WO2006031985A2 - Procede d'identification de nouveaux modulateurs de la proliferation cellulaire et de la mort cellulaire - Google Patents

Procede d'identification de nouveaux modulateurs de la proliferation cellulaire et de la mort cellulaire Download PDF

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WO2006031985A2
WO2006031985A2 PCT/US2005/032991 US2005032991W WO2006031985A2 WO 2006031985 A2 WO2006031985 A2 WO 2006031985A2 US 2005032991 W US2005032991 W US 2005032991W WO 2006031985 A2 WO2006031985 A2 WO 2006031985A2
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cell
viability
cell growth
regulator
proliferation
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PCT/US2005/032991
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WO2006031985A3 (fr
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Josephine Harada
Sumit Chanda
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Irm Llc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • the present invention generally relates to methods for identifying modulators of proliferation and cell death, and to therapeutic applications of such modulators. More particularly, the invention pertains to cDNAs that modulate cell growth and apoptotic activities, and to methods of using such cDNAs to identify novel compounds that regulate cell proliferation.
  • Apoptosis is a highly conserved cell suicide program essential for development and tissue homeostasis of all metazoan organisms. Changes to the apoptotic pathway that prevent or delay normal cell turnover can be just as important in the pathogenesis of diseases as are abnormalities in the regulation of the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, cell death is similarly regulated under normal circumstances by the interaction of gene products that either prevent or induce cell death.
  • the invention provides methods for identifying an agent that modulate proliferation and viability of a cell.
  • the methods involve assaying a biological activity of a cell growth and viability regulator identified by the present inventors (one encoded by a polynucleotide listed in Tables 1-3) in the presence of a test agent to identify one or more modulating agents that modulate the biological activity of the regulator. This is followed by testing one or more of the modulating agents for ability to modulate proliferation and viability of the cell.
  • the cell growth and viability regulator stimulates cell growth and viability, and is encoded by a polynucleotide selected from the members listed in Table 1.
  • the modulating agents inhibit the biological activity of the regulator.
  • the cell growth and viability regulator is LRRKl, 2810429O05Rik, 5033414D02Rik, or Ankrd25.
  • the cell growth and viability regulator inhibits cell growth and viability, and is encoded by a polynucleotide selected from the members listed in Tables 2-3.
  • the modulating agents stimulate the biological activity of the regulator.
  • the testing comprises examining the modulating agents for ability to inhibit proliferation of a tumor cell.
  • the methods can further comprise testing modulating agents for ability to modulate proliferation of a non-tumor control cell.
  • the cell growth and viability regulator is an enzyme, and the biological activity assayed is its enzymatic activity.
  • the cell growth and viability regulator can be a kinase.
  • the biological activity assayed is cellular level of the cell growth and viability regulator.
  • the biological activity assayed is a binding activity of the cell growth and viability regulator, and the modulating agents identified are compounds that can bind to the regulator.
  • the invention provides methods for identifying a modulator of cell proliferation.
  • the methods entail (a) contacting a test agent with a cell growth and viability regulator encoded by a polynucleotide listed in Tables 1-3; (b) detecting a change in a biological activity of said cell growth and viability regulator in the presence of the test agent relative to the biological activity in the absence of the test agent; and (c) detecting a change of proliferation activity of a cell in the presence of the test agent identified in (b) relative to proliferation activity of the cell in the absence of the test agent.
  • the cell growth and viability regulator stimulates cell growth, and is encoded by a polynucleotide selected from the members listed in Table 1.
  • the cell growth and viability regulator is LRRKl, 2810429O05Rik, 5033414D02Rik, or Ankrd25.
  • the invention provides methods for modulating proliferation of a cell. These methods entail contacting the cell with a cell growth- modulating compound, thereby modulating proliferation of the cell.
  • the cell growth- modulating compound employed in such methods is identified by (a) assaying a biological activity of a cell growth and viability regulator encoded by a polynucleotide listed in Tables 1-3 in the presence of a test agent to identify one or more modulating agents that inhibit the biological activity of the regulator; and (b) testing one or more of the modulating agents for ability to modulate proliferation activity of the cell.
  • Some of these methods are directed to tumor cells.
  • the tumor cells can be present in a subject.
  • Figures IA- ID show validation of growth-activating and transforming abilities of hit cDNAs:
  • Figure IA shows that overexpression of LRRKl, Ankrd25,
  • Figure IB shows that LRRKl, Ankrd25, 5033414D02Rik,
  • FIG. 1 C shows that CEF infected with 2810429O05Rik-expressing
  • RCAS vector grown under serum rich agar displayed increased saturation density compared to uninfected or vector-infected controls.
  • Figure 1 D shows that CEF infected with LRJRKl , Ankrd25,
  • 5033414D02Rik, 2810429O05Rik, and Ptges-expressing avian retroviruses are capable of anchorage independent growth in soft agar suspension culture.
  • Figures 2A-2E show that 5033414D02Rik is required for cell growth and viability under growth factor-depleted conditions:
  • Figure 2A shows confirmation of target mRNA knockdown by
  • Figure 2B shows confirmation of target mRNA knockdown by
  • Figure 2C shows that 5033414D02Rik expression is required for growth under reduced serum conditions.
  • Figure 2D shows that abrogation of 5033414D02Rik expression at low serum conditions increases cell death.
  • Figure 2E shows that siRNA-mediated depletion of 5033414D02Rik activates primary effector caspase(s) -3 and -7.
  • the invention is predicated in part on the discoveries by the present inventors of cDNAs that positively or negatively regulate cell proliferation and cell death ("cell growth and viability regulators"). Specifically, the present inventors employed a genome functionalization platform towards a systematic identification of apoptotic and cell cycle regulatory factors in the human and mouse genomes. As detailed in the Examples below, the impact of approximately 7,000 individually arrayed cDNAs on the cellular proliferation and death of U2OS cells was assessed through high content imaging. The study yielded a spectrum of activities that both positively and negatively affected cell growth or viability under serum-depleted conditions.
