AU2002326111A1 - Methods for the treatment and prognosis of leukemia and other cancer types - Google Patents

Description

METHODSFORTHETREATMENTANDPROGNOSISOFLEUKEMIAAND

OTHERCANCERTYPES

FIELDOFTHEINVENTION

This invention relates to metliods and pharmaceutical compositions for mducing cell death by selectively inducing apoptosis in a cell wliich does not express or contain low amounts of ARTS and to methods and pharmaceutical compositions for inducing cell death in leukemic cells.

BACKGROUND OF THE INVENTION

[oooi] Genetic abnormalities associated with hematological malignancies alter the normal structure and function of genes that control cell growth, differentiation or death either in a positive or negative manner (1). The genes involved can be grouped into two general categories. The first group involves the structural alternation of a normal cellular gene, named proto-oncogen, whose protein product induce uncontrolled proliferation or loss of contact inhibition. The second group consists of genes, whose loss of function is associated with malignant transformation, and are referred to as tumor suppressor genes (2). There are several tumor suppressor genes known to be important in evolution of acute leukemia. Both pi 5 and pi 6 have been found to be homozygously deleted in 6% to 28% of B lineage acute lymphoblasfic leukemia (ALL) patients, while pl6 was found to be deleted in 41% to 83% in T cell ALL (3). Others showed that pl6 can be functionally inactivated by either point mutations or hypermethylation of the pi 6 promoter region (4). Another tumor suppressor gene, retinoblastoma gene, was reported to be inactivated at a low frequency in T cell ALL patients (5). P53 another important tumor suppressor gene, was found to be functionally inactivated in 50% of samples from patients at relapse, suggesting -that p53 mutations may be important in disease progression (6). Others hypothesized that loss of a putative tumor suppressor named TEL may promote leukemogenesis by affecting cell growth and/or by altering cell adhesion (7).

[0002] Recently, a novel human apoptosis inducing protein (ARTS), wliich induces cell killing by proapoptotic inducers such as TNF β, Fas, etoposide, arabinoside (ara-c) and TNF α , was identified. ARTS is a member of the septin family of proteins and is encoded by 823 base pair cDNA sequence, encoding a predicted polypeptide of 274 amino acids. ARTS contains a P-loop GTP-binding domain, conserved in different classes of ATP/GTPases, including CED-4 and Apaf-1, which are major regulators of apoptosis.

SUMMARY OF THE INVENTION [0003] The invention provides a method of selectively inducing apoptosis in a cell which either contains basal levels or does not express or contain ARTS protein, comprising the step of contacting the cell with a ligand, which is efficient to increase the expression of the ARTS protein so as to increase the ARTS protein level or is efficient to activate proapoptotic inducer signal pathway in which ARTS participates, thereby selectively inducing apoptosis in cells.

[0004] In another embodiment, the invention provides a method of selectively inducing cell death in a subject in need comprising the steps of contacting cells with an effective amount of a ligand which specifically binds to the ARTS protein receptor or increases the expression of the ARTS protein so as to increase the amount of ARTS protein in the cell, or to activate the ARTS molecule thereby selectively inducing cell death in a subject.

[0005] In another embodiment, the invention provides a pharmaceutical composition comprising an amount of a ligand, which increases the expression of ARTS protein so as to increase the amount of ARTS protein in a cell, or activates ARTS pharmaceutically acceptable carrier.

[0006] In another embodiment the present invention provides a method for inhibiting pathologically cell proliferation comprising the step of immunizing a subject in need with a ligand which is efficient to increase the expression of the ARTS protein so as to increase the ARTS protein level or to activate proapoptotic inducer signal pathway in which ARTS participates, thereby selectively inducing apoptosis in cells

BRIEF DESCRIPTION OF THE DRAWINGS [0007] The present invention will be understood and appreciated more fully from the following detailed description taken in conjunction with the appended drawings in which: [0008] Figure 1 demonstrates the ARTS protein is absent in certain leukemic cells, yet present in hematopoetic stem cells, hrrn iiiistochemistry results using anti-ARTS specific polyclonal antibody on normal lymphocytes (1A) ALLl cells (IB) ATI cells (1C) and CD34+ stem cells (ID) showing the expression of ARTS in normal lymphocytes, ATI and CD34+ stem cells. ARTS protein is not expressed in ALLl leukemic cells (IB). [0009] Figure 2 shows that ALLl cells do not express ARTS. Reintroduction of ARTS into this cells restores apoptosis. RT-PCR results of RNA from ALLl cells show that although they express the control RNA BCR, they lack expression of ARTS ( 2A). Western blot analysis of protein lysate from ALLl cells showed no expression of ARTS protein in these cells (2B, two left lanes), and the appearance of ARTS protein in cells transfected with ARTS expression vector (2B, two right lanes). BCR protein showing in all lanes serves as positive and loading control.

[00010] ALLl cells transfected with either empty vector or ARTS, -/+ treatment with ara-C were incubated with annexin-V coated magnetic beads and separated using appropriate columns (Macs, Mitenyi- Biotec). Number of alive or apoptotic cells ( l 0⁶) counted is presented in 2C.

[oooii] Figure 3 shows that overexpression of ARTS induces apoptosis in leukemic cells: transfection of ARTS into K562, ATI, and HL-60 cells induces apoptosis. In all cell lines. The ability of ARTS to induce apoptosis, is at least equal to the effect of ara-C alone in all tested cells. In both ATI and HL-60 cells the effect of ARTS alone was higher then ara-C. In all cells apoptosis was more pronounced when the cells were both transfected with ARTS and treated with ara-C (3A-C). Kinetics of apoptosis was measured 24, 36 and 48 hours after transfection in K562 cells, and ara-C was added for two and four hours in each time point. Maximum effect of ARTS with ara-C was seen after two hours of treatment (3C). RT-PCR results in HL-60 cells showing that ARTS RNA is absent in these cells, as compared to ARTS

RNA present in K562 cells (3D) bcr, genomic DNA and RNA were shown to be present in HL-60 cells, as controls for the presence of DNA and RNA in this assay. Each graph represents the average results of 3 experiments done in duplicates.

