instructions.

Opticon real-time PCR instrument (MJ Research) and set experimental parameters are defined by creating a master file for each operation. The master file has the following three components.
1 Plate file 2 Protocol file 3 Option file.

  Plate file: Set up a plate layout that allocates wells as samples, standards, blanks (controls without template), or empty wells.

  Protocol file: Set PCR parameters and plate read steps to allow incorporation of melting curve profiles.

  Option file: Set data processing and quantification options. This includes the following:

  1. Apply standard and zero: normalization of signal variation between wells by applying normalization constants to the data from each well.

  2. Blank subtraction: Data from wells designated as blanks can be subtracted from the sample data.

  3. Subtract baseline signal.

  4). Threshold: Set the C (T) cycle threshold line used for quantification. It can be manually or automatically set to various standard deviations above the mean value for the specified cycle range.

  Option settings can be adjusted during post-operation data analysis.

The relationship between the quantification starting copy number and the number of cycles before detection can be used to calculate the initial amount of template in the sample. First, the threshold or position of the C (T) line must be defined on the graph of fluorescence versus cycle number. Usually this is set to the point where the sample exceeds the background noise and begins to increase. Next, the threshold cycle for an individual sample is defined as the cycle at which the fluorescent trace and the C (T) line intersect. By including a quantification standard, a graph of the standard curve of quantity versus C (T) cycle can be plotted. The initial template amount in the unknown sample can then be calculated by applying the sample threshold cycle to the standard curve.

  To correct for differences in RT reaction efficiency, quantitative PCR is performed using primers against a reference housekeeping gene (eg, GAPDH or RPS13) and the results are expressed as the ratio of target to reference gene template amount.

• Standards Both RNA standards and DNA standards are commonly used in quantitative PCR. In these experiments, a DNA standard consisting of a PCR product cloned into a pMSCV vector (Clonetech) was used. In this way, a large amount of reference material can be generated, which can be quantified spectrophotometrically. The plasmid is linearized because such a form more closely simulates the efficiency of cDNA amplification. Determine the range encompassing the range of test cDNA results, including 10-fold dilutions of standards from 100 pg / ml to 1 fg / ml.

  The QPCR procedure is summarized as follows: The cDNA template is amplified with target-specific QPCR primers using the QuantiTect SYBR Green PCR kit (Qiagen 204143) on the DNA Engine Opticon System (MJ Research). Amplification conditions include a 95 ° C. step for 15 minutes for initial activation of HotStarTaq DNA polymerase, followed by 35 cycles (94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds). The fluorescence of the sample at 521 nm is measured between the annealing and extension steps of the protocol. Melting curves are calculated at the end of 35 cycles to confirm product homogeneity.

  The standard curve uses the logarithm [template amount] of the RPS13 control template dilution (as above) and the fluorescence intensity measured in that well exceeds the level specified by the cycle threshold parameter (C (T) value). Plot against the number of cycles at the time. An estimate of the initial template amount of the treated sample is determined from this plot, and the amount of target mRNA present in each sample is normalized between samples by calculating the ratio of target to RPS13 for each sample. .

PCR primers Primers for the target gene were designed using Genetool software (Biotools), and the sequences are shown in Table 4.

Differential expression of the target between normal and cancerous tissues As measured by QPCR , differential expression of the target gene is observed across a range of corresponding normal and cancerous tissues. Table 5 shows the difference magnification between the corresponding pairs. The results presented show that the identified genes (eg, GPR86, GRAF, EKI, NTKL, CDC42, RBSK, EDG6, PRK / CNK, FLT4, and PSKH1) are differentially expressed compared to adjacent normal tissues. And / or correlate with cancer. This indicates a correlation between the deregulated expression of these genes and the expression of the cancer phenotype.

  These examples demonstrate that the identified apoptosis-related gene has a positive correlation with the cancer phenotype.

Gene targets are expressed in a series of cancer cell lines Table 6 shows the differential expression of candidate genes across a wide range of cancer cell lines obtained from various cancer types. Absolute levels were measured using QPCR and normalized to the housekeeping gene (RPS13).

  Target gene expression is detected in some or all of a wide range of cancer cell lines isolated from various parts of the body. Expression levels vary considerably depending on the cancer cell type.

B) Further expression profiles Expression profiling was extended using another QPCR method for the further range of cancer cell lines specified in Table 7.

Purification of total cellular RNA from cell lines The cell lines shown in Table 7 are grown logarithmically. RNA is prepared using the RNeasy midi kit (Qiagen) approximately as described above. Before adding the sample to the RNeasy column, the DNA is sheared by homogenization using a rotor-stator homogenizer. RNA elutes in a total of 400 μl RNase-free H ₂ O. Using DNA-free DNase (Ambion) according to the manufacturer's instructions, the contaminated genomic DNA is removed by treatment for 15 minutes, and DNase activity is removed by resin-based inactivation. RNA was ethanol precipitated, washed in 70% ethanol, and then suspended in RNA storage buffer (Ambion).

Preparation of cDNA for QPCR analysis RNA (10 μg) primed with random hexanucleotide primers was first strand in a reaction containing reverse transcriptase, RT buffer and dNTPs using cDNA Archive kit (ABI). Reverse transcribe to cDNA. The reaction is incubated at 25 ° C. for 10 minutes, 37 ° C. for 2 hours and then stored at −70 ° C. until use.

Quantitative PCR
Quantitative PCR (QPCR) profiling of gene expression is performed using SYBR Green PCR2x Master Mix Kit (Abgene) according to the manufacturer's instructions. PCR primers are designed using either Primer Express software (ABI) or web-based Gene Tools software (Stratagene). All primers were designed to produce a 90-120 bp amplicon at an annealing temperature of 60 ° C., and to identify the concentration at which target sequence amplification was optimal and specific, in a standard QPCR reaction They are tested at 50 nM, 100 nM, and 200 nM. Table 8 shows the primer sequences and the determined optimum concentrations.

Amplification is performed according to the manufacturer's instructions using the ABI7000 sequence detection system. Each PCR included appropriate primers and 1 × SYBR Green master mix in a total volume of 25 μl along with cDNA obtained from 50 ng RNA. Gene expression levels are expressed relative to the GAPDH housekeeping gene using a comparative Ct (cycle threshold) method according to standard methods. Briefly, for each cell line sample, the Ct value obtained from the GAPDH QPCR is subtracted from the Ct of the target gene to obtain a value called ΔCt. An internal control in each plate normalizes each ΔCt to eliminate variations between QPCR experiments, thereby calculating a value called ΔΔCt. The expression of the target gene in each sample compared to GAPDH is calculated using the equation relative expression = 2− ^ΔΔCt .

Representative data expression profiling data showing target gene expression in cancer cell lines is shown in FIG. 5 as a “heat map”. In the heat map, the expression level of each target gene is represented by different shadows. The expression of each gene is described relative to the expression of the GAPDH housekeeping gene.

Data Summary This expression profiling data shows that the 12 identified target genes are expressed across a range of cell lines representative of various types of human tumors. BAI2 is expressed in the log 3 range compared to GAPDH in all cell lines tested and does not show a significant incidence in certain cancer types. This is also true for the expression of DAGK-θ and EKI. The expression of GPR12 mRNA can be detected in 2/3 of the cell line panel, and the expression level shows a high value range of 5 logarithm. It is noteworthy that GPR12 expression is present primarily in cell lines representative of breast cancer, colorectal cancer, lung cancer, pancreatic cancer, and prostate cancer. This is very different from melanoma and ovarian cell lines, where GPR12 expression is low or undetectable. Of the cell lines that express GPR12, 3 breast cell lines, 1 lung cell line, and 2 pancreatic cell lines express GPR12 mRNA higher than the mean level, ie, this gene is overexpressed. It is thought that it is expressed. The GPR86 gene exhibits a wide range of expression with a log 5 range compared to GAPDH throughout its series of cancer cell lines and is not particularly prevalent in any cancer cell type. The GRAF gene is expressed in all tested cell lines and shows a narrow distribution of expression within the log 3 range. Although GRAF is expressed at relatively high levels throughout a series of cell lines, it is noteworthy that GRAF expression is particularly prevalent in cell lines representative of colorectal and pancreatic cell lines. ITPKC expression is detectable in all cell lines tested, but expression levels are distributed in a log 4 range. ITPKC expression is particularly high in cell lines obtained from breast and prostate cancer. NTKL expression profiling demonstrates that this gene is expressed at relatively high levels throughout this series of cancer cell lines and exhibits a particularly narrow distribution spanning only a log 2 range. RBSK is expressed in the log 4 range across this series of cell lines. It is noteworthy that the majority of breast, colorectal, and prostate cell lines express relatively high levels of RBSK compared to the average expression across this cell line group. The ROCK1 and ULK1 genes are expressed throughout a range of cell lines and do not show a significant prevalence in any particular cancer type. UKH is expressed at relatively high levels across a log 3 range across a range of cancer cell lines. In particular, UKH is expressed at very high levels in many cell lines representative of breast cancer, lung cancer, and ovarian cancer.

Example 4: Overexpression of identified genes functionally modulates apoptosis The erythroleukemia TF1 cell line is growth factor dependent and requires GM-CSF for survival in culture. By eliminating GM-CSF, TF1 cells spontaneously undergo apoptosis. The effects of candidate regulators of the apoptotic process, such as EDG6 and TLK2, can be determined in these cells by introducing candidate genes into these cells followed by measuring the extent of apoptosis.
Analysis of TF1 Apoptosis by Light Scattering Analysis Light scattering analysis utilizes the ability to measure cell size (forward scatter) and particle size (side scatter) by using a flow cytometer laser beam. Morphological changes associated with apoptosis, such as size reduction (reduction) and particle size reduction, affect these parameters. As a result, apoptotic cells will migrate from the parameters of the healthy population to the left and slightly downward.

TF1 cells are plated in 24-well plates (2 × 10 ⁵ / ml) and cultured for 48 hours in the presence or absence of GM-CSF (2 ng / ml). The cells are then collected by centrifugation (1000 rpm, 10 minutes) and washed with PBS. The pellet is resuspended in PBS (2 × 10 ⁵ cells / ml) and obtained by FacsCalibre. Forward and side scatter parameters are evaluated using Cell Quest software.

Analysis of TF1 apoptosis by Sub-G1 phase analysis TF1 cells are cultured for 48 hours in the presence or absence of GM-CSF (2 ng / ml). Thereafter, the cells are collected by centrifugation (1000 rpm, 10 minutes) and washed with PBS. The pellet is resuspended in PBS (2 × 10 ⁵ cells / ml) and then 1 ml is added dropwise, vortexing continuously to ice cold 70% EtoH. The cells are then permeabilized by incubating at −20 ° C. for 24 hours. To stain the DNA, the cells are centrifuged and washed with PBS. Cells were resuspended in 500 μl propidium iodide buffer (propidium iodide (50 μg / ml); RNase A (50 μg / ml)) and placed in the dark for 15 minutes before using FacsCalibre (Becton Dickinson) Perform flow cytometry analysis. Analysis is performed using Cell Quest software.