  • cDNA hits of interest include those which caused a significant retardation in cell growth or induced a significant proportion of cells to undergo cell death both under serum withdrawal, and normal serum conditions ("negative cell growth and viability regulators"). Novel cDNA activities that conversely enhanced the proliferative potential of U2OS cells were also identified (“positive cell growth and viability regulators").
  • the inventors further characterized a few of the positive cell growth and viability regulators in a series of validation assays and confirmed their ability to positively affect cell growth in multiple cellular backgrounds, and additionally demonstrated their oncogenic potency in primary cells.
  • RNAi methodologies were employed to show that one of the genes is required for cell survival under growth factor-restricted conditions.
  • the present invention provides methods for identifying modulators of cell proliferation and cell death.
  • the invention also provides methods for modulating apoptosis and for treating various diseases or conditions mediated by abnormal cellular differentiation, e.g., tumors, in human or non-human subjects.
  • the following sections provide guidance for making and using the compositions of the invention, and for carrying out the methods of the invention.
  • agent includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” can be used interchangeably.
  • analog is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.
  • contacting has its normal meaning and refers to combining two or more molecules (e.g., a test agent and a polypeptide) or combining molecules and cells (e.g., a test agent and a cell).
  • Contacting can occur in vitro, e.g., combining two or more agents or combining a test agent and a cell or a cell lysate in a test tube or other container.
  • Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate.
  • a heterologous sequence or a “heterologous nucleic acid,” as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that, although being endogenous to the particular host cell, has been modified. Modification of the heterologous sequence can occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous nucleic acid.
  • homologous when referring to proteins and/or protein sequences indicates that they are derived, naturally or artificially, from a common ancestral protein or protein sequence.
  • nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology.
  • a "host cell,” as used herein, refers to a prokaryotic or eukaryotic cell that contains heterologous DNA that has been introduced into the cell by any means, e.g., transfection, electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like.
  • cell growth and viability regulator or "regulator of cell growth and viability” encompasses "cell growth and viability-modulating genes” and “cell growth and viability-modulating polypeptides.” It refers to genes or their encoded polypeptides that positively or negatively regulate proliferation and death of a cell (e.g., a tumor cell). It includes both positive and negative cell growth and viability regulators. Negative cell growth and viability regulators cause a significant retardation in cell growth or induce a significant proportion of cells to undergo cell death under serum withdrawal and/or normal serum conditions. Conversely, negative cell growth and viability regulators enhance proliferative potential of a cell (e.g., U2OS tumor cell). Examples of such modulators are shown in Tables 1-3.
  • sequence identity in the context of two nucleic acid sequences or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
  • a “comparison window” refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math.
  • the polypeptides herein are at least 70%, generally at least 75%, optionally at least 80%, 85%, 90%, 95% or 99% or more identical to a reference polypeptide, e.g., a cell growth and viability regulator encoded by a polynucleotide in Table 1, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters.
  • a reference polypeptide e.g., a cell growth and viability regulator encoded by a polynucleotide in Table 1, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters.
  • nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to a reference nucleic acid, e.g., a polynucleotide in Table 1, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters.
  • a reference nucleic acid e.g., a polynucleotide in Table 1, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters.
  • a "substantially identical" nucleic acid or amino acid sequence refers to a nucleic acid or amino acid sequence which comprises a sequence that has at least 90% sequence identity to a reference sequence using the programs described above (preferably BLAST) using standard parameters.
  • the sequence identity is preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)). Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.
  • modulate with respect to a biological activity of a reference protein or its fragment refers to a change in the expression level or other biological activities of the protein.
  • modulation may cause an increase or a decrease in expression level of the reference protein, enzymatic modification (e.g., phosphorylation) of the protein, binding characteristics (e.g., binding to a target polynucleotide), or any other biological, functional, or immunological properties of the reference protein.
  • the change in activity can arise from, for example, an increase or decrease in expression of one or more genes that encode the reference protein, the stability of an mRNA that encodes the protein, translation efficiency, or from a change in other biological activities of the reference protein.
  • the change can also be due to the activity of another molecule that modulates the reference protein (e.g., a kinase which phosphorylates the reference protein).
  • Modulation of a reference protein can be up-regulation (i.e., activation or stimulation) or down-regulation (i.e. inhibition or suppression).
  • the mode of action of a modulator of the reference protein can be direct, e.g., through binding to the protein or to genes encoding the protein, or indirect, e.g., through binding to and/or modifying (e.g., enzymatically) another molecule which otherwise modulates the reference protein.
  • subject includes mammals, especially humans. It also encompasses other non-human animals such as cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys. These subjects are all amenable for treatment with cell growth-modulating compounds that can be identified in accordance with the present invention.
  • a "variant" of a reference molecule refers to a molecule substantially similar in structure and biological activity to either the entire reference molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical.
  • cDNAs were incubated with a non-liposomal transfection reagent (Fugene ⁇ , Roche Applied Science, Indianapolis, IN) and pZs-Green-Cl, a construct containing the cytomegalovirus immediate early gene promoter driving a human codon-optimized variant of the Zoanthus sp. green fluorescent protein (Clontech).
  • pZs-Green-Cl was used as a marker for transfected cells.
  • U2OS osteosarcoma cells were then introduced into each well to complete the transfection process. After 36 hours of incubation at 37°C and 5% CO 2 , normal culturing media (DMEM+10% fetal calf serum) was exchanged for phenol red-free DMEM+0.5% FCS (i.e. serum starved conditions). Media was supplemented with Hoechst 33342 (Molecular Probes, Eugene, OR) to permit visualization of cell nuclei. Cell images were acquired at this time using the Q3DM EIDAQlOO TM (Q3DM, Sorrento Valley, CA) high content imaging system affixed with a 10X, 0.5 NA objective (read 1).