DETAILED DESCRIPTION OF THE INVENTION

[00012] The invention is based on the unexpected findings obtained in cells derived from leukemic patients showing either complete absence of both the ARTS protein and the

ARTS RNA in these cells, or basal level of the ARTS protein and the ARTS RNA. Thus, based on the finding that ARTS is functioning as a tumor suppressor gene. The invention relates to methods of inducing cell death by selectively induce apoptosis in a cell which expresses low amounts of ARTS or does not express or contain ARTS protein. The apoptosis is induced by administration of a ligand which will increase the level of ARTS protein or will activate proapoptotic inducer signal pathway in which ARTS participates in a cell wliich has low level of ARTS or a cell which does not express ARTS protein. [00013] In another embodiment the cell is a leukemic cell, breast cell or a colon cell. [00014] The term "low level, or low amounts, or basal level, or basal amount of ARTS" refers interchangeably, hereinafter in the claims and the specification to a level of ARTS protein which is two to ten fold lower than the level of ARTS protein presented in a non malignant cell, such as for example, cell derived from healthy volunteer, or to cell that do not express ARTS at all, as determined by immunohistochemical methods for detecting protein level.

[00015] ARTS is a novel human apoptosis related protein which is a member of the septin family of proteins and possesses unique C-terminus and which effectively mediates apoptosis and modulates the actions of TGF- β on cells. The ARTS amino sequence is as follows:

Table 1: ARTS Protein Amino Acid Sequence

SEQ. ID. No. 1 : MIKRFLEDTT DDGELSKFVK DFSGNASCHP PEAKTWASRP QVPEPRPQAP DLYDDDLEFR PPSRPQSSDN QQYFCAPAPL SPSARPRSPW GKLDPYDSSE DDKEYVGF AT LPNQVHRKSV KKGFDFTLMV AGESGLGKST LVNSLFLTDL YRDRKLLGAE ERIMQTVEIT KHAVDIEEKG VRLRLTIVDT PGFGDAVNNT ECWKPVAEYI DQQFEQYFRD ESGLNRKNIQ DNRVHCCLYF ISPFGHGYGP SLRLLAPPGA VKGTGQEHQG QGCH

[00016] The invention provides a method for inducing apoptosis by introducing a ligand into malignant cells so as to increase its ARTS amounts or to activates ARTS. The activation of ARTS may be in another embodiment by activation of proapoptotic inducer signal pathway in which ARTS participates. [00017] In one embodiment the cell is a leukemic cell.

[00018] In another embodiment, the ligand could be an agonist, that acts as ARTS protein and will activate and induce apoptosis through the TGF- β apoptotic pathway, or any other apoptotic pathway triggered by the specific proapoptotic factor used. The cells that will be effected by the ARTS protein administration are cells which express low level of ARTS protein or do not express ARTS protein and which are sensitive to either "TGF- β induced apoptosis" or apoptosis induced by any other agent or factor used on the cells. The term "TGF- β induced apoptosis" means that the cells when exposed to at least 1 ng/ml TGF- β undergo apoptosis.

[00019] The invention provides a method of inducing apoptosis in cells and in particular leukemic cells by introducing ARTS into cells to increase its fntracellular amounts, or contacting with a ligand that can cause an increase in the expression of ARTS protein level in the leukemic cell. In another embodiment the ligand is any ligand which can activate proapoptotic inducer signal pathway in which ARTS participates.

[00020] According to the invention, the cell either does not express or contain ARTS protein, or expresses low amounts of ARTS, as detected by using anti ARTS antibodies and immunohistochemisty procedures, (see Experimental Procedures). The term "does not express or contain ARTS " means hereinafter in the Specification and in the Claims section that the cell express or contain either low level of ARTS protein or RNA that can be hardly detected by the existing immunohistochemistry methods / or by the methods of measuring RNA, or that ARTS protein or RNA levels in the cells is between two to twenty fold less than the level exist in a normal cell.

[00021] The term "apoptosis" relates hereinafter to cell death which is characterized by caspase activation and/or annexin-v binding, and/or nuclear condensation and subsequent fragmaentation of the DNA in nucleus. Apoptosis can be tested by visualizing the dead cells to identify whether they are shrinked, or using assays and kits which exist in the market for detecting DNA fragmentation, detecting of proteins which are related to Apoptosis such as BCL2 and detecting membrane alterations, such as asymmetry of the plasma membrane