GM-CSF exclusion reduces the proportion of cells in the live gate. When TF1 cells are cultured in the absence of GM-CSF, cell death is induced. This death causes the morphological changes seen in the forward and side scatter parameters, which can be detected by FacsCalibre analysis. As a result, by observing the proportion of cells within the survival gate (which is an arbitrary region pre-set for a healthy population of TF1 cells grown in the presence of GM-CSF (2 ng / ml)) A decrease in the proportion of cells with these light scattering parameters is observed when cytokines are eliminated.

  FIG. 6 shows that with 48 hours of factor exclusion, the percentage of cells in the survival gate has dropped from 77% to 35%, which represents a reduction of about 50% over this period.

  FIG. 7 shows that in TF1 cells grown for 48 hours in the absence of GM-CSF, cells with a sub-G1 profile indicating apoptotic cells are increased from 5% to about 55%. .

Expression of the control apoptosis regulator BCL2 inhibits apoptosis of TF1 cells To date, for a number of gene products, it has been shown to regulate the efficacy of apoptotic stimuli on cells. A typical example of this is BCL2 protein expression, which has already been shown in many cellular systems that apoptosis is inhibited by overexpression of this gene (Hockenbury D, Nunez G, Milliman C, Schreiber RD). Korsmeyer SJ, Nature, 348 (6299), 334-6, 1990). We have transduced TF1 cells with the human BCL2 gene and tested its effect on survival after GM-CSF exclusion.

  To assess the anti-apoptotic effect of BCL2 overexpression in TF1 cells, cells are transduced with a retrovirus expressing the full-length BCL2 coding sequence or a control vector lacking BCL2. Cells are then induced to cause apoptosis by eliminating GM-CSF for 48 hours. Record the percentage of transduced cells remaining in the survival gate (any area previously set for a healthy population of TF1 cells grown in the presence of GM-CSF (2 ng / ml)). The index of anti-apoptotic activity is calculated by computing the difference in the sample +/− GMCSF.

  As can be seen from FIG. 8, removal of GM-CSF from the culture medium induces apoptosis of control cells as measured by the number of cells migrating out of the survival gate (indicated by the gate). In contrast, expressing BCL2 reduces the number of cells that migrate from the survival gate. The ability of BCL2 to block apoptosis induced by elimination of GM-CSF has been reported previously (Ito, Overexpression of BCL2 suppresses apoptosis in the human leukemia cell line TF1, Rinho 1997 ), Pp. 628-37).

  These data demonstrate the reasonable nature of this assay for measuring apoptotic or survival activity.

Identified GPCR Overexpression of EDG6 reduces EGF6 apoptotic activity, which reduces the apoptosis of TF1 cells induced by exclusion of GM-CSF . A control vector is used to transduce TF1 cells. The EDG6 coding sequence encoding the amino acid sequence listed in Table 1B is cloned into the retroviral expression vector pMSCV (Clonetech). The plasmid is transfected into the retroviral packaging cell line Phoenix 293 (Standardford) using the CalPhos mammalian transfection kit (Clonetech) according to the manufacturer's instructions. Retroviruses are recovered after 72 hours. Retrovirus is transduced into TF1 cells by incubating for 18 hours. The medium is changed and the cells are incubated for an additional 48 hours.

  Transduced cells are cultured for an additional 48 hours in the absence or presence of GM-CSF (2 ng / ml) and then examined for viability using forward scatter / side scatter parameters. FIG. 9 shows that elimination of GM-CSF in control TF1 cells reduced viability to 65%, whereas cells overexpressing EDG6 had viability greater than 79%, It is shown that EDG6 expression leads to survival in TF1 cells.

Overexpression of the identified kinase TLK2 induces apoptosis in TF1 cells. To evaluate the apoptotic activity of TLK2, the retrovirus expressing the TLK2 coding sequence, or a control vector lacking TLK2, was used in TF1 cells. Transduce. The TLK2 coding sequence encoding the amino acid sequence listed in Table 1B is cloned into the retroviral expression vector pMSCV (Clonetech). The plasmid is transfected into the retroviral packaging cell line Phoenix 293 (Standardford) using the CalPhos mammalian transfection kit (Clonetech) according to the manufacturer's instructions. Retroviruses are recovered after 72 hours. Retrovirus is transduced into TF1 cells by incubating for 18 hours. The medium is changed and the cells are incubated for an additional 96 hours.

  After 96 hours, cell viability is tested using forward scatter / side scatter parameters. FIG. 10 shows that the control cell population is still viable (46.33%), whereas almost all TLK2 transfected cell populations are dead (0.48% viable cells). Show. These data demonstrate that TLK2 mediates a strong pro-apoptotic effect in TF1 cells.

Example 5: Knockdown of identified genes in cancer cell lines The functional impact of silencing these genes is investigated using small interfering RNA oligomers. This silencing effect is measured using viability and apoptosis assays as described in Examples 6-8.

  In order to assess the ability of each siRNA oligonucleotide to knock down the intended target, each siRNA was evaluated in a cell line known to express that target. Knockdown efficacy was measured using QPCR in two separate experiments, A) and B).

A)
Design of siRNA oligonucleotides The kinase / GPCRS coding sequence, a novel identified gene, is screened for AAN19TT siRNA target sequences with a GC content of 40-55% (http://www.ambion.com/techlib/misc/ siRNA finder. html). Candidate oligonucleotides are subjected to a BLAST search against the Genbank database to ensure that the selected sequence does not share significant homology with any other human gene. In addition, siRNAs for a subset of genes were designed by Ambion using their proprietary design algorithms. The selected oligonucleotides are shown in Table 9. The siRNA duplex is chemically synthesized by Qiagen as N19 (RNA) + TT (DNA) and purified to the Qiagen HPP standard. These oligos are supplied in an annealed state. The oligo was resuspended in siRNA buffer (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) at a concentration of 20 M, 1 minute at 90 ° C., then 1 hour at 37 ° C. Incubate. SiRNA designed by Ambion is chemically synthesized by Ambion and purified by HPLC. These siRNA duplexes are resuspended at 20 μM in H ₂ O. Double-stranded siRNA is stored at −20 ° C. until needed.

Validation of siRNA efficacy in silencing gene expression In order to evaluate each siRNA oligonucleotide for its ability to knock down its intended target, each siRNA was evaluated in a cell line known to express that target. In this example, the gene was tested on PC3 cells. For PC3 cells, expression profiling showed that all target genes were expressed. The effectiveness of knockdown was measured using QPCR. Cells are plated into each well of a 24-well plate at a cell density of 2 × 10 ⁴ and incubated overnight at 37 ° C. SiRNA is transfected into cells using RNAiFect reagent (Qiagen) and siRNA at a concentration of 50 nM according to the manufacturer's instructions. Cells in separate wells are transfected with 50 nM non-silencing control (Qiagen). The latter sample serves as a negative control for RNAi and also demonstrates the specificity of knockdown by siRNA. Transfected cells are harvested 24 hours after transfection and RNA is extracted using an RNeasy miniprep column with a DNase digestion step on the column. RNA elutes in 30 μl of RNase free H ₂ O. cDNA is synthesized from 12 μl of this RNA using anchor oligo (dT) primer (Sigma) and Transscriptor reverse transcriptase (Roche) in a total volume of 20 μl at 55 ° C. according to the manufacturer's instructions. The resulting cDNA is diluted with H ₂ O to a total of 200 μl and 10 μl is used in each QPCR reaction. Transcript levels are compared by QPCR compared to non-silencing controls. QPCR is performed using the same primers, conditions, and techniques used for expression profiling. See Table 8. All reactions were performed in duplicate experiments and average Ct values were calculated. For each sample, the Ct value for each target gene was normalized to GAPDH. Relative expression between target-transfected cells and non-silencing siRNA-transfected cells, normalized to GAPDH expression, is determined by the comparative Ct method. Table 10 shows the percentage of target gene knocked down in PC3 cells compared to control cells (in this case cells transfected with non-silencing siRNA).

B)
A second batch of siRNA was generated roughly as described above and was chemically synthesized by Eurogentec (Belgium) as N19 (RNA) + TT (DNA). The purity of siRNA oligos is described by a 0.2 μmol synthesis scale and purified by PAGE. This oligo was annealed at a concentration of 20 μM in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90 ° C., followed by 1 hour at 37 ° C. Let Double-stranded siRNA is stored at −20 ° C. until needed. Table 11 shows the sequences of selected oligonucleotides.

Verification of siRNA efficacy in silencing gene expression These siRNA oligos are first tested for efficacy in cell lines in which the gene is expressed, eg, Hela, U251, TF1, or DU145 cell lines. Cells were plated at a density of 7.5-10 × 10 ⁴ in each well of a 6-well plate and DMEM medium (1 × Dulbecco's Modified Eagle Medium (Sigma D2554) containing 10% fetal calf serum (FCS), Incubate overnight at 37 ° C. in 0.004% folic acid, 4 mM L-glutamine, 0.37% sodium bicarbonate, 0.1 mM sodium pyruvate). 2 μg of double-stranded siRNA is transfected into cells using DMRIE-C reagent (Invitrogen 10459-014) according to the method described in US Patent Application No. 60 / 498,488. Briefly, to prepare 1 ml of transfection solution, 2 μg of siRNA duplex is added to 1 ml of Optimem (Gibco), to which 8 μl of DMRIE C reagent is added. The transfection mixture is added directly to the cells and incubated for 4 hours at 37 ° C. After this 4 hour incubation, the transfection solution is removed and the cells are fed with fresh media and incubated for an additional 24 hours. Additional media changes are necessary at this point to minimize the toxicity of the transfection procedure. Cells are harvested for analysis of mRNA expression 24 or 48 hours after transfection. Additional wells of cells are transfected with 2 μg of missense control as described above.

For TF1 cells, 2 × 10 ⁵ cells / well in each well of a 24-well plate are transfected with 2 μg of siRNA using 8 μl of DMRIE-C reagent (InVitrogen) per ml of Optimem (Gibco). . Cells are co-transfected with control missense siRNA in parallel. At the time of transfection, 2 ng / ml recombinant GM-CSF is added to OPTI-MEM medium. The transfection medium is replaced after 4 hours with fresh RPMI 1640 containing 2 μg / ml GMSCF and 10% FCS and incubated at 37 ° C. Twenty-four hours after transfection, the medium is changed as follows: RPMI 1640 + 10% FCS containing 2 μg / ml GMSCF is added to 3 wells and RPMI 1640 + 10% FCS without GMSCF is added to the other 3 wells. Add to. These cells are harvested for QPCR and phenotypic assays 48 and 72 hours after transfection. Non-transfected control TF1 cells are treated similarly. Samples are named T48 + G, T48-G depending on the time collected after transfection and the presence (+ G) or absence (-G) of GM-CSF in the medium. As described above, RNA is isolated, reverse transcribed into cDNA, and QPCR is performed. The short term (72 hours after transfection) cell viability assay / apoptosis assay used is discussed below.