  • Q3DM EIDAQlOO TM Q3DM, Sorrento Valley, CA
  • CytoshopTM an integrated image analysis and data visualization solution which generates numerical descriptions of cellular assay data from image sets. CytoshopTM first locates each cell in an image by segmenting images into regions of individual cells. These regions of pixels are then used for the extraction of an assortment of cellular measurements including cell number, size, shape, and intensity and distribution of labeled molecules. Measurements are subsequently collated into a relational database (image table) which may then be analyzed according to desired measurements. CytoshopTM further provides a data visualization tool set to enable rapid plotting and sorting of cell data according to specified metrics, and permits the user to access images of cell subpopulations characterized by specific measurement ranges.
  • mice 2810429O05Rik from Table 1 and also mouse prostaglandin E synthase Ptges (accession number BC024960) which demonstrated potent growth-inducing abilities in the primary screen were evaluated in additional cellular assays. As shown in Example 2 below, all these gene activities were found to augment cellular proliferation rates in primary cells, and further demonstrated tumorigenic potential in multiple transformation assay types. A further analysis of the requirement for 5033414D02Rik in cell growth under reduced serum conditions led to its characterization as a putative arbiter of cell survival.
  • test agents are first assayed for their ability to modulate a biological activity of a cell growth and viability regulator encoded by the cDNAs shown in Tables 1-3 ("the first assay step”). Modulating agents thus identified are then subject to further screening for ability to modulate cell growth or cell death, typically in the presence of the cell growth and viability regulator ("the second testing step”). Depending on the cell growth and viability regulator employed in the method, modulation of different biological activities of the cell growth and viability regulator can be assayed in the first step. For example, a test agent can be assayed for binding to the cell growth and viability regulator.
  • the test agent can be assayed for activity to modulate expression of the cell growth and viability regulator, e.g., transcription or translation.
  • the test agent can also be assayed for activities in modulating the cellular level or stability of the cell growth and viability regulator, e.g., post-translational modification or proteolysis.
  • the cell growth and viability regulator has a known biological or enzymatic function (e.g., kinase activity or protease activity)
  • the biological activity monitored in the first screening step can be the specific biochemical or enzymatic activity of the cell growth and viability regulator.
  • enzymatic activity of a kinase e.g., LRRKl encoded by BC005408 in Table 1
  • a phosphatase e.g., encoded by BC004666 in Table 1
  • the cell growth and viability regulator is LRRKl
  • test agents are first screened for ability to modulate the kinase's activity in phosphorylating a substrate.
  • the substrate to be used in the screening can be a molecule known to be enzymatically modified by the enzyme (e.g., a kinase), or a molecule that can be easily identified from candidate substrates for a given class of enzymes.
  • a kinase substrates are available in the art. See, e.g., www.emdbiosciences.com; and www.proteinkinase.de.
  • a suitable substrate of a kinase can be screened for in high throughput format.
  • substrates of a kinase can be identified using the Kinase-Glo® luminescent kinase assay (Promega) or other kinase substrate screening kits (e.g., developed by Cell Signaling Technology, Beverly, Massachusetts).
  • test agents can be screened for ability to either up-regulate or down-regulate a biological activity of the cell growth and viability regulator in the first assay step. Once test agents that modulate the cell growth and viability regulator are identified, they are typically further tested for ability to modulate cell growth or cell death (e.g., apoptosis). This further testing step is often needed to confirm that their modulatory effect on the cell growth and viability regulator would indeed lead to enhanced cell proliferation or cell death.
  • test agent which inhibits a biological activity of a positive cell growth and viability regulator (e.g., one shown in Table 1) needs to be further tested in order to confirm that such modulation can result in suppressed or reduced proliferation of a cell harboring the cell growth and viability regulator (e.g., a tumor cell such as U2OS cell).
  • a test agent which modulates (e.g., stimulating) a biological activity of a negative cell growth and viability regulator e.g., one shown in Table 2 or 3 can also be further tested to confirm that it can lead to cell growth suppression or reduction.
  • test agents are screened to identify compounds that specifically inhibit growth of tumor cells.
  • the modulating agents identified from the first screening step are further screened for lack of significant effect on the growth of a normal non-tumor control cell. This additional step could ensure that the antitumor agents identified with the screening methods of the invention are specific for tumor cells.
  • both the first assaying step and the second testing step either an intact cell growth and viability regulator, or a fragment thereof, may be employed.
  • Analogs or functional derivatives of the cell growth and viability regulator could also be used in the screening.
  • the fragments or analogs that can be employed in these assays usually retain one or more of the biological activities of the cell growth and viability regulator (e.g., kinase activity if the cell growth and viability regulator employed in the first assaying step is a kinase). Fusion proteins containing such fragments or analogs can also be used for the screening of test agents.
  • Functional derivatives of a cell growth and viability regulator usually have amino acid deletions and/or insertions and/or substitutions while maintaining one or more of the bioactivities and therefore can also be used in practicing the screening methods of the present invention.
  • a functional derivative can be prepared from a cell growth and viability regulator by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art.
  • the functional derivative can be produced by recombinant DNA technology by expressing only fragments of a cell growth and viability regulator that retain one or more of their bioactivities.
  • Test agents or compounds that can be screened with methods of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, polypeptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Some test agents are synthetic molecules, and others natural molecules.
  • Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds.
  • Combinatorial libraries can be produced for many types of compound that can be synthesized in a step-by-step fashion.
  • Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642.
  • Peptide libraries can also be generated by phage display methods (see, e.g., Devlin, WO 91/18980).
  • Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field.
  • Known pharmacological agents can be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidif ⁇ cation to produce structural analogs.