[00022] The ARTS protem is located in the mitochondria and in certain cells the apoptotic induction is correlated with translocation of ARTS to the nucleus during the apoptotic process. The method of inducing apoptosis is based on the unexpected dramatic data showing that cells derived from leukemic subjects express less ARTS protein or do not express at all ARTS protein. This was examplified in Example 4, where leukemic cells lines that were transfected with ARTS have demonstrated apoptosis in contrast to cells that were transfected with empty vectors. [00023] The term "contacting" is defined here in the specification and in the claims to administering the cell, or exposing the cell to a ligand, which causes an increase in the level of expression of ARTS protein or in the level of ARTS protein in the cell so as to activate TGF-? induced apoptosis pathway or any other apoptotic pathway in which ARTS participates. [00024] In another embodiment the ligand can be a peptide, a protein, an agonist, an antisense, an antibody, or a nucleic acid construct. [00025] ' The "protein" or the "peptide" refer hereinabove in the claims and in the specification to ARTS protein and variants of the ARTS protein. The variants include any amino acid sequence, in any length from 5-20 amino acids derived from the ainino acid sequence provided in SEQ. ID. No. 1. For Example without being limited, the peptide can be: LPNQVHRKSV KKGFDFTLMV, or LVNSLFLTDL, or MIKRFLEDT or DFSGNASCHP PEAKTWAS or KHAVDI. [00026] The ARTS can be produce by transfecting cells with a vector containing nucleic acid which encodes for ARTS protein, or by a direct peptide synthesis using solid phase techniques. The ARTS protein can be recovered from culture medium or from cell lysate. In another embodiment the ARTS is purified from recombinant cell proteins or polypeptides for example by fractionation on ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on cation-exchange resin such as DEAE, SDS-PAGE and the like. [00027] T e term "agonist" refers hereinabove in the specifications and in the claims to any molecule which, when bound or activate the protein or the receptor , increases the amount or the duration of the effect of the biological or immunological activity of protem. The molecule can be a peptide or a nucleic acid construct. The agonist can be also an enzyme or a hormone.

[00028] The term "DNA construct" refers hereinabove in the Specification and in the claims to any nucleic acid sequence wliich increases the expression of the ARTS protein in the cell or that cause activation of ARTS protein. The aά ninisτration of such a DNA construct will result in one embodiment in higher amount of ARTS in the cell mitochondria,cytoplasm and /or nucleus. The term "DNA construct" covers DNA that has the sequence of part or a full sequence of naturally occurring genomic DNA molecule, a separate molecule such as cDNA, a genomic fragment, a fragment produced by a polymerase chain reaction (PCR), a restriction fragment or a recombinant nucleic acid sequence that is a part of a hybrid gene. The length of the probe can be 20 to 80 bases.

[00029] In another embodiment the "DNA construct" is a nucleic acid sequence encoding ARTS variants or ARTS from different species. The nucleic acid sequence encoding ARTS is set forth in SEQ. ID. No. 2

SEQ. ID. No. 2

A TGATCAAGCG TTTCCTGGAG GACACCACGG

ATGATGGAGA ACTGAGCAAG TTCGTGAAGG ATTTCTCAGG AAATGCGAGC TGCCACCCAC CAGAGGCTAA GACCTGGGCA TCCAGGCCCC AAGTCCCGGA GCCAAGGCCC CAGGCCCCGG ACCTCTATGA TGATGACCTG GAGTTCAGAC CCCCCTCGCG GCCCCAGTCC TCTGACAACC AGCAGTACTT CTGTGCCCCA GCCCCTCTCA GCCCATCTGC CAGGCCCCGC AGCCCATGGG GCAAGCTTGA TCCCTATGAT TCCTCTGAGG ATGACAAGGA GTATGTGGGC TTTGCAACCC TCCCCAACCA AGTCCACCGA AAGTCCGTGA AGAAAGGCTT TGACTTTACC CTCATGGTGG CAGGAGAGTC TGGCCTGGGC AAATCCACAC TTGTCAATAG CCTCTTCCTC ACTGATCTGT ACCGGGACCG GAAACTTCTT GGTGCTGAAG AGAGGATCAT GCAAACTGTG GAGATCACTA AGCATGCAGT GGACATAGAA GAGAAGGGTG TGAGGCTGCG GCTCACCATT GTGGACACAC CAGGTTTTGG GGATGCAGTC AACAACACAG AGTGCTGGAA GCCTGTGGCA GAATACATTG ATCAGCAGTT TGAGCAGTAT TTCCGAGACG AGAGTGGCCT GAACCGAAAG AACATCCAAG ACAACAGGGT GCACTGCTGC CTGTACTTCA TCTCACCCTT CGGCC ATGGG TATGGTCCAA GCCTGAGGCT CCTGGCACCA CCGGGTGCTG TCAAGGGAAC AGGCCAAGAG CACCAGGGGC AGGGCTGCCA CTAG [00030]In another embodiment the DNA construct is a cis acting gene is integrated upstream to an endogenous ARTS gene and therefore induce increase in the level of ARTS protein. [00031] In another embodiment the DNA construct cis acting gene which is integrated to any element which activate proapaototic inducer signal pathway in which ARTS participates. [00032] he term "cis-acting" is used to describe a genetic region that serves as an attachment site for DNA-binding proteins (e.g. enhancers, operators and promoters) thereby affecting the activity of genes on the same chromosome. In another embodiment [00033] The DNA construct is linked to a suitable control sequence capable of effecting expression of the nucleic acid sequence hi a suitable host, i.e. expression vector. The expression vector can be in a form of a plasmid, a cosmid, a viral particle or a phage. [00034] The term "antisense" refers to any composition containing a nucleic acid sequence, which is complementary to the "sense" strand of a specific nucleic acid sequence. Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation "negative" can refer to the antisense strand, and the designation "positive" can refer to the sense strand. [00035] In another embodiment the method of inducing apoptosis in the cell further comprising the use of cell killing agent. There are many processes by which cell death is achieved and some of these can lead to apoptosis. One of these is the absence of metabolism and another is the denaturation of enzymes. In either case vital stains will fail to stain these cells. These endpoints of cell death have been long understood and predate the current understanding of the mechanisms of cell death. Furthermore, there is the extinction between cytotoxic effects where cells are killed and cytostatic effects where the proliferation of cells are inhibited. The term "cell killing agent" refers to any agent, which cause either apoptotic or cytotoxic effects or cytostatic effects i the cells.