The missense control serves as a negative control demonstrating that activation of the RNAi machinery itself is not toxic to the cell. Missense controls also show the specificity of knockdown induced by siRNA oligomers. The sequence of the missense control siRNA oligo used was as follows:
MS
Sense: UGAGAAUGUGAUGCGCGUCTT
Antisense: GACGCGCAUCACAUUCUCATT.

The missense control outlined above is ELAV-1 (accession number XM It is designed to introduce a 4 base pair mismatch substitution into the siRNA oligo sequence for 008947; gi1477591). The siRNA oligos were confirmed to be unable to reduce ELAV-1 expression by these substitutions, and therefore the substitutions make MS siRNA a true negative control.

  To date, additional genes described in the literature as being associated with apoptosis have been selected as control genes in RNAi assays. For example, Survivin (Survivin) is a known major apoptosis modulator whose expression is deregulated in cancer and is a known drug target in cancer development. When its expression (maintaining survival and cancer phenotype) is reduced by survivin specific siRNA oligos, the cancer cells die (as described by the cell viability assay discussed below). Thus, Survivin knockdown provides a positive control for the induction of cytotoxicity and is also identified herein when the target gene also induces a cytotoxic response in cancer cells upon knockdown. The function and potential role in enhancing the cancer phenotype for any of the target genes can be judged by analogy from Survivin knockdown. The sequences of these controls and the siRNA oligomers used against them are shown below.

Survivin (Survivin B, SurB, SURB, SUR)
Sense: GAACUGGCCCUCUCUGAGAGtt
Antisense: CUCCAAGAAGGGCCAGUCUCtt
PI3KR1
Sense: AUGACUCGAUGUGACACGUUtt
Antisense: AAACGUGCACAUCGAUCAUtt
BCL2
Sense: GUACAUCCAUAUAUAGCUGtt
Antisense: CAGCUUAUAUGGAUGUAACTt
c-Raf (CRAF)
Sense: UAGUUCAGCAGUUUGGGCUAtt
Antisense: UAGCCAAAACUGCUGAACUAtt
Cells are harvested as described above after DMRIE transfection using siRNA oligos for apoptosis-related genes or controls as specified herein, 24 hours or 48 hours after transfection. Thereafter, after cell lysis on a KIAshredder column (Qiagen 79654), RNA is extracted using an RNeasy Miniprep column (Qiagen 74104). RNA is quantified by spectroscopic analysis and 1 μg is reverse transcribed to cDNA using SuperScript II RNAse H-reverse transcriptase (Invitrogen 18064-014). Transcript levels are compared by QPCR against missense controls as described in detail in Example 3. The QPCR primers are designed to amplify a PCR product from CDS about 100-300 nucleotides in length according to the guidelines outlined in Qiagen's Quantitt SYBR Green PCR handbook.

  The selected QPCR primers are summarized in Table 4 as described above.

  Two template standards are used for quantitative PCR assessment of mRNA levels.

1. Control RPS13, ie ribosomal protein subunit 13 (NM) diluted in [1000, 100, 10, 1 and 0.1 fg, respectively] [001017] Amplified PCR products are used to quantify transcript levels.
2. RPS13 is amplified from the sample template in separate PCR reactions for normalization purposes.

  A cDNA template obtained from a suitable cell line transfected with a gene-specific positive control, eg Survivin, or a missense control siRNA, in the DNA Engineer Opticon system (MJ Research), QuantTectSYBR Green PCR kit (Qiagen 204143) Is used to amplify with gene-specific QPCR primers (as described in detail in Example 3). Amplification conditions include a 15 minute step at 95 ° C. for initial activation of HotStarTaq DNA polymerase followed by 35 cycles (94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds). The fluorescence of the sample at 521 nm is measured between the annealing and extension steps of the protocol. Melting curves are calculated at the end of 35 cycles to confirm product homogeneity.

  The standard curve uses the logarithm [template amount] of the RPS13 control template dilution (as above) and the fluorescence intensity measured in that well exceeds the level specified by the cycle threshold parameter (C (T) value). Plot against the number of cycles. An estimate of the initial template amount in the treated sample is determined from this plot, and the amount of target mRNA present in each sample is normalized between samples by calculating the ratio of target to RPS13 for each sample.

  Normalized expression values are used to calculate the percentage of target knockdown compared to transfected control cells (in this case cells transfected with missense siRNA).

By targeting a specific gene that is effective for reducing target gene expression with the siRNA oligonucleotide, the siRNA oligonucleotide efficiently reduces the mRNA expression of the targeted gene. This is done by comparing the relative levels of specific mRNA present in the cells before and after treatment with each siRNA oligo, and the level of specific mRNA in cells transfected with the missense control oligo Is measured by comparing. The level of knockdown achieved varies for each oligo, within the range of cell lines examined.

  Table 12 shows the percentage of target gene reduction after treatment of 4 different cell types with siRNA.

siRNA Does Not Induce Interferon Response Inhibition of gene expression using siRNA has been associated with non-specific induction of interferon from target cells (Sledz CA et al., Nat Cell Biol, September 2003, 5th ( 9) Volume, pages 834-9. Since induction of interferon can affect biological assays, we control the effect of the siRNA oligonucleotides of the invention on the induction of interferon. This is examined by quantifying the mRNA levels of two interferon response genes, OAS1 and GBP1, both before and after siRNA transfection.

The QPCR primer was designed by MWG Biotech as described above, and is as follows.

FIG. 11 shows two interferon response genes, OAS1 (NM) in seven cell lines (HCT15, A549, SKOV3, DU145, PC3, A498, and U251) after siRNA transfection with MS or GAPDH siRNA. 002534) and GBP1 (NM 002503) mRNA levels (by QPCR quantification as described above). This demonstrates that in HCT15, A549, SKOV3, DU145, and U251 cells, transfection of siRNA does not enhance interferon response gene mRNA levels compared to non-transfected cells. For PC3 and A498 cells, some increase in interferon-responsive gene mRNA was observed, which has a sufficient impact in biological assays, especially considering the results with the missense control (MS) It is not considered to be scale.

  The expression levels of OAS1 and GBP1 in cells transfected with siRNA are expressed as a percentage of the expression level in untransfected control cells.

Example 6: Knockdown of identified genes in cells functionally modulates apoptosis as measured by short-term assays This example demonstrates that identified "new" genes can be found in various cell types. Demonstrate that it plays a fundamental role in the apoptotic pathway. We demonstrate that disruption of the functional expression of these genes modulates apoptosis and cell viability over a 72 hour period in a wide range of cancer cell lines.

  Following confirmation of siRNA activity, introduction of siRNA duplexes into cancer cell lines, knockdown of target gene mRNA expression, apoptosis / cell viability, and other characteristics related to cancer phenotype (implementation) Test whether (as discussed in Examples 7 and 8) is modulated. Test cell lines include Hela, TF1, U251, SKOV3, OVCAR3, MCF7, PC3, HCT15, 786-0, HT29, M-14, H460, LnCap, DU145, A549, A498, HCT116, PanC1, MDA-MB-231 And MDA-MB-468.

  Except where otherwise indicated, the methods used for transfection and subsequent analysis by QPCR or cell viability / apoptosis assays only vary depending on the experimental scale used and the cell line used. Briefly, test cell lines are seeded 24 hours prior to transfection at a density such that they are 40-70% confluent upon transfection, depending on the cell line and the required experimental scale. To do. Cells are transfected with 2 μg siRNA + 8 μl DMRIE C reagent (Invitrogen) per ml Optimem (Gibco). Cells are transfected simultaneously with a control missense siRNA in parallel. Transfection medium is replaced after 4 hours with fresh medium containing 10% FCS and incubated at 37 ° C. for 24 hours. After this 24 hour incubation, further media changes are required to minimize toxicity associated with the transfection procedure. These cells are harvested for QPCR analysis 24 or 48 hours after transfection. As described above, RNA is isolated, reverse transcribed into cDNA, and QPCR is performed. For our short-term cell viability / apoptosis and phenotype assays, cells were harvested 48 or 72 hours after transfection and our long-term viability assay (as in Examples 7 and 8). For discussion purposes, cells are harvested 10-14 days after transfection.

  TF1 cells are cultured as described above. The cell viability / apoptosis assay utilized is discussed below.

  The sensitivity of the survival test to detect apoptosis induction / reduction of cell viability due to RNAi knockdown of a particular gene was determined by Survivin and / or BCL2 (apoptosis / viability) in each experiment as discussed in detail above. Evaluate by including siRNA oligos against (control) and missense negative control oligos.

In order to measure the impact of target knockdown on cell survival over 72 hours, i.e., the effect of knocking down the expression of the target gene by the short-term cell viability assay RNAi, the viability of cells with reduced target mRNA was determined by Investigate using the various assays described below. In addition, a number of apoptosis assays are performed to demonstrate that the decrease in viability was caused by apoptosis.

The cell viability and apoptosis assays utilized are as follows:
1. MTT high-throughput screening (MTT HTS)
The MTT assay measures the ability of cells to convert soluble MTT salts into insoluble blue formazan crystals. This conversion requires the activity of mitochondrial succinate dehydrogenase in living cells. Therefore, dead cells cannot convert MTT. As a result, the amount of color change is directly proportional to the level of viability of the cell population.

  Comparing the number of cells surviving 72 hours after transfection with siRNA oligos against our target gene with the number of cells subsequently surviving after transfection with missense siRNA oligos; For each of our target genes, the percent cytotoxicity in the examined cell line is confirmed.

Cells are seeded in 96-well plates and allowed to attach overnight at 37 ° C. in a humidified atmosphere containing 5% CO ₂ in air. The wells are seeded with an appropriate number of cells depending on the cell line. The cells are then transfected using siRNA oligomers for the gene of interest at a concentration of 2 μg. The transfection mixture consists of 2 μg siRNA duplexes in 1 ml Optimem containing 8 μl DMRIE-C reagent as detailed above. Transfection is continued for 4 hours, after which the transfection solution is removed and the cells are fed with fresh media and incubated overnight. Additional media changes 24 hours after transfection are essential to minimize toxicity of the transfection reagent. The cells are then incubated at 37 ° C. for an additional 48 hours after transfection, as described above, followed by MTT quantification of cell number.