  • Combinatorial libraries of peptides or other compounds can be fully randomized, with no sequence preferences or constants at any position.
  • the library can be biased, i.e., some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, or to purines.
  • test agents can be naturally occurring proteins or their fragments.
  • test agents can be obtained from a natural source, e.g., a cell or tissue lysate.
  • Libraries of polypeptide agents can also be prepared, e.g., from a cDNA library commercially available or generated with routine methods.
  • the test agents can also be peptides, e.g., peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about -15 being particularly preferred.
  • the peptides can be digests of naturally occurring proteins, random peptides, or "biased" random peptides.
  • the test agents are polypeptides or proteins.
  • the test agents can also be nucleic acids. Nucleic acid test agents can be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be similarly used as described above for proteins.
  • the test agents are small molecule organic compounds, e.g., chemical compounds with a molecular weight of not more than about 1,000.
  • high throughput assays are adapted and used to screen for such small molecules.
  • combinatorial libraries of small molecule test agents as described above can be readily employed to screen for small molecule compound modulators of cell growth.
  • a number of assays are available for such screening, e.g., as described in Schultz (1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) MoI Divers. 3:61-70; Fernandes (1998) Curr Opin Chem Biol 2:597-603; and Sittampalam (1997) Curr Opin Chem Biol 1 :384-91.
  • Libraries of test agents to be screened with the claimed methods can also be generated based on structural studies of the cell growth and viability regulators discussed above or their fragments. Such structural studies allow the identification of test agents that are more likely to bind to the cell growth and viability regulators.
  • the three- dimensional structures of the cell growth and viability regulators can be studied in a number of ways, e.g., crystal structure and molecular modeling. Methods of studying protein structures using x-ray crystallography are well known in the literature. See Physical Bio-chemistry, Van Holde, K. E. (Prentice-Hall, New Jersey 1971), pp. 221-239, and Physical Chemistry with Applications to the Life Sciences, D. Eisenberg & D. C.
  • Modulators of the present invention also include antibodies that specifically bind to a cell growth and viability regulator in Tables 1-3.
  • Such antibodies can be monoclonal or polyclonal.
  • Such antibodies can be generated using methods well known in the art.
  • the production of non-human monoclonal antibodies, e.g., murine or rat can be accomplished by, for example, immunizing the animal with a cell growth and viability regulator in Tables 1-3 or its fragment (See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York).
  • Such an immunogen can be obtained from a natural source, by peptides synthesis or by recombinant expression.
  • Humanized forms of mouse antibodies can be generated by linking the
  • Human antibodies against a cell growth and viability regulator can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991). Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using a cell growth and viability regulator or its fragment.
  • test agents are first screened for ability to modulate a biological activity of a cell growth and viability regulator identified by the present inventors.
  • a number of assay systems can be employed in this screening step.
  • the screening can utilize an in vitro assay system or a cell-based assay system.
  • test agents can be screened for binding to a cell growth and viability regulator, altering expression level of the cell growth and viability regulator, or modulating other biological activities of the cell growth and viability regulator.
  • binding of a test agent to a cell growth and viability regulator is determined in the first screening step. Binding of test agents to a cell growth and viability regulator can be assayed by a number of methods including e.g., labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.), and the like. See, e.g., U.S.
  • test agent can be identified by detecting a direct binding to the cell growth and viability regulator, e.g., co- immunoprecipitation with the cell growth and viability regulator by an antibody directed to the cell growth and viability regulator.
  • the test agent can also be identified by detecting a signal that indicates that the agent binds to the cell growth and viability regulator, e.g., fluorescence quenching or FRET.
  • Competition assays provide a suitable format for identifying test agents that specifically bind to a cell growth and viability regulator.
  • test agents are screened in competition with a compound already known to bind to the cell growth and viability regulator.
  • the known binding compound can be a synthetic compound. It can also be an antibody, which specifically recognizes the cell growth and viability regulator, e.g., a monoclonal antibody directed against the cell growth and viability regulator. If the test agent inhibits binding of the compound known to bind the cell growth and viability regulator, then the test agent also binds the cell growth and viability regulator.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay see Stahli et al., Methods in Enzymology 9:242-253, 1983
  • solid phase direct biotin-avidin EIA see Kirkland et al., J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see, Harlow and Lane, "Antibodies, A Laboratory Manual,” Cold Spring Harbor Press, 3 rd ed., 2000); solid phase direct label RIA using 125 I label (see Morel et al., MoI. Immunol. 25(1):7-15, 1988); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552, 1990); and direct labeled RJA (Moldenhauer et al., Scand. J. Immunol. 32:77-82, 1990).
  • such an assay involves the use of purified polypeptide bound to a solid surface or cells bearing either of these, an unlabelled test agent and a labeled reference compound.
  • Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test agent.
  • the test agent is present in excess.
  • Modulating agents identified by competition assay include agents binding to the same epitope as the reference compound and agents binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference compound for steric hindrance to occur.
  • a competing agent is present in excess, it will inhibit specific binding of a reference compound to a common target polypeptide by at least 50 or 75%.
  • the screening assays can be either in insoluble or soluble formats.
  • One example of the insoluble assays is to immobilize a cell growth and viability regulator or its fragment onto a solid phase matrix.
  • the solid phase matrix is then put in contact with test agents, for an interval sufficient to allow the test agents to bind. After washing away any unbound material from the solid phase matrix, the presence of the agent bound to the solid phase allows identification of the agent.
  • the methods can further include the step of eluting the bound agent from the solid phase matrix, thereby isolating the agent.
  • the test agents are bound to the solid matrix and the cell growth and viability regulator is then added.