As was shown in Example 4, ARTS protein not only induce apoptosis but also increase the sensitivity of the cell to other apoptotic agents such as, without limitation, ara-C or cell killing agents.

[00036] hx another embodiment there is provided a method of selectively inducing mammalian cell death by admirristering to the subject in need, a therapeutically effective amount of a ligand, which specifically activated ARTS, or increases the expression or the amount of the ARTS protein . In another embodiment the selectively inducing cell death in a subject in need can b performed by inducing its processing or other structural or biochemical changes leading to ARTS mediated apoptosis in cell,

[00037] The term" effective amount" or "therapeutically effective amount" refers hereinafter refer to dosage that provides the specific pharmacological response for which the drug is administered in a significant number of subjects in need of such treatment. The "therapeutically effective amount" may vary according, for example, the physical condition of the patient, the age of the patient and the severity of the disease. [00038] Thus, the invention methods and ligands can be further extended to treat mammals and especially humans with diseases involving pathological proliferation of mammalian cells such as cancer. To "treat" refers to: (i) preventing a disease, occurring in an animal that may be predisposed to the disease, disorder and/or condition, but has not yet been diagnosed as having it; (ii) inhibiting the disease i.e., arresting its development; and (iii) relieving the disease i.e., causing regression of the disease. [00039] In another embodiment there is provided a pharmaceutical composition comprising a ligand, which specifically activated the TGF-β apoptotic pathway, and/or other apoptotic pathways in which ARTS participates or increases the expression or the amount of the ARTS protein in the cell , or activates proapoptotic inducer signal pathway in which ARTS participates (in particular in leukemic cell) and a pharmaceutically acceptable carrier.

[00040] Hereinafter, the teπns "pharmaceutically acceptable carrier" and

"physiologically acceptable carrier" which may be interchangeably use, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. [00041] It should be noted in this respect that tlie drug can be also a prodrug, which is inserted to the body in an inactive form and is activated upon n internal or external triggers. [00042] The pharmaceutical compositions utilized in this invention may be admimstered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means. [00043] Alternatively, the composition may be in dry form, for reconstitution before use with an appropriate sterile liquid.

[00044] hi addition to tl e active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical

Sciences (Maack Publishing, Easton Pa.). [00045] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. [00046] Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate. [00047] Ovagee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage. [00048] Pharmaceutical preparations, which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. [00049] Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.

[00050] Vox topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [00051] Tlie pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. [00052] The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. [00053] After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. [00054] Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

[00055] Vox any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to deteπnine useful doses and routes for administration in humans. Effective dose is determined also in clinical trials in humans.

[00056] hi another embodiment the present invention provides a method for inhibiting pathologically cell prohferation by immunizing a subject in need with a ligand which is efficient to increase the expression of the ARTS protein so as to increase the ARTS protein level or to activate proapoptotic inducer signal pathway in which ARTS participates, thereby selectively inducing apoptosis in cells. The subject in need can be any subject who may develop cancer and in particular is predicted to develop leukemia.

[00057] In another embodiment tl e invention provides method of diagnosing and/or predicting hematological malignancy in a subject comprising the steps of: i. obtaining a sample from the subject. The sample may be is a lympohocyte, a bone marrow cell or a stem cell. ii. contacting said sample with an antibody directed to ARTS protein, thereby to form an antigen-antibody complex; iii. detecting the an antigen-antibody complex level, thereby determining the level of said ARTS protein in said sample; and iv. Comparing said level of said ARTS to predetermined ARTS protein level of samples derived from healthy subj ects.

[00058] The antibodies are agents which can bind to ARTS protein. They can be monoclonal or polyclonal and can be prepared in any mammal. In another embodiment the antibodies can be bound to a radioisotope, fluorescent colorimetric or chemilumiscent compound such as without limitation rhodamine or luciferin or an enzyme such as for example horseredish peroxidase. The cells can be also stained by immunohistochemical methods and visulaized than in a microscope, [00059] In another embodiment, the protein of the cells is extracted. The crude protein is separated for example on SDS PAGE and is than transferred onto nitrocellulose. The resulting Western Blots are screened with a primary antibody which is direct to ARTS and another antibody which bind to the primary antibody and to bound to a radioisotope, fluorescent colorimetric or chemilumiscent compound such as without limitation rhodamine or luciferin or an enzyme such as for example horseredish peroxidase. The Blots are than exposed to a film and the level of thickness or darkenss of the bands is detected and compared to the control level derived from healthy volunteers.

[00060] In another embodiment there is provided a method of diagnosing and/or predicting a hematological malignancy in a subject comprising the steps of: i. obtaining a sample from the subject; ii. obtahiing cDNA from said sample; iii. contacting said cDNA with a specific primer for open reading frame of the ARTS protein so as to form a complex; iv. amplifying said complex so as to obtain an amplified product; detecting said amlified product; and v. comparing said amplified product level to predetermined amplified product level derived from samples from healthy subjects. As was demonstrated in Example 3, some of the samples derived from leukemic patients are characterised by the absence of ARTS mRNA. In another embodiment, ARTS mRNA can be measured by southern or northern blot, dot blotting or in-situ hybridization with a labled probe based on the known sequences of ARTS. [00061] The term "RNA" refers to an oligonucleic in which the sugar is ribose, as opposed to deoxyribose in DNA. RNA is intended to include any nucleic acid, which can be entrapped by ribosomes and translated into protein. The term "mRNA" refers to messenger RNA.