  Cell viability is determined using the MTT assay. Briefly, 10 μl of a 5 mg / ml MTT solution in PBS (Sigma) is added to a 100 μl aliquot of cells, mixed well and incubated at 37 ° C. for 2.5-4 hours. Viable mitochondrial succinate dehydrogenase converts soluble MTT salts into insoluble blue formazan crystals. The medium is then removed from the wells by aspiration and the formazan crystals are solubilized by adding 100 μl DMSO. Cell viability correlates with the conversion of MTT to formazan crystals that produce a dark purple solution upon solubilization with DMSO. Plates are read at 570 nm with a Molecular Devices Emax precision microplate reader.

2. Forward Scatter / Side Scatter (FSC / SSC) Analysis The FSC / SSC analysis used in this example is an apoptosis assay that can accurately distinguish cells undergoing apoptosis or necrosis from viable cells. This is based on characteristic changes in cell size and granularity associated with the morphology of living, apoptotic and necrotic cells. To confirm the characteristic cell size and granularity of the cell population, cells transfected with the missense control are analyzed using a flow cytometer. The position of this cell population on the FSC and SSC scale is recognized by isolating this population at the gate. The impact of siRNA oligotransfection on our target gene on cell viability is confirmed by observing the migration of the cell population from the pre-recorded live cell gate. Necrotic cells grow in size before they collapse, and therefore initially show a transition upward and to the right of the viable cell population, and then, when disrupted, a cell debris location on the FSC / SSC plot ( Completely move to the bottom left). However, apoptotic cells simply migrate to the left side of the viable cell population with gradual cell size reduction, and then gradually shift down the SSC scale as cell size increases in late apoptosis. .

  Thus, changes in FSC / SSC parameters can be used to identify cells undergoing apoptosis after siRNA transfection due to changes in cell size and granularity associated with apoptosis. 24-72 hours after transfection of model cells with missense siRNA oligonucleotides, the percentage of cells with a forward scatter / side scatter (FSC / SSC) profile of viable cells is determined as described above. The transfection procedure was performed exactly as described above. Cells are harvested at these time points, centrifuged (1000 rpm, 10 minutes) and washed with PBS (Sigma). The pellet is resuspended in PBS or media and cells are obtained immediately after FacsCalibre (HP Biosciences). Forward and side scatter parameters are evaluated using Cell Quest software. The position determined by the FSC / SSC parameters of the cell population transfected with siRNA oligos against our target gene is compared to the position of cells transfected with the aforementioned missense control siRNA. The percentage (%) of cells remaining in the viable cell gate after transfection with the target siRNA oligo is ascertained for each gene target across the cell lines examined.

3. Sub G1 analysis of apoptosis Quantification of the percentage (%) of cells in each phase of the cell cycle is achieved by permeabilizing the cell suspension and staining the nuclear DNA of each cell with propidium iodide. . This is a fluorescent stain, and therefore the intensity of the fluorescence detected by the flow cytometer correlates with the amount of DNA in the cell. Analysis of cells transfected with our missense siRNA oligos reveals the% of the viable cell population relative to the expected cell population at each phase of the cell cycle. Cells in the G1 phase have a diploid amount of DNA, ie “2n”, and cells with a DNA amount> 2n are in the synthesis phase, ie, “S” phase of the cell cycle. Cells that are “4n” are in the G2M phase of the cell cycle and are just about to divide into two cells. However, cells undergoing apoptosis have DNA of <2n, that is, Sub-G1 amount (less than G1 amount) because the DNA is fragmented and leaks out from the cell nucleus. Therefore, by quantifying the percentage of cells with Sub-G1 amount of DNA, the percentage of apoptotic cells in the population can be quantified. This population quantification is performed 24-72 hours after transfection. Therefore, the proportion of cells in the Sub-G1 phase of the cell cycle is the percentage of cells in the cell population after transfection with siRNA oligomers against the newly identified gene compared to cells transfected with the missense control. Indicates the scale. Cell cycle analysis / SubG1 parameters were resuspended in buffer (0.1% sodium citrate, 0.1% Triton X-100, 5 mg / ml propidium iodide 200 μl, total volume 20 ml with PBS). Cloudy and obtained by staining cells for 7-24 hours at 4 ° C. in the dark. Thereafter, PI-stained cells are obtained with a flow cytometer. Analysis of FL2 fluorescence is performed with Cell Quest software to quantify the Sub-G1 phase of the cells.

  FIGS. 12-31 demonstrate that target gene knockdown by RNAi modulates apoptosis as detected by MTT HTS, FSC / SSC, and Sub-G1 analysis. These figures reveal the differential induction of apoptosis by the target gene compared to the missense (MS), BCL2, and / or Survivin controls across the range of cell lines examined for each gene using these apoptosis assays To do.

4). JC-1 analysis of mitochondrial membrane depolarization Analysis of the mitochondrial membrane potential of a cell population determines the percentage (%) of apoptotic and viable cells in the population. Mitochondrial membrane potential is quantified using JC-1 dye obtained from Molecular Probes according to the manufacturer's instructions. Cells are transfected as described above using target specific siRNA oligomers, our positive control or Survivin, negative control or missense (called MS). Briefly, cells are harvested and incubated with JC1 dye 48-72 hours after transfection with siRNA oligonucleotides. When incubated with viable cells, the dye remains in an aggregated form that emits red fluorescence when activated using an argon laser. However, when apoptosis is occurring, the JC-1 dye transitions to a monomer-dominated form that emits green fluorescence upon activation. Thus, a transition from a red fluorescent population to a red / green or green fluorescent population indicates an apoptotic population. Quantification of apoptosis occurring in the examined population of cells is performed by performing fluorescence analysis with Cell Quest software. The percentage (%) of cells undergoing apoptosis after transfection with target-specific siRNA oligos is directly compared to apoptosis induced by siRNA missense oligos.

  FIG. 25 demonstrates that knockdown of the target gene by RNAi modulates apoptosis as detected by JC-1 analysis. This demonstrates the differential induction of apoptosis by the target gene in the U251 cell line compared to missense (MS), BCL2, and / or Survivin controls.

5. Role of caspase activation and analysis of apoptosis using the caspase inhibitor z-vad fmk Apoptosis induced by transfection with siRNA oligomers against newly identified genes and detected by analysis of the Sub-G1 population To determine if it is dependent, transfection experiments followed by 72 hours of incubation are performed at +/− z-vad fmk. Common caspase inhibitors reduce apoptosis only when apoptosis is induced by a caspase-dependent pathway.

  FIG. 25 shows that knockdown of the target gene by RNAi induces apoptosis as detected by Sub-G1 analysis and can further depend on caspase activation in a gene and cell line specific manner. This example demonstrates the differential induction by target genes of apoptosis in a caspase-dependent or independent pathway in the U251 cell line compared to missense (MS), BCL2, and / or Survivin controls.

6). WST-1 Viability Screening The Wst1 assay measures the ability of cells to convert wst1 salts into soluble blue formazan. This conversion requires the activity of mitochondrial succinate dehydrogenase in living cells. Therefore, dead cells cannot convert wst-1. As a result, the amount of color change is directly proportional to the level of viability of the cell population.

Cells (Pancl, DU145, PC3, MDA-MB-231, MDA-MB-468, HCT116) were plated at a density of 3000 cells / well in 100 μl medium in a 96-well plate and contained 5% CO ₂ . Incubate overnight at 37 ° C in a humidified atmosphere. Adherent cells are then transfected with siRNA at a concentration of 25 nM or 50 nM using RNAiFect reagent (Qiagen) according to the manufacturer's instructions. Briefly, 1 μg siRNA is added to a total volume of 100 μl EC-R buffer (Qiagen), and an appropriate volume of RNAiFect depending on the cell line is added to the cells. After 15 minutes incubation at room temperature, the siRNA-RNAiFect complex is diluted to 275 nM or 550 nM with EC-R buffer. The diluted siRNA complex is then added to the cells to a final concentration of 25 nM (MDA-MB-231 and MDA-MB-468) or 50 nM (Pancl, DU145, PC3, HCT116). After 4 hours, the transfection solution is removed from the cells by inversion and fresh growth medium is added. All transfections were performed in triplicate wells, which included mock transfection (transfection reagent only), negative control siRNA (Qiagen non-silencing siRNA) and positive control targeting BCL2, Survivin, and C-Raf. Including.

  The Wst1 cell viability assay is performed 72 hours after transfection. To each well of the transfected cells, add Wst1 reagent (Roche) at 11-fold dilution (10 μl Wst1 in 100 μl cell growth medium) and mix well in a 96-well plate. Incubate at 37 ° C for a certain period of time. During this incubation, the tetrazolium salt WST-1 (4-[-3 (4-iodophenyl) -2 (4-nitrophenyl) -2H-5 tetrazolio] -1,3-benzenedisulfonate) is present in the mitochondria of living cells. Converted to formazan by activity. The released formazan dye is proportional to the number of viable cells and is measured at 450 nM using a scanning multiwell spectrophotometer. The relative number of viable cells in each transfected cell population is expressed as the percent reduction in absorbance from the mock transfected cells.

  As is apparent from FIG. 32, knockdown of the target gene by siRNA modulates cell death detected by Wst-1 conversion. This example demonstrates the differential induction of cell death by knocking down our target gene compared to missense and negative controls. The results of targeting other apoptotic genes (BCL2, c-Raf, and Survivin) that have been characterized in detail are also shown.

FIG. 32 shows the decrease in cell viability of cancer cell lines transfected with siRNA that silences the target gene. Each graph shows the mean decrease in WST-1 absorbance compared to mock-transfected cells obtained from independent experiments with n = 3 or n = 4 as described above. Error bars indicate standard error. Significance tests were performed using Student's t-test comparing each gene with a negative control at ^* P <0.05, ^** P <0.01, and ^*** P <0.001. Statistical analysis is performed on all samples. However, not all genes were recorded as causing significant effects due to the limited sample size and the inclusion of sample outliers that increased standard error. However, these genes can also cause significant biological effects if the sample size increases.

7. Cell proliferation assay (BrdU)
Proliferating cells can incorporate bromodeoxyuridine into their DNA during DNA synthesis. The BrdU assay can measure the incorporation of bromodeoxyuridine into the DNA of cells undergoing DNA synthesis and can therefore quantify the relative number of viable cells in S phase in the cell population.

  Transfection of cells is performed as described above for the WST-1 assay, but using a 96-well plate with a white wall and a clear bottom. The BrdU assay is performed 72 hours after transfection using a BrdU chemiluminescence proliferation assay kit (Roche) according to the manufacturer's instructions. Briefly, BrdU labeling solution is added to each well of transfected cells and the cells are incubated for a period of 2-4 hours depending on the cell line. The medium is removed from the cells by inversion and the cells are fixed with FixDenat solution for 30 minutes, followed by addition of anti-BrdU-POD solution and standing for an additional 60 minutes. The antibody solution is removed by inversion and the wells are rinsed 3 times with wash buffer. After washing, substrate solution is added to each well and the relative light units are measured with a microplate luminometer following a 3 minute incubation. The relative number of proliferating cells in each transfected cell population is expressed as a reduction in relative light units derived from mock transfected cells.