  • Soluble assays include some of the combinatory libraries screening methods described above. Under the soluble assay formats, neither the test agents nor the cell growth and viability regulator are bound to a solid support. Binding of a cell growth and viability regulator or fragment thereof to a test agent can be determined by, e.g., changes in fluorescence of either the cell growth and viability regulator or the test agents, or both. Fluorescence may be intrinsic or conferred by labeling either component with a fluorophor.
  • either the cell growth and viability regulator, the test agent, or a third molecule can be provided as labeled entities, i.e., covalently attached or linked to a detectable label or group, or cross-linkable group, to facilitate identification, detection and quantification of the polypeptide in a given situation.
  • detectable groups can comprise a detectable polypeptide group, e.g., an assayable enzyme or antibody epitope.
  • the detectable group can be selected from a variety of other detectable groups or labels, such as radiolabels (e.g., 125 1, 32 P, 35 S) or a chemiluminescent or fluorescent group.
  • the detectable group can be a substrate, cofactor, inhibitor or affinity ligand.
  • Binding of a test agent to a cell growth and viability regulator provides an indication that the agent can be a modulator of the cell growth and viability regulator. It also suggests that the agent may modulate cell growth and cell death through, e.g., binding to and modulating the cell growth and viability regulator.
  • a test agent that binds to a cell growth and viability regulator can be further tested for ability to modulate proliferative activity of a cell, e.g., a tumor cell (i.e., in the second testing step outlined above).
  • a test agent that binds to a cell growth and viability regulator can be further examined to determine whether it modulates another biological activity (e.g., an enzymatic activity) of the cell growth and viability regulator.
  • another biological activity e.g., an enzymatic activity
  • the existence, nature, and extent of such modulation can be tested by an activity assay.
  • Such an activity assay can confirm that the test agent binding to the cell growth and viability regulator indeed modulates the cell growth and viability regulator. More often, such activity assays can be used independently to identify test agents that modulate activities of a cell growth and viability regulator (i.e., without first assaying their ability to bind to the cell growth and viability regulator).
  • the methods involve adding a test agent to a sample containing a cell growth and viability regulator in the presence or absence of other molecules or reagents which are necessary to test a biological activity of the cell growth and viability regulator (e.g., enzymatic activity if the cell growth and viability regulator is an enzyme), and determining an alteration in the biological activity of the cell growth and viability regulator.
  • a biological activity of the cell growth and viability regulator e.g., enzymatic activity if the cell growth and viability regulator is an enzyme
  • Methods for monitoring various enzymatic activities are well known in the art. For example, assays for monitoring a number of protein kinase activities are described in, e.g., Chedid et al., J. Immunol. 147: 867-73, 1991 ; Kontny et al., Eur J . Pharmacol.
  • the activity assays also encompass in vitro screening and in vivo screening for alterations in expression level of the cell growth and viability regulator. These assays can be performed using methods well known and routinely practiced in the art, e.g., Samrbook et al., supra; and Ausubel et al., supra. D. Testing modulating agents for ability to regulate cell growth and viability
  • a modulating agent Once a modulating agent has been identified to bind to a cell growth and viability regulator and/or to modulate a biological activity (including expression level) of the cell growth and viability regulator, it can be further tested for ability to modulate cell proliferation and cell death.
  • this screening step is performed in the presence of the cell growth and viability regulator on which the modulating agent acts.
  • this screening step is performed in vivo using cells that endogenously express the cell growth modulating polypeptide.
  • effect of the modulating agents on the proliferative activity of a cell that does not express the cell growth and viability regulator can also be examined.
  • the screening methods are directed to identifying compounds that specifically inhibit proliferation of a tumor cell, effect of the modulating agents on the growth of a non-tumor normal cell can also be examined as a control.
  • a tumor cell line preferably a cell line from that specific tumor
  • a detectable marker e.g., GFP
  • GFP GFP
  • the cell growth and viability regulator against which the modulating agents are identified in the first screening step can be either expressed endogenously by the cell or expressed from second expression vector.
  • the cell growth and viability regulator e.g., encoded by a mouse gene
  • the cell line e.g., a human cell line
  • a second vector expressing the polypeptide can be introduced into the cell.
  • tumor cell lines include human glioblastoma cell line U373 (ATCC); melanoma cell line SK-MEL-2; ovarian cancer cell line OVCAR-4; leukemia lines HL60 and RPMI-8226; lung cancer cell lines NCI-H322M and NCI-H460; colon lines COLO 205 and HCC-2998; brain tumor lines SF-539 and SNB-75; and breast cancer lines MCF7 and HS 578T (Monks et al., Anticancer Drug Des 12: 533-541, 1997; and Boyd and Paull, Drug Dev Res 34: 91-109, 1995).
  • U373 ATCC
  • melanoma cell line SK-MEL-2 ovarian cancer cell line OVCAR-4
  • leukemia lines HL60 and RPMI-8226 lung cancer cell lines NCI-H322M and NCI-H460
  • colon lines COLO 205 and HCC-2998 colon lines COLO 205 and HCC-2998
  • Non-tumor cell lines include, e.g., human embryonic kidney cell line (HEK293); human umbilical vein endothelial cell line (HUVEC); epithelial cell line MCF-IOA (Soule et al., Cancer Res. 50: 6075-6086, 1990); colon cell line (CCD-I8C0) and ovarian cell line (NOV-31 (Hirasawa et al., Cancer Research 62, 1696-1701, March 15, 2002).
  • ATCC provides many tumor/normal cell line pairs that are used to elucidate the underlying causes of cancers. They can also be employed to screen modulating agents of the present invention to identify selective anti-tumor agents.