[00062] RNA can be extracted from cells or tissues according to methods known in the art. In a preferred embodiment, RNA can be extracted from monolayers of mammalian cells grown in tissue culture, cells in suspension or from mammalian tissue. RNA can be extracted from such sources by, e.g., treating the cells with proteinase K in the presence of SDS. In another embodiment, RNA is extracted by organic solvents. In yet another embodiment, RNA is extracted by differential precipitation to separate high molecular weight RNA from other nucleic acids. RNA can also be extracted from a specific cellular compartment, e.g., nucleus or the cytoplasm. In such methods, tlie nucleus is either isolated for purification of RNA therefrom, or the nucleus is discarded for purification of cytoplasmic RNA. Further details regarding these and other RNA extraction protocols are set forth, e.g., in Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989). [00063] For instance, RNA can be extracted by a method using guanidium thiocyanate and purification of the RNA on a cesium chloride gradient. Accordingly, tissue or cells are lysed in the presence of guanidium thiocyanate and the cell lysate is loaded on a cushion of cesium chloride (CsCl) and centrifuged at high speed, such that the RNA is recovered in the pellet and the DNA is left in the supernatant after the centrifugation. The RNA can then be recovered by ethanol precipitation. This method is set forth in details, e.g., in Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989). [00064] hi order to prevent RNA from being degraded by nucleases, e.g., by RNAases, that may be present, the extraction of RNA, and reactions involving RNA are performed in "RNAase free conditions". Various methods known in the art can be used to maintain RNAase free conditions. For example, during RNA extraction, potent denaturing agents, such as guanidium hydrochloride and guanidium thiocyanate can be used to denature and thereby inactivate nucleases. Reducing agents, e.g., ? -mercaptoethanol, can also be used to inactivate ribonucleases. This combination of agents is particularly useful when isolating RNA from tissues rich in ribonucleases, e.g., pancreas (Chirgwin et al. (1979) Biochemistry 18:5294).

[00065] Othex reagents that can be added to a solution containing RNA to prevent degradation of the RNA include RNAase inhibitors, also referred to herein as "protein inhibitor of RNAases", e.g., Rnasin RTM which can be obtained, from

Promega Corp. (Madison, Wis.) (e.g., Cat #N2514). Protein inhibitors of RNAases are preferably not included during extraction of RNA using potent denaturing agents (since these will also denature the protein inhibitor of RNAases). However, it is preferable to include such protein inhibitors of RNAases during RNA extraction using more gentle methods of cell lysis and RNAse inhibitors are preferably present at all stages during the subsequent purification of RNA. [00066] et another reagent that can be added to a solution containing RNA to prevent degradation of the RNA include vanadyl-ribonucleoside complexes. The complexes formed between the oxovanadium IV ion and any of tlie four ribonucleosides are transition-state analogs that bind to many RNAases and inhibit their activity almost completely. The four vanadyl-ribonucleoside complexes are preferably added to intact cells and preferably used at a concentration of 10 mM during all stages or

RNA extraction and purification. Yet in another embodiment, macaloid is used to absorb RNAases. [00067] In one embodiment, cDNA is synthesize from the mRNA. This step is performed, without being limited, by Reverse Transcriptase Polymerization Chain Reaction (RT/PCR), which produce single stranded DNA molecule using RNA as a template.

The technique of reverse transcription can be used to amplify cDNA transcribed from mRNA encoding for secreted and transmembrane proteins. The method of RT/PCR is well known in the art (for example, see Watson and Fleming,) and can be performed as follows: Total cellular RNA is isolated by, for example, the standard guanidium isothiocyanate method and the total RNA is reverse transcribed. The reverse transcription method involves synthesis of DNA on a template of RNA using a reverse transcriptase enzyme and a 3' end primer. Typically, the primer contains an oligo(dT) sequence.

[00068] The cDNA is than contacting with a specific primer for open reading frame of the ARTS protein so as to form a complex.

[00069] The term "complex" is refer hereinabove in the specification and in the claims section to cDNA which is hybridizes to contacting with a specific primer for open reading frame of the ARTS protein. The complex is then amplified using the PCR method and the above described first and second specific primers. (Belyavsky et al, Nucl Acid Res 17:2919-2932, 1989; Krug and Berger, Methods in Enzymology,

Academic Press, N.Y., Vol.152, pp. 316-325, 1987 which are incorporated by reference). [00070] An oligonucleic acid molecule is "hybridizable" to another oligonucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the oligonucleic acid molecule can anneal to the other oligonucleic acid molecule under tlie appropriate conditions of temperature and solution ionic strength (see Sambrook et al.). The conditions of temperature and ionic strength determine the

"stringency" of the hybridization. The hybridization portion of the hybridizing nucleic acids is at least 15 nucleotides in length and at least 80% identical to the sequence if SEQ ID No:2. [00071] In one embodiment for preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55°C, can be used,

(e.g., 5 times SSC, 0.1% SDS, 0.25%) milk, and no formamide; or 30% formamide, 5 times SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5 times or 6 times SCC. High stringency hybridization conditions correspond to the highest Tm e.g., 50%> formamide, 5 times or 6 times SCC.

[00072] Hybridization requires that the two oligonucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing oligonucleic acids depends on the length of the oligonucleic acids and the degree of complementation, variables well known in the art. For hybridization with shorter oligonucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide deteimines its specificity (see Sambrook et al., 11.7-11.8). In one embodiment the length for a hybridizable oligonucleic is at least about 10 nucleotides. [00073] In a specific embodiment, the hybridization conditions are as described in the methods section: step 1- 94°C - 4 minutes, step 2- 40 cycles of 94°C for 30 seconds,

42°C for 60 seconds, 72°C for 20 seconds and step 3- 72°C for 5 minutes.