  FIG. 33 demonstrates that knockdown of our target gene by siRNA modulates cell proliferation detected by bromodeoxyuridine incorporation. The BrdU assay measures the number of viable cells attached to a tissue culture surface that have undergone DNA synthesis within a defined 2 hour period. This example demonstrates the differential reduction in cell death by knocking down our target gene compared to missense and negative controls. The results of targeting other apoptotic genes (BCL2, c-Raf, and Survivin) that have been characterized in detail are also shown.

FIG. 33 shows a reduction in cell proliferation of cancer cell lines transfected with siRNA that silences the target gene. Each graph shows the mean decrease in relative light units derived from BrdU incorporation compared to mock transfected cells, as described above, obtained from independent experiments with n = 3 or n = 4. Error bars indicate standard error. Significance tests were performed using Student's t-test comparing each gene with a negative control at ^* P <0.05, ^** P <0.01, and ^*** P <0.001. Statistical analysis is performed on all samples. However, not all genes were recorded to cause a significant effect due to the limited sample size and the inclusion of sample outliers that increased standard error. However, these genes can also cause significant biological effects if the sample size increases.

8). Annexin V Apoptosis Assay To demonstrate that the decrease in cell viability is due to the induction of apoptosis, the apoptotic state of cells whose targets are silenced is measured by the binding of annexin V to cell surface apoptosis markers. Examine using assay. Apoptosis or programmed cell death is an important and effective regulatory pathway of cell growth and proliferation. Cells respond to specific evoked signals by initiating intracellular processes that result in characteristic physiological changes. These include phosphatidylserine (PS) externalization to the cell surface, cleavage and degradation of certain cellular proteins, compaction and fragmentation of nuclear chromatin, and loss of membrane integrity (late stage) There is. Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS), a membrane component that is normally localized on the inner surface of the cell membrane. PS molecules migrate to the outer surface of the cell membrane early in the apoptotic pathway, where annexin V can easily bind to them.

  The annexin V assay is performed using a guabanoxin assay that utilizes annexin V-PE to detect PS outside the membrane of apoptotic cells. Cell impermeable dye 7-AAD is excluded from living and early apoptotic cells, but penetrates late apoptotic and dead cells and is included in this assay as an indicator of membrane structural integrity. The assay used a Guava Technologies personal cytometer and a guabanexin kit (Catalog No. 4500-0010, Guava Technologies), and transfected cells with siRNA in a 24-well plate. The manufacturer's protocol was modified to perform the assay after 48, 72, and 72 hours.

Cells (DU145, PC3, and HCT116) were plated at a density of 20000 cells / well in 500 μl medium in a 24-well plate and incubated overnight at 37 ° C. in a humidified atmosphere containing 5% CO _2. To do. Subsequently, adherent cells are transfected with siRNA (EKI, ROCK1, NTKL, and RBSK) at a concentration of 50 nM using RNAiFect reagent (Qiagen) according to the manufacturer's instructions. Briefly, 1 μg siRNA is added to a total volume of 100 μl EC-R buffer (Qiagen), and an appropriate volume of RNAiFect depending on the cell line is added to the cells. After 15 minutes incubation at room temperature, the siRNA-RNAiFect complex is diluted to 550 nM with EC-R buffer. The diluted siRNA complex is then added to the cells to a final concentration of 50 nM. After 4 hours, the transfection solution is removed from the cells and 500 μl of fresh growth medium is added. All transfections included mock transfection (transfection reagent only) and negative control siRNA (Qiagen non-silencing siRNA).

500 μl of medium is decanted from the cells and combined with 150 μl PBS used to wash the cells. Using 100 μl trypsin, the attached cells are detached, neutralized and the cells are resuspended in 500 μl medium / PBS mixture. Cells are centrifuged, washed with 500 μl of 1 × Nexin buffer and resuspended in buffer at a concentration of 2-10 × 10 ⁵ cells / ml. To 40 μl of cell suspension, 2.5 μl of annexin V-PE and 2.5 μl of 7-AAD are added and the cells are incubated on ice for 20 minutes with light shielding. Thereafter, 450 μl of 1 × nexin buffer is added to the cells and the cell population is analyzed using a Guava Technologies personal cytometer according to the manufacturer's instructions. Therefore, using this assay, the following three cell populations were used: (i) non-apoptotic cells (annexin V (−) and 7-AAD (−)), (ii) early apoptotic cells (annexin V (+) and 7− AAD (−)), (iii) late apoptotic cells, and dead cells (Annexin V (+) and 7-AAD (+)) can be distinguished. The total number of early and late apoptotic cells is measured and the relative number of apoptotic cells is calculated.

  FIG. 34 demonstrates that target gene knockdown by siRNA induces apoptosis as measured by an increase in phosphatidylserine positive cells in various cell lines. This example demonstrates the induction of apoptosis by knocking down our target gene compared to missense and mock controls (transfection reagent only).

  FIG. 34 shows that cells transfected with siRNAs that silence the EKI, ROCK1, NTKL, and RBSK genes undergo apoptosis. Each graph shows the percentage of transfected DU145, HCT116, or PC3 cells stained with the apoptosis-specific marker Annexin V relative to the mock transfected (transfection reagent only) cell population. The results from two independent experiments are shown.

Example 7: Knockdown of identified genes in cells functionally modulates apoptosis as measured by a long-term viability assay. In this example, siRNA oligomers, and siRNA efficacy studies performed on each siRNA oligomer are , As described in Example 5.

Inhibition of target gene expression in cancer cell lines by siRNA modulates long-term cancer cell viability as measured by the clonogenic assay (Clonogenicity Assay) is a cancer cell line for long-term logarithmic growth. A cell viability screen that allows assessment of cytotoxic damage at various seeding densities so that the final effect of the test substance can be accurately quantified. In this case, the “cytotoxic injury” to be tested involves transfection of a novel target kinase and siRNA oligomers that knock down the expression of the GPCR, as outlined above. Ten to 14 days after siRNA transfection, cell numbers are quantified by MTT conversion as described in detail in Example 6.

Clonogenicity assay In this example, the effect of knocking down target gene expression on various cancer cells is measured over 14 days. Model cell lines include Hela, DU145, TF1, U251, SKOV3, OVCAR3, MCF7, PC3, HCT15, 786-0, HT29, M-14, H460, LnCap, PC3, A498, and A549.

  The seeding number in the clonogenic assay is optimized for each cell line examined. The seeding numbers are different for each cell line, and explain the differences in cell size, growth rate, and susceptibility to transfection-induced toxicity for each cell line in question. Cell lines that are models for this example include HCT15, MCF7, A549, SKOV3, DU145, U251, PC3, A498, and HeLa. As described in detail in Examples 5 and 6, the siRNA oligo against Survivin serves as a positive control for toxicity and the missense control oligomer serves as a negative control in this assay. As mentioned above, c-Raf, BCL2, and PI3K siRNA oligomers serve as additional controls as described above. The clonogenic assay is performed as outlined below.

10-14 Day Clonogenic Assay / Long Term Cytotoxicity Screening Method Cells are seeded in 96-well plates as described below, at 37 ° C. in a humidified atmosphere containing 5% CO ₂ in air. Allow the cells to attach overnight. Seeding is performed with the appropriate number of cells (depending on the cell line) in the well of column 1 of a 96-well plate, and this cell suspension is represented on the 96-well plate from column 1 to 6 (results on the x-axis). ), Dilute to 1/2. Next, the cells are transfected with siRNA oligomers for the gene of interest at a concentration of 2 μg / ml. Transfection is continued for 4 hours, after which the transfection solution is removed and the cells are fed with fresh media and incubated overnight. Twenty-four hours after transfection, additional media changes are performed to minimize toxicity of the transfection reagent. The cells are then incubated at 37 ° C. as described above for a period of 10-14 days after transfection. The medium is changed once a week. Subsequently, the quantification of MTT is performed as described in Example 5.

Inhibition by siRNA of target gene expression in cancer cell lines is determined by an anchorage-independent / soft agar assay that modulates long-term cancer cell viability as measured by proliferation in an anchorage-independent / soft agar assay. The effect of modulation of gene expression on striatum formation can be assessed by observing and quantifying tumor-like body morphology and long-term logarithmic colony counts, thereby ascertaining whether any cell death has been induced.

  The ability of cancer cells to form tumor-like bodies in semi-solid agar is unique to the aggressive tumor cell phenotype. In this example, the functional consequences of knocking down the target gene are examined in this anchorage independent cell assay.

  Prior to initiating siRNA experiments, the ability of each cancer cell line to perform anchorage independent tumor-like body formation in soft agar is assessed. Visual observation of tumors formed after a 6-day incubation period allows PC3, U251, Hela, MCF7, HCT15, and A549 cell lines to form well-defined tumors in this assay, and appropriate for this cancer phenotype analysis. It was shown to function as a model. However, all cancer cell lines outlined above can serve as models for this cancer cell property because they are highly compatible in RNAi.

  The ability of this assay to successfully identify cytotoxicity when knocking down gene expression with RNAi is verified by the identification of siRNA against Survivin as a cytotoxic agent in a test cell line. Thus, using an anchorage-independent / soft agar assay, we evaluated the effect on tumor-like body formation of reducing the expression of our kinase / GPCRS of interest using specific siRNA oligomers, The results are compared with the cytotoxicity induced by Survivin. The soft agar assay is performed as outlined below.

Soft Agar Assay Method Cells are seeded in 6-well plates and allowed to attach overnight at 37 ° C. in a humidified atmosphere containing 5% CO ₂ in air. Thereafter, the cells are transfected with siRNA oligomers for the gene of interest at a concentration of 2 μg. Transfection is continued for 4 hours, after which the transfection solution is removed and the cells are fed with fresh media and incubated overnight. Transfected cells are harvested for seeding as follows in a soft agar assay. Cells were seeded at 5 × 10 ⁴ cells per well in 6-well plates and incubated for 12/14 days, while 0.05% agar at 37 ° C. in a humidified atmosphere containing 5% CO _{2 in} air. / Grow in medium cell suspension. Subsequently, the formed colonies are stained with crystal violet, their morphology is evaluated, and the formed colonies are counted at 4 × magnification.

  The cytotoxic effect of each gene is determined by a decrease in the number of colonies compared to our MS control, but any effect on colony morphology is recorded.

Reduced expression of target genes by siRNA reduces cancer cell viability in a 10/14 day clonogenic assay-Part I
This example shows a decrease in target gene mRNA expression compared to the apoptosis / viability controls Survivin, BCL2, c-Raf, PI3K, and missense (MS) controls on 10/14 days of clonogenicity. It demonstrates that the assay can induce cell death as measured by MTT analysis. The control gene Survivin induces a significant reduction in the viability / clonogenicity of the cell lines tested in this assay in these cell lines compared to the MS control.