  • tumor/normal cell line pairs include non-small cell lung cancer cell line (ATCC No. CCL-256) and normal peripheral blood cell line ATCC No. CCL-256.1; adenocarcinoma cell line ATCC No. CRL-5868 and normal peripheral blood cell line ATCC No. CRL-5957; malignant melanoma cell line ATCC No. CRL- 1974 and normal cell line ATCC No. CRL- 1980; basal cell carcinoma cell line ATCC No. CRL-7762 and normal skin cell line ATCC No. CRL-7761 ; colorectal adenocarcinoma cell line ATCC No. CCL-228 and normal lymph node cell line ATCC No. CCL-227; and giant cell sarcoma cell line ATCC No. CRL-7554 and normal bond cell line ATCC No. CRL-7553. Any of these cell line pairs can be used to screen the modulating agents for compounds that selectively inhibit proliferation of tumor cells.
  • Modulation of cell growth by a test agent can be examined with a number of methods known to the skilled artisans. For example, many fluorescence-based microplate assays can be employed to monitor effect of compounds on cell proliferation and cell death. See, e.g., Jones et al., J Immunol Methods. 254:85-98, 2001; Stagoj et al., Biotechniques. 36:380-2, 2004; Blaheta et al., J Immunol Methods 142: 199-206, 1991; Lewinsohn et al., J Immunol Methods. 110:93-100, 1988; and the methods detailed in the Examples below.
  • test agents are examined by high content imaging using cell harboring an assayable marker, e.g., a marker protein such as GFP expressed from a vector, as demonstrated in the Examples below.
  • cell proliferation can be monitored with a luminescence based viability assay, e.g., CellTiter-GloTM Luminescent Assay as detailed in the Examples below.
  • a luminescence based viability assay e.g., CellTiter-GloTM Luminescent Assay as detailed in the Examples below.
  • cells can be plated onto 96-well, 384-well, or 1536-well plates prior to administering the test compounds.
  • absorbance of each well is measured with a content imaging system or a microplate reader (e.g., an Acquest Plate Reader or a Labsystems reader).
  • a content imaging system or a microplate reader e.g., an Acquest Plate Reader or a Labsystems reader.
  • the image sets or absorbance values are then analyzed and translated into quantitative data , corresponding to the number of live or viable cells in each well, as exemplified in the Examples below.
  • the tumor cells are first administered with the modulating compounds identified in the first screening step.
  • Ability of the compounds to inhibit tumor cell growth is then examined.
  • cytotoxic activity of the compounds on a tumor cell can be monitored by measuring the ICso value (i.e., the concentration of a compound which causes 50% cell growth inhibition) of each of the modulating compounds.
  • an antitumor compound identified from this screening step will have an IC 50 value less than l ⁇ M on one or more of tumor cell lines. More preferably, the IC 50 value of antitumor compounds identified in accordance with the present invention is less than 25OnM.
  • the antitumor agents have an IC 50 value of less than 5OnM, less than 1OnM on at least one of the above described tumor cell lines. Most preferably, the antitumor agents obtained from this screening step will have an IC5 0 value that is less than InM.
  • Methods of determining IC50 values of compounds in inhibiting cultured cell lines are well known in the art. These were described in the art, e.g., Remington, The Science and Practice of Pharmacy, Mack Publishing Co., 20 th ed., 2000; and US Patent No. 6,552,027.
  • the modulating agents identified in the first screening step are further tested for cell growth-modulating activity on non-tumor control cells. This additional step is performed to identify compounds that selectively inhibit proliferation of tumor cells while having little or no effect on growth of normal cells. Any of the above noted non-tumor cell lines may be used in this test step.
  • the present invention provides novel methods and compositions for modulating cell proliferation and cell death.
  • the methods and compositions of the present invention find therapeutic applications in treating various clinical conditions or disease states that are linked to abnormal cell proliferation, e.g., various forms of tumors. Modulation of cell growth activities is also useful for preventing or modulating the development of such diseases or disorders in a subject suspected of being, or known to be, prone to such diseases or disorders.
  • Prevention or treatment of various forms of tumors, benign or malignant can be achieved by targeting one or more of the cell growth and viability regulators listed in Tables 1-3 (especially human genes).
  • a subject in need of treatment is administered with a pharmaceutical composition comprising one or more of the antitumor compounds identified in accordance with the present invention.
  • a great number of diseases and conditions are amenable to treatment with methods and compositions of the present invention.
  • tumors that can be treated with methods and compositions of the present invention include both solid tumors or metastatic tumors.
  • Cancers that can be treated by the compositions and methods of the invention include cardiac cancer (e.g., sarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma); lung cancer (e.g., bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma); various gastrointestinal cancer (e.g., cancers of esophagus, stomach, pancreas, small bowel, and large bowel); genitourinary tract cancer (e.g., kidney, bladder and urethra, prostate, testis; liver cancer (e.g., hepatoma, he
  • Disease states other than cancer which can be treated by the methods and compositions also include restenosis, autoimmune disease, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures such as surgery, angioplasty, and the like.
  • cells not in a hyperproliferation state are the subject of treatment.
  • the cells may be proliferating "normally", but proliferation enhancement may be desired.
  • cells may be in a "normal” state, but proliferation modulation may be desired to enhance a crop by directly enhancing growth of a crop, or by inhibiting the growth of a plant or organism which adversely affects the crop.
  • therapeutic applications of the present invention include treatment of individuals or agricultural crops with any one of these disorders or states.
  • modulators of cell proliferation e.g., inhibitors
  • modulators of cell proliferation can be directly administered under sterile conditions to the subject to be treated. They can be administered alone or as the active ingredient of a pharmaceutical composition.
  • Therapeutic composition of the present invention can be combined with or used in association with other therapeutic agents. For example, a subject may be treated concurrently with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment.
  • chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyure
  • compositions of the present invention typically comprise at least one active ingredient together with one or more acceptable carriers thereof.
  • Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered (e.g., nucleic acid, protein, modulatory compounds or transduced cell), as well as by the particular method used to administer the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject.
  • This carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral, sublingual, rectal, nasal, or parenteral.