[00074] The polymerization is catalyzed by a DNA-Taq-Polymerase in the presence of four deoxynucleotide triphosphates, one of which is radioactive, or nucleotide analogs to produce double-stranded DNA molecules. The double strands are then separated by any denaturing method including physical, chemical or enzymatic. Commonly, the method of physical denaturation is used involving heating the oligonucleic, typically to temperatures from about 80°C to 105°C for times ranging from less than 1 to 10 minutes. The process is repeated for the desired number of cycles. [00075] The resulting amplified product is subjected to gel electrophoresis or other size separation techniques and may be detected by ethidum bromide staining (Sambrook, et al, 1989). [00076] Detection of the resulting bands is usually accomplished by exposure of the gel to

X-ray film (autoradiography). The amplified products that are obtained from the sample is compared to amplified products from healthy samples and from pateint with hematological malignancy, so as to detect the presence or the absence of hematological malignancy in the subject . [00077] It will be appreciated that the present invention is not limited by what has been described hereinabove and that numerous modifications, all of which fall within the scope of the present invention, exist.

EXAMPLES Experimental Procedures [00078] Immunohistochemistry: [00079] Immunohistochemistry was done using Histostain plus- Kit (Zymed) according to manifacturers instructions, with anti-ARTS specific primary antibodies. [00080] Bone marrow sampling:

[00081] Bone marrow sampels were taken from leukemic patients or people at diagnosis of other pathological states as a part of their medical assesment. Small portion of those sampels were used for ARTS staining.

[00082 !*] A DNA isolation: [00083] RNA was isolated using Tri-Reagent (MRC), DNA from blood samples was isolated using DNA isolation kit (Boeljxmger-Mannheim). [00084] Apoptosis assays: [00085] Detection of apoptotic cells was done using several metliods: Separation of apoptototic cells using MACS- magnetic beads system (Mitenyi- Biotec). Annexin-V conjugated to magnetic beads were incubated with cells transfected either with empty vector or with ARTS expression construct, -/+ treatment with ara-C. The apoptotic cells which were bound to the annexin-V conjugated magnetic beads were eluted from the beads according to manufacturer's instructions, and counted. Efficiency of transfection was evaluated in each experiment by counting the number of cells transfected with vector-GFP construct which were visualized using a fluorescence microscope provis AX70 (Olympus).

[00086] Caspase 3 activity assay (Boehringer-Mannheim) were used with lysates of transfected cells, according to the manufacturers' protocols. Results were read using FL-600 microplate fluorescence reader (Bio-Tek). Quantitation of apoptotic cells and immunolocalization of ARTS was performed using immunofluorescence detection; cells were blocked with 5% BS A in PBS for 30 minutes, then incubated with the indicated antibodies, followed by secondary antibodies conjugated with either fluorescein or rhodamine. After washes, a drop of DAPI containing mounting solution (Vector) was added to each slide. [00087] Transfection methods:

[00088] Transient transfections : Transfections of leukemic cell lines were done by electroporation -using Gene-Pulser (Bio-Rad).

Example 1 Detection of the presence of ARTS protein in bone marrow derived from acute leukemic patients and healthy controls

[00089] An immunohistochemistry method was used to deteπ ine the presence of ARTS protein in bone marrow of normal controls versus bone marrow from patients with leukemia by using specific antibody to ARTS. [00090] Samples from 12 patients with acute leukemia and 10 healthy controls were assessed for the presence of ARTS protein. The results clearly show the presence of ARTS protein in the cytoplasm of cells from the myeloid lineage in bone marrow samples derived from the healthy control. Cells of the erythroid lineage were not stained. In contrast, ARTS protein was not detected in 9 out of 12 the samples derived from the patients with acute leukemia.

Example 2 Detection of the presence of ARTS protein in bone marrow derived from acute lymphoblastic leukemic patients (ALL) and healthy control

[0009i]Bone marrow samples from 11 acute lymphoblastic leukemia (ALL) diagnosed patients, and a sample from one patient with acute promyelocytic leukemia (A.PL) were assessed for the presence of ARTS protein. In tl e lyphoblasts of 9 out of the

11 ALL samples, ARTS protein was not detected. The remaining two samples showed weak presence of ARTS in their nuclei. In the APL sample, ARTS was found in the cytoplasm of the myeloblast. In contrast, in lymphocytes from peripheral blood of 10 healthy controls, ARTS protein was detected in the cytoplasm of ~50 % of the lymphocytes (Fig 1A) in all assessed samples.

[00092]In addition, in a purification of enriched CD34 stem cells of the hematopoetic system, high expression of ARTS protein was detected (Fig ID), suggesting that the absence of ARTS in the leukemic cells is not related to their differentiation stage, but rather to their malignant state.

Example 3 ARTS RNA levels in patients with leukemia [00093] The percentage of leukemic patients which are not expressing ARTS RNA was assessed. Samples (blood or bone marrow) diagnosed with ALL were tested for the presence or absence of ARTS RNA, using specific primers for the ORF (open reading frame) of ARTS. So far, tested 16 samples of ALL patients, out of 7 (43.7%) did not express the ARTS RNA at all, 4 (25%) showed significantly reduced amount of ARTS RNA (estimated as at least 10 fold lower amount) ,as compared to 7 samples from healthy or non leukemic control subjects out of which 6 (85.7%>) that showed high ARTS RNA expression and one -negative. Moreover, when leukemic samples were tested for the presence of exon 6, the unique sequence which codes for the specific C terminus of ARTS, it was found that four samples (19%) out of 21 sample derived from ALL patients did not contain this exon. Other genetic aberrations were found in the leukemic patients, like for example, one patient with intact exon 6 that had no expression of ARTS RNA transcript, or another patient wliich was lacking both exon 5 and 6. These exciting results strongly suggest that loss of ARTS expression, for example through mutational inactivation, provides a selective advantage to transformed leukemic cells by making them less susceptible to apoptosis. These results also strongly attest to ARTS being a novel tumor suppressive gene in leukemia.