  Example of results / clonogenic profile obtained from a clonogenic assay of a gene that has a cytotoxic effect upon knockdown (STK6) and another gene that does not induce cytotoxicity upon knockdown (FLJ13351) The U251 cell line is shown in FIG.

  Targeting many genes showed induction of toxicity throughout the series of cell lines used, while other genes showed cytotoxicity induction in a cell type-specific manner. The maximum cytotoxicity induced by the knockdown of each gene was determined from the clonogenic profile in all three independent experiments as described below, and Table 13 (ag) for each cell line. Summarized in

FIG. 35 shows: (a) Transfection with siRNA oligos against STK6 reduces viability in the U251 cell line as measured by clonogenic assay. Note that seeding density is reduced from 3.5 × 10 ³ to 0.1 × 10 ³ cells on the x-axis, and the y-axis represents the number of cells quantified by MTT conversion 10-14 days after transfection. I want to be. This reduction is substantially greater than the apoptotic gene Survivin; (b) transfection with siRNA oligos against FLJ13351 increases cytotoxicity in the U251 cell line as measured by clonogenic assays. Do not induce. It is noted that the seeding density decreases from 3.5 to 0.1 × 10 ³ cells on the x-axis, and the y-axis represents the number of cells quantified by MTT conversion at 10-14 days after transfection. I want.

Note:
In order to be considered cytotoxic, siRNA oligos against gene targets are observed in a cell line so that a significant difference is observed between the target gene and MS, ie no overlap of standard error bars is detected. Thus, reduced viability must be induced at at least two seeding densities in the clonogenic assay. The average percentage of viability recorded relative to missense for the dilution rate at which maximum toxicity was induced is shown in Table 13.

  Table 13 Clonogenic assay-This table shows the average viability of the indicated cell lines in the clonogenic assay compared to missense for 3 independent experiments. As an internal standard, “Survivin”, BCL2, c-Raf, and PI3KR1, which are known survival genes, were also targeted with siRNA. When no cytotoxic response is detected, “−” is recorded.

  These examples demonstrate that knockdown of target gene mRNA results in functional modulation of apoptosis and cell death in certain cancer cells and does not induce apoptosis in cells derived from other tumor types . Cancer cell types that are affected by down-regulation of gene expression depend on each “newly” identified apoptosis regulator. Thus, since inhibition of these same identified genes results in cytotoxicity of specific cancer cell types, this example will show a direct therapeutic application of inhibition of those genes to therapeutic treatment of specific cancers. Prove it.

Reduced expression of target genes by siRNA reduces cancer cell viability in a 14-day clonogenic assay-Part II
The effect of target gene silencing is measured over 14 days using a clonogenic assay. In this clonogenic assay, clonogenicity is measured by counting colonies.

Cells (MDA-MB-231, MDA-MB-468, Panc1, A549, and HCT116) are plated at a density of 20000 cells / well in 500 μl medium in a 24-well plate and contain 5% CO ₂ . Incubate overnight at 37 ° C. in a humidified atmosphere. The adherent cells are then transfected with siRNA at a concentration of 50 nM using RNAiFect reagent (Qiagen) according to the manufacturer's instructions. Briefly, 1 μg siRNA is added to a total volume of 100 μl EC-R buffer (Qiagen), and an appropriate volume of RNAiFect depending on the cell line is added to the cells. After 15 minutes incubation at room temperature, the siRNA-RNAiFect complex is diluted to 550 nM with EC-R buffer. The diluted siRNA complex is then added to the cells to a final concentration of 50 nM. After 4 hours, the transfection solution is removed from the cells and 500 μl of fresh growth medium is added. All transfections include mock transfection (transfection reagent only), negative control siRNA (Qiagen non-silencing siRNA), and positive control siRNA (targeting against BCL2, Survivin, and C-Raf). It was.

  Twenty-four hours after transfection, transfected cells are removed from tissue culture plates by trypsinization and neutralization in cell growth media. The density of viable cells was measured according to the manufacturer's instructions using a ViaCount assay used in combination with a Guava personal cytometer. Cells are diluted to a density of 2500 cells / ml and a 50 μl aliquot of this cell suspension is added to seed 125 cells per well in a triple well of a 6 well plate containing 2.5 ml of cell growth medium. . Cells are incubated for 14 days. Cell fixation and staining is performed by removing the medium and adding 50:50 ethanol: water containing 0.5% (w / v) methylene blue so that the cells are immersed. Cells are incubated for 30 minutes and the staining solution is removed by immersing the plate in water. Count colonies.

  FIG. 36 shows reduced viability in a 14-day clonogenic assay in cells transfected with siRNA that silences the target gene. Each graph shows the average number of colonies from cells transfected with siRNA. Data are from triple wells from a single experiment. Error bars indicate standard error.

Soft Agar Assay The soft agar assay demonstrates that reduced expression of target genes by RNAi induces apoptosis / cell death of cancer cell lines, as measured by the reduced ability of cancer cells to form tumor-like bodies.

  Table 14 outlines examples of genes detected by soft agar assay that have cytotoxic effects upon knockdown. Table 14 shows the percentage survival compared to missense survival in an anchorage independent assay for a given cell line after transfection with siRNA oligomers against the target gene. As an internal standard, Survivin, a known survival gene, was also targeted with siRNA. This table shows the magnitude of the cytotoxicity induced by knockdown of each target gene compared to the MS control, i.e., the number of colonies formed, stained with crystal violet and counted visually as discussed above. It is shown.

  Examples of soft agar results for the PSKH1 and TLK2 genes are also shown individually in FIGS. 37 and 38. The cytotoxic response rate described in Table 14 was determined from the graphs shown in FIG. 37 and FIG. Together, these results demonstrate that in a range of cancer cell types, viability decreases after knocking down target gene expression.

  FIG. 37 shows that knockdown of the PSKH1 gene by RNAi modulates apoptosis in cancer cell lines as measured by an anchorage independent soft agar assay. FIG. 38 shows that knockdown of the TLK2 gene by RNAi does not modulate cancer cell line apoptosis as measured by an anchorage-independent soft agar assay. Cytotoxicity is quantified as the reduction in the number of tumoroids / colonys formed by the cancer cell line after transfection with PSKH1 siRNA oligos in logarithmic growth over 14 days. As an internal standard, Survivin, a known survival gene, was also targeted with siRNA.

  Of the targets shown, the test kinases with the highest cytotoxicity as measured by the soft agar assay are MPSK1 and PSKH1, which induce significant cell death in all cell lines. MKNK induced cell death consistently in two of the test cell lines and also in only one experiment but in the HCT15 cell line. PRK / CNK consistently induced HCT15 cell lines and also induced toxicity in the A549 cell line, which was also a single experiment. Survivin induced cell death in all cell lines and served as a reproducible control in this experiment, whereas c-Raf induced cell death only in the A549 and HCT15 cell lines.

  This example demonstrates that a large number of target genes can induce death in the indicated cell lines in a 14 day anchorage independent assay. These examples show that knockdown of the “newly identified” “apoptotic regulator” mRNA results in a functional modulation of apoptosis in certain cancer cells, whereas cells obtained from other tumor types Let us demonstrate that it does not induce apoptosis. Cancer cell types that are affected by down-regulation of gene expression depend on each “newly” identified apoptosis regulator. Thus, since inhibition of these same identified genes results in cytotoxicity of specific cancer cell types, this example demonstrates the direct therapeutic application of inhibition of those genes to the therapeutic treatment of specific cancers It is.

Notes on colony morphology:
The cell line shown in this example forms tumoroids of various sizes and shapes in a soft agar assay. However, it should be noted that the morphology of the tumor-like bodies formed by all cell lines, especially A549 and HCT15, changes when knockdown of a particular gene affects cell viability. Tumor-like bodies transfected with MS show a dispersed / scattered form, and most of the tumor-like bodies present in these samples are in a dispersed form, but when a toxic gene is transfected (eg in A549) For MKNK and MPSKI), the morphology often changed very dramatically from a dispersed form to a solid tumor that was generally smaller in size and round. These round solid tumors dominate when cytotoxicity is induced by transfection with siRNA.

RNAi knockdown of target gene expression reduces long-term viability of cancer cells. Evaluation of long-term viability of cancer cells using a combination of soft agar assay and clonogenic assay is effective for specific genes as candidate drug targets. Demonstrate what is possible (see “Summary” section). These examples show that knockdown of the “new” identified “apoptosis regulator” mRNA, along with the addition of stress from cell cloning and anchorage-independent growth, is the function of apoptosis in cells across generations of cells. To demonstrate dynamic modulation. Furthermore, since inhibition of these same identified genes results in cytotoxicity of certain cancer cells, this example demonstrates the direct therapeutic application of inhibition of those genes to the therapeutic treatment of cancer .

Example 8: Knockdown of identified genes in cells functionally modulates cancer cell migration Cancer cell migration is one of the most malignant features in tumors. In the following examples, we demonstrate that migration can be blocked by suppressing the expression of the indicated target genes. In this example, we use a commercially available cell migration assay.

Cell Migration Assay Cells are seeded in 6-well plates and allowed to attach overnight at 37 ° C. in a humidified atmosphere containing 5% CO ₂ in air. The cells are then transfected with siRNA oligonucleotides for the gene of interest at a concentration of 2 μg / ml as described in detail in Example 5. Transfection is continued for 4 hours, after which the transfection solution is removed and the cells are fed with fresh media and incubated overnight. Transfected cells are harvested for seeding in migration assays as described below.

  A Chemicon cell migration assay (ECM550) is used to quantify the number of PC3 cells that have successfully migrated across the Boyden chamber towards the chemotactic agent during the 24 hour incubation period. Assessment of PC3 cell migration is performed by routine techniques 48 hours after siRNA transfection. The effect of reducing the expression of the gene of interest is compared to that due to the missense control oligomer and the effect of UPA knockdown. It has been reported in the literature that UPA knockdown reduces PC3 cell migration. Migration assays are performed according to manufacturer's instructions. The results are illustrated in FIGS.

  FIG. 39 shows that knockdown of target genes by siRNA in PC3 cells reduces their ability to migrate.

  FIG. 40 shows that knockdown of target genes by siRNA in PC3 cells increases their ability to migrate.

  FIG. 41 shows that knockdown of target genes by siRNA in PC3 cells does not modulate their ability to migrate.

  Cells were plated and transfected with a given siRNA as described above. PC3 cell migration was followed using a fluorescent dye. As a result, an increase in fluorescence units has a positive correlation with an increase in migration. The effect of targeting a particular gene was compared to targeting either a missense control or urokinase plasminogen activator (UPA). UPA has been previously demonstrated to be a gene that modulates PC3 cell migration.