  • the antitumor compound can be complexed with carrier proteins such as ovalbumin or serum albumin prior to their administration in order to enhance stability or pharmacological properties.
  • compositions of the present invention include syrup, water, isotonic saline solution, 5% dextrose in water or buffered sodium or ammonium acetate solution, oils, glycerin, alcohols, flavoring agents, preservatives, coloring agents starches, sugars, diluents, granulating agents, lubricants, and binders, among others.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • concentration of therapeutically active compound in the formulation may vary from about 0.1-100% by weight.
  • Therapeutic formulations are prepared by any methods well known in the art of pharmacy.
  • the therapeutic formulations can be delivered by any effective means that can be used for treatment.
  • the suitable means include oral, rectal, vaginal, nasal, pulmonary administration, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) infusion into the bloodstream.
  • parenteral administration antitumor agents of the present invention may be formulated in a variety of ways.
  • Aqueous solutions of the modulators may be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art.
  • the compounds of the present invention may also be administered encapsulated in liposomes.
  • compositions may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension.
  • the hydrophobic layer generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such a diacetylphosphate, stearylamine, or phosphatide acid, and/or other materials of a hydrophobic nature.
  • the therapeutic formulations can conveniently be presented in unit dosage form and administered in a suitable therapeutic dose.
  • a suitable therapeutic dose can be determined by any of the well known methods such as clinical studies on mammalian species to determine maximum tolerable dose and on normal human subjects to determine safe dosage. Except under certain circumstances when higher dosages may be required, the preferred dosage of an antitumor agent of the present invention usually lies within the range of from about 0.001 to about 1000 mg, more usually from about 0.01 to about 500 mg per day.
  • the preferred dosage and mode of administration of an antitumor agent can vary for different subjects, depending upon factors that can be individually reviewed by the treating physician, such as the condition or conditions to be treated, the choice of composition to be administered, including the particular antitumor agent, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the chosen route of administration.
  • the quantity of an antitumor agent administered is the smallest dosage which effectively and reliably prevents or minimizes the conditions of the subjects. Therefore, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention.
  • Mammalian Gene Collection were carried out essentially as described in Huang et al., Proc Natl Acad Sci U S A, 101 : 3456-61, 2004; and Chanda et al., Proc Natl Acad Sci U S A, 100: 12153-8, 2003.
  • This library consisting of approximately 7,000 full length mammalian cDNAs in the mammalian expression vector pCMV-Sport 6, was spotted into 384-well plates such that each well contained 62.5 ng of an individual cDNA of known identity.
  • cDNAs were incubated with a non-liposomal transfection reagent (Fugene ⁇ , Roche Applied Sciences, Indianapolis, IN) and 20 ng of pZs-Green-Cl, a construct containing the cytomegalovirus immediate early promoter driving a human codon-optimized variant of the Zoanthus green fluorescent protein (Clontech, Palo Alto, CA).
  • pZs-Green-Cl a construct containing the cytomegalovirus immediate early promoter driving a human codon-optimized variant of the Zoanthus green fluorescent protein (Clontech, Palo Alto, CA).
  • U2OS human osteosarcoma cells ATCC, Manassas, VA
  • pZs-Green- Cl was used to mark the co-transfected population.
  • CytoshopTM a set of software modules used for generating quantitative cellular assay data from image sets in an automated, high-throughput fashion.
  • CytoshopTM systematically locates every cell over a selected range of wells, producing an image database (image table) that is the basis for creating cell-by-cell measurements over images, wells, plates, or plate sets. CytoshopTM further enables accurate measurement of facets present in a focused set of cells in a mixed population.
  • CellTiter-GloTM reagent was then added to cells according to the manufacturer's specifications, and luminescence output measured using the Acquest Plate Reader (LJL Biosystems, Sunnyvale, CA). The relative luminescence recorded in each well, which correlates with the number of metabolically active cells, was normalized to pcDNA ⁇ (Invitrogen, Carlsbad, CA) vector-transfected control wells.
  • Viral supernatants were harvested from stable lines and used to infect fresh CEF. After serially passaging the infected cells three times, growth kinetic analyses were performed using the Beckman-Coulter/Q3DM EIDAQ 100TM quantitative high- throughput fluorescence microscopy system. In brief, cells were seeded at a density of ⁇ 500 cells per well in 384-well plate format, and maintained in culture for 5 days at 37 ° C in Ham FlO cloning media. Cells were visualized by staining with Hoechst 33342, and growth monitored by imaging daily using a 1OX, 0.5 NA objective, and Cohu CCD camera. Image analysis was performed using CytoshopTM, and the effect of stably expressing select growth-activator cDNA clones quantitated by gating image data on Hoechst staining intensity.
  • Focus formation and soft agar assays in primary chicken embryo fibroblasts were performed essentially as described in Bister et al., Virology 82: 431-48, 1977; and Bos et al., Genes Dev 4: 1677-87, 1990. Briefly, CEFs were seeded at a density of 5 x 10 cells/well of a 6-well plate and infected with activator-expressing viruses. Cells were then overlaid with agar medium and maintained in culture for 3-4 weeks. Foci were subsequently visualized by staining with crystal violet. Soft agar assays were performed as described in Bister et al.
  • a synthetic siRNA SMARTpool comprised of a pool of 4 independent
  • siRNA transfection efficiencies were determined by performing parallel transfections with a Cy3-conjugated siRNA targeting the GL2 firefly luciferase gene (Dharmacon).
  • SMARTpool, activator-cDNA clone 5033414D02Rik was FLAG-epitope tagged and transfected in the presence or absence of the indicated SMARTpool/siRNA into NIH3T3 mouse embryo fibroblasts (ATCC).
  • NIH3T3 mouse embryo fibroblasts ATCC.