Example 4

Experiments in cell lines

Introduction of ARTS into leukemic cell lines causes pronounced apoptosis

[00094] To investigate the implications of the absence of ARTS protein in the leukemic patients, leukemic cells were transfected in vitro.

[00095] The experiment was performed in four different leukemic cell lines, ATI and K562 which contain ARTS protein and ALLl and HL-60 wherein the ARTS protein is absent. After transfecting the cells with ARTS expression vector, the ability of ARTS to promote apoptosis and to affect the responses of leukemic cells to pro-apoptotic stimuli was tested.

[00096] LLl cells which contains the Ph+ chromosome ,ATI containing the t(12:21) translocation, K562 containing the t(9:22) translocation and Hl-60, containing no known genetic aberration were studies. It was found that ARTS is not expressed in ALLl cells, both on RNA and protein level (Fig 2A,B). Moreover, transfection of ARTS into these cells caused a dramatic induction of apoptosis, which was even increased when the ARTS transfected cells were treated with ara -C, restoring apoptotic response to these transformed cells (Fig 2B,C). [00097]hi all our experiments cells were transfected with either empty vector or ARTS containing expression vector, and tested for apoptosis following treatment with ara-C. Apoptosis was measured by counting the apoptotic fraction of cells binding to annexin -V antibody -coated beads, separated by magnetic sorter (MACS), and by testing caspase 3 activity of the transfected cells (Roche).

[00098] In all tested leukemic cell lines, there was an induction of apoptosis following ARTS overexpression. [00099] Moreover, in both Hl-60 and K562 cells, the apoptotic effect of ARTS was as high as the ara-C effect on these cells (a 2.5 to 5 fold increase in apoptosis in HL-60 and K562 cells, respectively) (Fig 3 A,B). In all tested cells the effect of ARTS alone was at least as potent in inducing apoptosis as ara-c (Fig 2C, Fig 3A-C). In ALLl and ATI cells tlie effect of ARTS was even stronger than the effect of ara-C alone (Fig3A, Fig 2C. In all these cells ARTS was found to induce caspase 3 activity (results shown for HL-60 cells in Fig 3B, similar results found with other cell lines, are not presented). These results are summarized in figure 3. Each graph represents the average results of 3 experiments done in duplicates. In all cell lines, apoptosis was much more pronounced when the cells were both transfected with ARTS and treated with ara-C (see figures 3 A,B). [OOOioojTo optimize the apoptotic effect of ARTS on tlie leukemic cells, kinetics of apoptosis was measured 24 ,36 and 48 hours after transfection, and ara-C was added for two and four hours in each time point. In k562 cells, the maximum effect of ARTS with ara-C was seen after two hours of treatment, and after four hours there was a decrease in apoptosis (figure 3C). ooww/Immunohistochemistry stains with anti-ARTS specific antibody in ATI and K562 cells showed the presence of ARTS protein in both these cell lines. In addition, western blot analysis of extracts from these two cell lines confirmed the immunohistochemistry results (data not shown). Surprisingly, the HL-60 extract did not contain the ARTS protein. Further examination of HL-60 cells using RT-PCR, revealed that ARTS is also absent at the RNA level in these cells (figure 3D (bcr, genomic DNA and RNA were shown to be present in HL-60 cells, as positive control for the experiment). K562 and ATI, showed normal levels of ARTS RNA using RT-PCR.

References

Varmus HE and Lowell CA. 1994 Blood 83,5-9.

Thandla S, Apian PD. Molecular biology of acute lymphocytic leukemia. 1997 Seminars in Oncology 24,45-56. Hebert J, Cayuela JM, Berkeley J, et al: Candidate tumor-suppressor genes MTS1 (pi 6 ^lllk4) and MTS2 (pl5^mk4B) display frequent homozygous deletions in primary cells from T but not from B-cell lineage acute lymphoblastic leukemias. Blood 84:4038-4046,1994. Merlo A, Herman JG, Mao L, et al: 5' CpG island methylation is associated with transcriptional silencing of the tumor suppressor pl6/CDKN2/MTSl in human cancers. Nature Medicine 1 :686-692, 1995.

Aliuja HG, Jat PS, Foti A, et al: Abnonnalities of the retinoblastoma gene in the pathogenesis of acute leukemia. Blood 78:3259-3268,1991. Yeargin J, Cheng J, Haas M. Role of the p53 tumor suppressor gene in the pathogenesis and in the suppression of acute lymphoblastic T-cell leukemia. Leukemia 6:85S-91S,1992 (suppl).

Fenrick R, Wang L, Nip J, et al: TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transfonned cells while repressing the transcription of stromelysin-1. Molecular and cellular biology 20:5828-5839,2000. Sarit Larisch, Youngsuk Yi, Rona Lotan, Hedviga Kerner, Sarah Eimerl, W. Tony

Parks, Gottfried Yossi, Stephanie Birkey Reffey, Mark P. de Caestecker, David Danielpour ,Naomi Book-Melamed, Rina Timberg, Colin Duckett, Robert J. Lechleider, Hermann Steller, Joseph Orly, Seong-Jin Kim & Anita B. Roberts. ARTS, a novel mitochondrial septin-like protein, mediates apoptosis dependent on its P-loop motif. Nature Cell Biology. 2:915-921. 2000.