Targeted gene targeted knockdown modulates migration in the PC3 prostate cancer cell line. In this example, prostate cell line PC3 is used to measure the effects of targeting various kinases and GPCRs with siRNA. The PC3 cell line is traditionally used for such chemotaxis assays.

  By reducing the expression of PCTAIRE, MPSK1, RS6PK, TLK2, MKNK, CDC42, EDG6, and FLT4, inhibition of migration by prostate cancer cells across the 8 μm porous filter in Boyden chambers was found to be a missense control and urokinase plasmino It is produced to varying degrees compared to both of the gene activator (UPA) control genes.

  In contrast, reducing the expression of MAK, PRK / CNK, and PSKH1 by siRNA in the PCi cell line with RNAi increases the ability of cancer cells to migrate.

  Changing the expression of MAPKK5 and P14KB by RNAi had no effect on the migration of this cell line. Therefore, these genes must play a role in the migration / chemotaxis of these prostate cancer cells.

  These examples demonstrate that knockdown of the “new” identified apoptosis regulator mRNA results in a functional modulation of the cancer phenotype of prostate cancer cells. By reducing the expression of these genes, we affect the ability of these aggressive cells to exhibit chemotaxis, thereby affecting the ability of cancer to metastasize. Cancer metastasis is a characteristic that contributes significantly to cancer recurrence, resistance to treatment, and mortality. Thus, since inhibition of these same identified genes can reduce metastasis of cancer cells, this example demonstrates the direct therapeutic application of inhibition of those genes to therapeutic treatment of cancer.

  All publications referred to in this specification, and references cited in the above publications, are hereby incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be covered by the appended claims.

FIG. 5 shows sub-G1 analysis of neutrophils cultured overnight in the presence or absence of GM-CSF before ratio analysis of cells with sub-G1 profile. From this profile, it is clear that significantly fewer neutrophils are undergoing apoptosis when cultured with GM-CSF than when cultured without. It was determined by the proportion of cells with a sub-G1 profile (9.7% present vs. 65% absent). FIG. 3 shows that GM-CSF in the absence of GM-CSF or in the presence of Ly204002 increases the amount of apoptosis compared to neutrophils cultured with GM-CSF alone. GM-CSF in the absence of GM-CSF or in the presence of an AKT inhibitor (1L-6-hydroxymethyl-chiloinositol 2- (R) -2-O-methyl-3-O-octadecyl carbonate) -Increases the amount of apoptosis compared to neutrophils cultured only with CSF. It is a figure which shows the comparative analysis of the expression data using a microarray analysis and QPCR. The shaded bar graph (dark color) is QPCR, and the shaded bar graph (light color) is Affymetrix. FIG. 6 shows a “heat map” representing the relative expression of 12 target genes for the entire group of 61 cell lines. Expression is relative to GAPDH. FIG. 6 shows forward scatter and side scatter analysis of TF1 population by flow cytometry. TF1 cells are cultured for 48 hours in the presence or absence of GM-CSF (2 ng / ml) prior to acquisition and analysis using a FacsCalibre flow cytometer. The area surrounded by the gate represents a living gate area. From this figure, it is observed that the number of cells exhibiting the side scatter parameter of healthy cells is reduced by about 50% in cells depleted of the factor. It is a figure which shows the cell cycle analysis by flow cytometry. Apoptotic cells have degraded DNA, resulting in increased low intensity staining, measured as a sub-G1 peak in the fluorescence histogram. FIG. 3 is a composite image of a dot plot showing typical results for TF1 cells treated under various conditions. Cells are transduced with retroviral vectors (C) containing the full length BCL2 coding sequence, or control vectors (A and B) without BCL2, and placed under the conditions indicated. The presence of BCL2 in TF1 cells allows more cells to survive under exclusion of GM-CSF, as judged by the proportion of cells within the survival gate (enclosed area). As described in the example description, transduced with a retroviral vector containing the full-length EDG6 coding sequence, or a control vector without EDG6, for 48 hours prior to viability analysis using FSC / SSC parameters It is a figure which shows the TF1 cell cultured in the presence or absence of GM-CSF. After GM-CSF exclusion, surviving cells expressing EDG6 were significantly more than those transduced with the control vector alone, indicating that EDG6 protects TF1 cells from apoptosis. Viability analysis using FSC / SSC parameters transduced with retroviral vectors containing the full-length TLK2 coding sequence, or control vectors without TLK2, as described in the example descriptions. FIG. 2 shows TF1 cells cultured for 96 hours prior to. In contrast, less than 1% of the cells transduced with TLK2 were able to continue to survive, whereas in contrast, the population transduced with the control vector alone had significantly more viable cells, This indicates that apoptosis is induced in TF1 cells by TLK2. Two interferon response genes, OAS1 (NM) after siRNA transfection with MS or GAPDH siRNA in seven cell lines (HCT15, A549, SKOV3, DU145, PC3, A498, and U251) 002534), and GBP1 (NM 002503) mRNA levels (by the described QPCR quantification). FIG. 12 shows the modulation of apoptosis by MAK siRNA knockdown. 12 to 31 are diagrams showing apoptosis detected by MTT, FSC / SSC, and Sub-G1 analysis. The MTT assay was performed at 72 hours. Optical density / absorbance has a positive correlation with cell viability. FSC / SSC analysis detects apoptosis by monitoring changes in cell morphology as determined by cellular forward and side scatter parameters. Viability% has a positive correlation with the% of the population remaining in the viable cell gate after transfection with the gene specific siRNA oligo compared to the missense control. The sub-G1 analysis measures DNA fragmentation. DNA fragmentation is one of the characteristics of cells undergoing apoptosis. The ratio (%) of sub-G1 has a positive correlation with the number of cells undergoing apoptosis. FIG. 25 further shows the results of the JC-1 assay and caspase assay described herein. Modulation of apoptosis induced by siRNA oligos is compared to that obtained using control MS, BCL2, and / or Survivin siRNA oligos. FIG. 13 shows the modulation of apoptosis by GRP86 siRNA knockdown. Others are as described in the column of FIG. FIG. 14 shows the modulation of apoptosis by siRNA knockdown of PCTAIRE. Others are as described in the column of FIG. FIG. 15 shows the modulation of apoptosis by siRNA knockdown of GRAF. Others are as described in the column of FIG. FIG. 16 shows the modulation of apoptosis by MPSK1 siRNA knockdown. Others are as described in the column of FIG. FIG. 17 shows modulation of apoptosis by siRNA knockdown of RS6PK. Others are as described in the column of FIG. FIG. 16 shows modulation of apoptosis by siRNA knockdown of TLK2. Others are as described in the column of FIG. FIG. 19 shows modulation of apoptosis by siRNA knockdown of EK1. Others are as described in the column of FIG. FIG. 20 shows the modulation of apoptosis by siRNA knockdown of MKNK. Others are as described in the column of FIG. FIG. 21 shows the modulation of apoptosis by siRNA knockdown of NTKL. Others are as described in the column of FIG. FIG. 21 shows the modulation of apoptosis by siRNA knockdown of NTKL. Others are as described in the column of FIG. FIG. 22 shows the modulation of apoptosis by siRNA knockdown of CDC42. Others are as described in the column of FIG. FIG. 23 shows the modulation of apoptosis by RBSK siRNA knockdown. Others are as described in the column of FIG. FIG. 24 shows the modulation of apoptosis by siRNA knockdown of EDG6. Others are as described in the column of FIG. FIG. 25 shows the modulation of apoptosis by siRNA knockdown of PRK. FIG. 25 further shows the results of the JC-1 assay and caspase assay described herein. Others are as described in the column of FIG. FIG. 26 shows the modulation of apoptosis by MAPKK5 siRNA knockdown. Others are as described in the column of FIG. FIG. 27 shows the modulation of apoptosis by siRNA knockdown of P14KB. Others are as described in the column of FIG. FIG. 27 shows the modulation of apoptosis by siRNA knockdown of P14KB. Others are as described in the column of FIG. FIG. 28 shows modulation of apoptosis by siRNA knockdown of FLT4. Others are as described in the column of FIG. FIG. 29 shows the modulation of apoptosis by siRNA knockdown of PSKH1. Others are as described in the column of FIG. FIG. 30 shows the modulation of apoptosis by ITPKC siRNA knockdown. Others are as described in the column of FIG. FIG. 31 shows the modulation of apoptosis by ROCK siRNA knockdown. Others are as described in the column of FIG. FIG. 31 shows the modulation of apoptosis by ROCK siRNA knockdown. Others are as described in the column of FIG. It is a figure which shows the result of a Wst1 viability assay. It is a figure which shows the result of a Wst1 viability assay. It is a figure which shows the result of a Wst1 viability assay. It is a figure which shows the result of a BrdU proliferation assay. It is a figure which shows the result of a BrdU proliferation assay. It is a figure which shows the result of a BrdU proliferation assay. FIG. 6 shows apoptosis of transfected cells measured by Annexin V. It is a figure which shows the sample result of a clonogenic assay. It is a figure which shows the result of a 14-day clonogenicity assay. It is a figure which shows the result of a 14-day clonogenicity assay. FIG. 4 shows knockdown of PSKH1 in an anchorage independent soft agar assay. FIG. 6 shows knockdown of TLK2 in an anchorage independent soft agar assay. It is a figure which shows the movement of the PC3 cell processed by siRNA. It is a figure which shows the movement of the PC3 cell processed by siRNA. It is a figure which shows the movement of the PC3 cell processed by siRNA. It is a figure which shows the movement of the PC3 cell processed by siRNA. Tables 1A and 1B showing a list of specific genes and their respective accession numbers. Tables 1A and 1B showing a list of specific genes and their respective accession numbers. Tables 1A and 1B showing a list of specific genes and their respective accession numbers. Table 2 showing the rate of change in the expression of the genes identified in Table 1 for the entire treatment group compared to untreated neutrophils. Table 3 listing the first range of cancer cell lines. Table 4 describing the QPCR primers for the first set of target genes. Table 5 showing QPCR results in normal / cancerous tissues. Expression in cancer tissue is expressed relative to normal cells from the same tissue. Table 6 showing the results of QPCR in cancer cells. Table 7 listing the second range of cancer cell lines. Table 7 listing the second range of cancer cell lines. Table 8 describing the QPCR primers for the second set of target genes. Table 9 showing the sequence of the first batch of siRNA oligonucleotides. Table 10 demonstrating the effectiveness of siRNA in the PC3 prostate cancer cell line. Table 11 showing the sequence of the second batch of siRNA oligonucleotides. ^1-A second alternative siRNA oligonucleotide is used in Example 7 as shown in the figure. ^2- Alternative siRNA oligonucleotides used for TLK2, as indicated. Table 12 demonstrating the effectiveness of siRNA for the second batch in several cell lines. Table 13 shows the viability of the indicated cell lines when using siRNA, compared to missense in clonogenic assays in three independent experiments. Table (a) U251. Table 13 shows the viability of the indicated cell lines when using siRNA, compared to missense in clonogenic assays in three independent experiments. Table (b) PC3. Table 13 shows the viability of the indicated cell lines when using siRNA, compared to missense in clonogenic assays in three independent experiments. Table (c) A549. Table 13 shows the viability of the indicated cell lines when using siRNA, compared to missense in clonogenic assays in three independent experiments. Table (d) DU145. Table 13 shows the viability of the indicated cell lines when using siRNA, compared to missense in clonogenic assays in three independent experiments. Table (e) SKOV3. Table 13 shows the viability of the indicated cell lines when using siRNA, compared to missense in clonogenic assays in three independent experiments. Table (f) A498. Table 13 shows the viability of the indicated cell lines when using siRNA, compared to missense in clonogenic assays in three independent experiments. Table (g) HCT15. Table 14 shows the viability of the indicated cell lines in an anchorage independent soft agar assay after transfection with siRNA oligomers.