  • Approximately 2 x 10 5 NIH3T3 cells were seeded into 6-well plates and either mock-transfected, or transfected with an siRNA targeting 5033414D02Rik or GL2. After incubating for 24 hours, medium was replaced, and cells were transfected with FLAG-5033414D02Rik using TransYT-3T3 reagent (Minis) according to the manufacturer's instructions.
  • Target mRNA knockdown was further validated using semi-quantitative
  • RNAs from siRNA-transfected cells were harvested using the RNeasy mini kit (Qiagen).
  • cDNA was then produced using Superscript IIITM reverse transcriptase (Invitrogen) with 1 ug total RNA as template. Ensuing cDNA products were then used in PCR reactions to amplify cytoplasmic beta actin and the RIKEN cDNA 5033414D02 gene.
  • Histograms evaluating total nuclear intensity and event frequency for representative wells were used to define fluorescence boundaries for sub-Gl, Gl, and S/G2/M populations. Cell fractions in each group were determined for all wells, and replicate wells averaged for each condition.
  • siRNA-transfected NIH3T3 cells were also evaluated for the induction of apoptosis using the Apo-ONETM Homogeneous Caspase-3/7 Assay kit (Promega) according to the manufacturer's instructions.
  • the Apo-ONE assay kit provides a pro- fluorescent substrate with an optimized bi-functional cell lysis and activity buffer to measure the activities of caspase-3 and -7.
  • Cells treated with 1 ⁇ g/ml recombinant TRAIL (Calbiochem, San Diego, CA) for 4 hours were analyzed in parallel to serve as a positive control (Aza-Blanc et al., MoI Cell, 12: 627-37, 2003).
  • Example 2 Identifying Novel Cell Growth and Viability Regulators [00101] To identify novel gene activities that may participate in regulating cell growth and viability, a genome-scale gain of function screen examining cell growth and survival under low serum conditions was performed. To this end, an arrayed cDNA expression library comprised of -7,000 full length mammalian cDNAs was introduced together with a green fluorescent protein (GFP) expression construct into U2OS human osteosarcoma cells by means of high-throughput transfection. Changes in cell growth conferred by the overexpression of individual cDNAs under serum-starved conditions were monitored across the cDNA matrix using a quantitative high throughput fluorescence microscopy platform. Imaging was performed on live cells in an automated fashion, and performed as a two-step process, prior (read 1) and subsequent (read 2) to withdrawal of serum from the growth medium.
  • GFP green fluorescent protein
  • cDNAs that, under reduced serum conditions, (i) permitted continued cell growth to a level > 3 standard deviations from the trimmed experimental mean (z-score) or (ii) sensitized cells to cell death by a z-score of ⁇ -3.25. Also identified are cDNAs which induce rapid cell death under normal serum conditions. Examples of these different classes of cDNAs are respectively shown in Tables 1-3 above.
  • Example 3 Characterization of Exemplary Cell Growth and Viability Regulators [00104] Five cDNAs which exhibited a robust growth-inductive phenotype were selected for further study. These included several gene products of unknown function including LRRKl, Ankrd25, 5033414D02Rik, and 2810429O05Rik, as well as prostaglandin E synthase (Ptges). Ptges is an inducible enzyme that functions downstream of cyclooxygenase-2 (COX-2) in the prostaglandin E2 (PGE2) biosynthetic pathway. Clinical, genetic, and biochemical studies have suggested that the elevation of PGE2 by the COX2-dependent pathway plays a critical role in the development of colorectal cancer.
  • COX-2 cyclooxygenase-2
  • PGE2 prostaglandin E2
  • RNA interference methodologies Given the ability of 5033414D02Rik overexpression to confer an increase in proliferative rates in both immortalized and primary cells, we further investigated its necessity for cell growth in NIH3T3 cells using RNA interference methodologies. Knock down of 5033414D02Rik expression was achieved utilizing synthetic small-interfering RNA oligonucleotides. As shown in Figure 2 A, siRNA to 5033414D02Rik specifically reduced target mRNA expression. To further validate the ability of 5033414D02Rik siRNA to reduce target protein levels, the expression of FLAG- epitope tagged 5033414D02Rik was evaluated by immunoblot analysis. As seen in Figure 2B, FLAG-5033414D02Rik levels were specifically reduced in the presence of the corresponding siRNA. 5033414D02Rik transcript and protein levels were unaffected by GL2 control siRNA.
  • NIH3T3 cells were transfected with 5033414D02Rik or control siRNA, and cell cycle distribution was measured by high content image analysis at 24 hour intervals post transfection. Treatment with 5033414D02Rik siRNA caused for a prominent sub-Gl fraction to appear at later times post transfection (Figure 2D).

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Abstract

La présente invention concerne de nouveaux ADNc codant pour des régulateurs de croissance et de viabilité cellulaire. Cette invention concerne aussi des procédés d'utilisation de ces ADNc dans la recherche de composés qui modulent la croissance et la viabilité cellulaire. Ces procédés consistent d'abord à rechercher des composés test de modulateurs de régulateurs de croissance et de viabilité cellulaire qui sont codés par ces ADNc, puis à rechercher à nouveau les agents modulateurs identifiés des modulateurs de régulateurs de croissance et de viabilité cellulaire. Cette invention concerne aussi des procédés et des compositions pharmaceutiques permettant de traiter des maladies et des états (par exemple des tumeurs) associés à une prolifération cellulaire anormale.
PCT/US2005/032991 2004-09-15 2005-09-15 Procede d'identification de nouveaux modulateurs de la proliferation cellulaire et de la mort cellulaire WO2006031985A2 (fr)

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REN M. ET AL.: 'Effects of Mutant Ran/TC4 Proteins on Cell Cycle Progression' MOLECULAR AND CELLULAR BIOLOGY vol. 14, no. 6, June 1994, pages 4216 - 4224, XP003004428 *

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