Claims (36)

What is claimed is:

1. A method of selectively inducing apoptosis in a cell which contains low basal of ARTS protein, comprising the steps of contacting the cell with a ligand, which is efficient to increase the expression of the ARTS protein, so as to increase the ARTS protein level or to activate ARTS molecule, thereby selectively inducing apoptosis in cells.

2. The method of claim 1, wherein the ligand is a peptide, a protein, an agonist, an antisense, an antibody, or a nucleic acids construct.

3. The method of claim 1, wherein said ARTS protein comprises the amino acid sequence as set forth in SEQ. ID. No. 1.

4. The method of claim 2, wherein said peptide or protein is a part of the amino acid sequence as set forth in SEQ. ID. No. 1.

5. The method of claim 1, wherein said DNA construct is a nucleic acid sequence encoding ARTS variants or ARTS from different species.

6. The method of claim 1, wherein said DNA construct is a cis acting gene which is integrated upstream to an endogenous ARTS gene and therefore induce increase in the level of ARTS protein.

7. The method of claim 1, further comprising contacting the cell with an amount of a cytotoxic drug.

8. The method of claim 1, wherein the cell is a leukemic cell, a breast cell, or a colon cancer cell.

9. A method of selectively inducing cell death in a subject in need comprising the steps of contacting cells with an effective amount of a ligand which specifically binds to the ARTS protein receptor or increases the expression of the ARTS protein so as to increase the amount of ARTS protein in the cell, or to activate the ARTS molecule, thereby selectively inducing cell death in a subject.

10. The method of claim 9, wherein the ligand is a peptide, a protein, an agonist, an antisense, an antibody, an anatgonist or a nucleic acids construct.

11. The method of claim 9, wherein said ARTS protein comprises the amino acid sequence as set forth in SEQ ID. No. 1.

12. The method of claim 10, wherein said peptide or protein is a part of the amino acid sequence as set forth in SEQ. ID. No. 1.

13. The method of claim 9, wherein said DNA construct is a nucleic acid sequence encoding ARTS variants or ARTS from different species.

14. The method of claim 9, wherein said DNA construct is a cis acting gene which is integrated upstream to an endogenous ARTS gene and therefore induce increase in the level of ARTS protein.

15. The method of claim 9, fiirther comprising contacting the cell with an amount of a cytotoxic drug.

16. The method of claim 9, wherein the cell is a leukemic cell or any other cancer cell

17. A pharmaceutical composition comprising an amount of a ligand of claim 1 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, further comprising an amount of a cell killing agent.

19. The pharmaceutical composition of claim 17, wherein the ligand is a peptide, a protein, an agonist, an antisense, an antibody, an anatgonist or a nucleic acids construct.

20. The pharmaceutical composition of claim 17, wherein said ARTS protein comprises the amino acid sequence as set forth in SEQ ID. No. 1.

21. The pharmaceutical composition of claim 19, wherein said peptide or protein is a part of the amino acid sequence as set forth in SEQ. ID. No. 1.

22. The pharmaceutical composition of claim 17, wherein the cell is a leukemic cell, a breast cell, or a colon cancer cell.

23. The method of claim 19, wherein said DNA construct is a nucleic acid sequence encoding ARTS variants or ARTS from different species.

24. A method for inhibiting pathologically cell proliferation comprising the step of immunizing a subject in need with a ligand which is efficient to increase the expression of the ARTS protein so as to increase the ARTS protein level or to activate proapoptotic inducer signal pathway in which ARTS participates, thereby selectively inducing apoptosis in cells.

25. The method of claim 24, wherein the ligand is a peptide, a protein, an agonist, an antisense, an antibody, an anatgonist or a nucleic acids construct.

26. The method of claim 24, wherein said ARTS protein comprises tlie amino acid sequence as set forth in SEQ ID. No. 1.

27. The method of claim 25, wherein said DNA construct is a nucleic acid sequence encoding ARTS variants or ARTS from different species.

28. The method of claim 25, wherein said peptide or protein is a part of the amino acid sequence as set forth in SEQ. ID. No. 1.

29. A method of predicting hematological malignancy in a subject comprising tlie steps of:

(a) obtaming a sample from the subject;

(b) obtaining cDNA from said sample;

(c) contacting said cDNA with a specific primer for an open reading frame of the ARTS protein so as to form a complex; (d) amplifying said complex so as to obtain an amplified product;

(e) detecting said amlified product; and

(f) comparing said amplified product level to predetermined amplified product level derived from samples from healthy subjects, tliereby diagnosing the presence of hematological malignancy in a subject

30. The method of claim 29, wherein the sample is a lympohocyte, a bone marrow cell or a stem cell.

31. The method of claim 29, wherein the hematological malignancy is an acute leukemia, a myeloid lineage, or an acute lymphoblastic leukemia.

32. The method of claim 29, wherein step e) further comprising the step of separating said amplified product.

33. The method of claim 32, wherein said step of separating further comprising the step of visualizing said amplified product.

34. A method of predicting hematological malignancy in a subject comprising the steps of: (a) obtaining a sample from the subject.

(b) contacting said sample with an antibody directed to an epitope on the ARTS protein, thereby to form an antigen-antibody complex; (c) detecting tlie an antigen-antibody complex level, thereby determining the level of said ARTS protein in said sample;

(d) comparing said level of said ARTS to predeteπnined ARTS protein level of samples derived from healthy subjects, thereby diagnosing tlie presence of heatological maliganncy in the subj ect.

35. The method of claim 33, wherein the sample is a lympohocyte, a bone marrow cell or a stem cell.

36. The method of claim 33, wherein the hematological malignancy is acute leukemia, myeloid lineage, or acute lymphoblastic leukemia or any other subtype of leukemia.