Claims (39)

A method of identifying an agent that modulates the function of an apoptosis-related polypeptide having the sequence set forth in Table 1B, comprising:
(A) providing a sample containing an apoptosis-related polypeptide having the sequence set forth in Table 1B and a candidate agent and incubating under conditions that allow the candidate agent to bind to the polypeptide;
(B) measuring the binding of the apoptosis-related polypeptide having the sequence described in Table 1B to the candidate agent in a sample; and (c) the apoptosis-related polypeptide having the sequence described in Table 1B Binding of the polypeptide having the sequence set forth in Table 1B to the control substance known not to bind to the polypeptide having the sequence set forth in Table 1B. Step to compare with,
Binding to the candidate agent in a sample by the apoptosis-related polypeptide having the sequence described in Table 1B as compared to binding to the control substance by the apoptosis-related polypeptide having the sequence described in Table 1B Wherein the candidate agent is shown to modulate the function of the apoptosis-related polypeptide having the sequence set forth in Table 1B.   A method for identifying an inhibitor of an apoptosis-related protein encoded by a gene selected from Table 1B, comprising the step of providing a preparation containing the encoded protein; Incubating the preparation with the test agent under conditions that allow binding; and by detecting the presence or absence of a signal generated from the interaction of the agent with the protein, Determining whether a test agent interacts with the protein and thereby determining whether the test agent inhibits the apoptosis-related protein.   3. The method of claim 1 or 2, wherein the preparation containing the protein comprises cells that express the protein.   A method for identifying an inhibitor of apoptosis-related protein kinase encoded by a protein kinase gene selected from Table 1B, comprising the step of providing a preparation containing said encoded protein kinase; Incubating the preparation with the test agent under conditions that allow the test agent to bind to the protein kinase; by detecting a change in the phosphotransferase activity of the protein kinase, And determining whether the test agent inhibits the apoptosis-related protein kinase.   A method for identifying an inhibitor of an apoptosis-related cell surface receptor protein encoded by a gene selected from Table 1B, comprising: an agent to a protein encoded by a cell surface receptor gene selected from Table 1B Providing a cell expressing the protein on its surface linked to a second component capable of providing a detectable signal in response to binding of the protein; under conditions that permit binding to the protein Contacting with a test agent; and determining whether the agent binds to and inhibits the protein by detecting the presence or absence of a signal generated from the interaction of the compound with the protein And thereby encoded by a gene selected from Table 1B above And that the activity of the apoptosis-associated cell surface receptor protein, comprising the step, it is determined whether the test agent inhibits. The second component capable of providing a detectable signal is G protein, and _{Gi, Go,} Gs, a G protein _{G 16,} G 15, Gq or _{G 12-13,,} according to claim 5 the method of.   A method for determining whether a chemical compound specifically binds to and inhibits an apoptosis-related protein encoded by a gene selected from Table 1B, which produces a second messenger response, and Table 1B Contacting a cell expressing a protein encoded by a gene selected from with a chemical compound under conditions suitable for protein inhibition, and the second messenger reaction in the presence and absence of the chemical compound. Such cells are those that normally do not express the protein, and the change in the second messenger response in the presence of the chemical compound is determined by the gene from which the compound is selected from Table 1B. Inhibits the encoded apoptosis-related protein It shows a method.   The second messenger reaction is activation of chloride ion channel, change of intracellular calcium ion level, release of inositol phosphate, release of arachidonic acid, GTPγS binding, activation of MAP kinase, accumulation of cAMP, intracellular potassium ion level Or a change in intracellular sodium ion levels.   8. The method according to claim 7, wherein the second messenger reaction is measured by a change in reporter gene activity, preferably selected from secreted alkaline phosphatase, luciferase, and β-galactosidase.   The method according to any one of claims 1 to 6, wherein the test agent is selected from a low molecular weight organic molecule, an antibody or antibody fragment, an antisense oligonucleotide, a small molecule inhibitory dsRNA, and a ribozyme. A method for detecting the presence of an apoptosis-related polypeptide having a sequence set forth in Table 1B in a sample comprising:
(A) contacting a biological sample containing DNA or RNA with a probe comprising a fragment of at least 15 nucleotides of a nucleic acid having a sequence set forth in Table 1B under hybridization conditions; and (b) the probe Detecting a double-stranded molecule formed between the nucleic acid in the sample,
Wherein the detection of double-stranded molecules indicates the presence of an apoptosis-related polypeptide having the sequence set forth in Table 1B in the sample. A method for detecting the presence of an apoptosis-related polypeptide having a sequence set forth in Table 1B in a sample comprising:
(A) providing an antibody capable of binding to the apoptosis-related polypeptide having the sequence shown in Table 1B;
(B) incubating a biological sample with the antibody under conditions that allow formation of an antibody-antigen complex; and (c) detecting an antibody-antigen complex comprising the antibody;
Wherein the detection of antibody-antigen complexes indicates the presence of an apoptosis-related polypeptide having the sequence set forth in Table 1B in the sample. A method for modulating apoptosis in a cell comprising:
(A) A step of introducing a double-stranded nucleic acid having the sequence shown in Table 1B or a complementary sequence thereof into the cell and transforming it, wherein the nucleic acid sequence is operably linked to a regulatory sequence. And (b) culturing the cell under conditions under which the nucleic acid sequence is expressed;
And thereby modulating apoptosis in said cells. A method for modulating apoptosis in a cell comprising:
(A) A step of introducing a double-stranded nucleic acid sequence encoding a polypeptide having the sequence shown in Table 1B into the cell for transformation, wherein the nucleic acid sequence is operably linked to a regulatory sequence. And (b) culturing the cell under conditions under which the nucleic acid sequence is expressed,
And thereby modulating apoptosis in said cells. A method for modulating apoptosis in a cell comprising:
(A) introducing a double-stranded nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to a polypeptide having the sequence shown in Table 1B into the cell and transforming it. The nucleic acid sequence is operably linked to a regulatory sequence; and (b) culturing the cell under conditions under which the nucleic acid sequence is expressed;
And thereby modulating apoptosis in said cells. A method for modulating apoptosis in a cell comprising:
(A) introducing and transforming into the cell an isolated nucleic acid molecule comprising a regulatory sequence operably linked to a nucleic acid sequence encoding a ribonucleic acid (RNA) precursor, wherein said step The precursor is
(I) a first stem portion comprising a 15-40 nucleotide long sequence that is identical to consecutive 15-40 nucleotides on the sequence set forth in Table 1B;
(Ii) a second stem portion comprising a 15-40 nucleotide long sequence that is complementary to a contiguous 15 to 40 nucleotides on the sequence set forth in Table 1B;
(Iii) a loop portion connecting the two stem portions;
And the first stem portion and the second stem portion can hybridize to each other to form a double-stranded stem; and (b) conditions under which the nucleic acid sequence is expressed Culturing the cells under,
And thereby modulating apoptosis in said cells.   The method according to any one of claims 13 to 16, wherein the nucleic acid sequence is a nucleic acid having the sequence shown in Table 1B or a complementary sequence thereof.   17. A method according to any of claims 13 to 16, wherein the nucleic acid sequence encodes a polypeptide having the sequence set forth in Table 1B.   17. The method according to any of claims 13 to 16, wherein the nucleic acid encodes a polypeptide having 80% sequence identity to a polypeptide having the sequence set forth in Table 1B.   17. A method according to any of claims 13-16, wherein apoptosis is reduced.   The method according to any of claims 13 to 16, wherein apoptosis is enhanced.   An RNA precursor encoded by a nucleic acid sequence having the sequence set forth in Table 1B.   A composition comprising the RNA precursor according to claim 22 as a biologically active ingredient.   A host cell transformed by the method according to claim 13.   A method for providing an anti-apoptotic protein to a mammal, the method comprising introducing a mammalian cell transformed by the method according to any one of claims 13 to 16 into the mammal.   23. A method of treating a disease or pathological condition characterized by abnormal apoptosis in a mammalian tissue, comprising contacting the tissue with an RNA precursor according to claim 22.   27. The method of claim 26, wherein the disease is cancer.   A pharmaceutical composition comprising an apoptosis-related nucleic acid having the sequence described in Table 1B as an active ingredient and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising an apoptosis-related polypeptide having the sequence described in Table 1B as an active ingredient and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising an antibody against an apoptosis-related nucleic acid having the sequence described in Table 1B as an active ingredient and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising an antibody against an apoptosis-related polypeptide having the sequence described in Table 1B as an active ingredient and a pharmaceutically acceptable carrier.   32. A method of diagnosing a disease or pathological condition characterized by abnormal apoptosis in a mammalian tissue, the method comprising contacting the tissue with an antibody according to claim 30 or 31 and detecting an antibody / antigen complex. And detecting that it is detected to indicate the disease or pathological condition.   A method of treating a disease or pathological condition characterized by abnormal apoptosis in a mammalian tissue, comprising contacting said tissue with an antagonist of an apoptosis-related polypeptide having a sequence set forth in Table 1B.   A method of treating a disease or pathological condition characterized by abnormal apoptosis in a mammalian tissue, comprising contacting the tissue with an agonist of an apoptosis-related polypeptide having a sequence set forth in Table 1B. A kit for treating a disease or pathological condition characterized by abnormal apoptosis in mammalian tissue comprising:
(A) a polypeptide encoded by a nucleic acid having the sequence set forth in Table 1B;
(B) a nucleic acid having the nucleotide sequence described in Table 1B; or (c) a kit comprising an antibody that recognizes an epitope of the polypeptide described in (a).   An array comprising at least two apoptosis genes each having a nucleic acid sequence having the sequence set forth in Table 1B.   38. The array of claim 36, wherein the nucleic acid sequence is a DNA sequence.   38. The array of claim 36, wherein the nucleic acid sequence is an RNA sequence.   An array comprising at least two apoptotic proteins each having a polypeptide sequence having the sequence set forth in Table 1B.

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