METHODS OF IDENTIFYING INDUCIBLE INHIBITORS OF TRANSFORMATION PROGRESSION

Abstract: This invention provides a method of identifying a progression suppressor gene which comprises: a) introducing DNA containing a progression gene into a population of transformed cells; b) treating the introduced transformed cells from step (a) so as to induce the expression of at least one progression suppressor gene and cause a subpopulation of the cells to revert to a characteristic phenotype; c) selecting cells which express the progression suppressor gene and exhibit the characteristic phenotype; d) isolating mRNA from the cells from step (c); e) comparing the mRNA so obtained with mRNA obtained from the uninduced transformed cells as to identify mRNA expressed only by the cells from step (c); and f) isolating the gene encoding such mRNA so as to thereby identify the progression suppressor gene.

Description: METHODS OF IDENTIFYING INDϋCIBLE INHIBITORS OF TRANSFORMATION PROGRESSION The invention disclosed herein was made with Government support under NIH Grant No. CA35675 and CA43201 from the Department of Health and Human Services. Accordingly, the U.S. Government has certain rights in this invention. Background of the Invention Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims. Protein kinase C (PKC) is a key component in signal transduction in eucaryotic cells and when specific PKC isoforms are over-expressed in immortal mammalian cells, they can induce transformation-associated properties. In the present study, applicants demonstrated that a cloned PKC β χ gene can induce an enhanced expression of the transformed phenotype in type 5 adenovirus (Ad5)- transformed rat embryo (RE) cells (clone Ell) , a process termed transformation progression. Ell cells expressing the PKC & gene, clone Bl/PKC, produce PKC β : mRNA and display enhanced PKC enzymatic activity and binding of [ 3 H]-phorbol-12, 13-dibutyrate (PDBu) to cell surface phorbol ester receptors. Bl/PKC cells grow with increased efficiency in agar in comparison with parental Ell cells, and anchorage-independence is further enhanced in both cell types by addition of the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) . A single-exposure of Bl/PKC cells to 5-azacytidine (AZA) , followed by growth in the absence of this demethylating agent, results in Bl/PKC-AZA clones which display a stable reversion of the progression phenotype to that of the unprogressed parental Ell clone. Loss of the progression phenotype corresponds with a reduction in PKC fii-induced biochemical and cellular changes. In contrast, progression-suppression does not involve an alteration in expression of the Ad5 transforming genes, E1A and E1B, or the endogenous PKCe gene. TPA cannot induce the progression phenotype in Bl/PKC-AZA cells, but it can reversibly induce an increase in the transcriptional rate and steady-state mRNA levels of PKC β x and c-jun and it increases AP-1 DNA-binding. These results indicate that the PKC B-, gene can serve as a transformation progression- inducing gene in rat embryo cells previously transformed by Ad5 and progression may be mediated by the inactivation by methylation of an AZA-sensitive "progression suppressor gene(s)". The suppression process in Bl/PKC cells is independent of expression of the Ad5-transforming genes but correlates directly with the reduced expression of the transfected PKC β x gene in AZA-treated Bl/PKC cells. The carcinogenic process is a consequence of a series of sequential changes in the phenotype of a cell resulting in the acquisition of new properties or a further elaboration of transformation-associated traits by the evolving tumor cell (reviewed in Fisher, 1984; Nowell, 1986; Nicholson, 1987; Weinstein, 1988; Bishop, 1991). The mechanism underlying tumor cell progression remains to be defined. Possible factors contributing to transformation progression include: activation of cellular genes that promote the cancer cell phenotype, i.e., oncogenes; loss or inactivation of cellular genes which function as inhibitors of the cancer cell phenotype, i.e., tumor suppressor genes; and/or combinations of these genetic changes in the same tumor cell (reviewed in Weinberg, 1985; Bishop, 1987, 1991; Sager, 1989; Marshall, 1991). Previous studies indicate that transformation of secondary rat embryo cells by Ad5 is often a sequential process resulting in the acquisition of and further elaboration of specific phenotypes by the transformed cell (Fisher et al., 1979 a,b,c). Progression in the Ad5-transformation model is characterized by the development of enhanced anchorage- independence and tumorigenic potential (as indicated by reduced latency time for tumor formation in nude mice) by progressed cells (Babiss et al., 1985). The progression phenotype in Ad5-transformed rat embryo cells can be induced by selection for growth in agar or tumor formation in a nude mouse (Fisher et al., 1979a,b,c), referred to as spontaneous-progression, or by transfection with the Ha-ras (T24) oncogene, referred to as oncogene mediated-progression (Duigou et al., 1989). Both spontaneous and oncogene mediated-progression are stable cellular traits that have remained undiminished in Ad5-transformed rat embryo cells even after > 100- passages in onolayer cultures. However, a single- treatment with the demethylating agent 5-azacytidine (AZA) results in a reversion in transformation progression in the majority of treated cellular clones (Babiss et al., 1985; Duigou et al., 1989; Reddy et al., 1993) . These observations, and additional gene expression studies (Duigou et al., 1991; Reddy et al., 1993) , suggest that progression is a reversible process that may be affected by the state of methylation of putative progression suppressor cellular genes. AZA can also induce both C3H 10T 1/2 and 3T3 cells to differentiate into muscle, fat and cartilage (Taylor & Jones, 1979; Jones 1985). This AZA-induced determination process in C3H 10 1/2 cells correlates with a resistance to transformation by 3-methylcholanthrene (Harrington et al., 1988). Similarly, non-terminal differentiation in 3T3 T cells induces resistance to both UV irradiation and 4-nitroquinoline oxide (Scott et al., 1989). Non¬ terminal differentiation also induces SV40-transformed 3T3 cells to repress expression of the large T antigen and to revert to a non-transformed state (Scott et al. , 1989) . It is not presently known if the anticancer activities demonstrated by AZA-treated and non-terminally differentiated mouse cells is analogous to the progression suppression phenotype induced by AZA in Ad5- transformed RE cells. The tumor promoting diterpene phorbol ester, TPA can enhance expression of the transformed phenotype in Ad5- transformed rat embryo cells (Fisher et al., 1979 a,b,c). Since the major cellular receptor for TPA is PKC, these results suggest that alterations in expression of this enzyme may contribute to the process of transformation progression. PKC is a multigene family of serine/threonine kinases that plays a pivotal role in regulating signal transduction processes in mammalian cells (Ohno et al., 1991; Nishizuka, 1992). Biochemical and molecular cloning studies indicate that the PKC family consists of at least ten subspecies, including the Ca 2+ -dependent conventional PKCs (cPKC: α, βl, B2, γ) and the Ca 2+ -independent novel PKCs (nPKC: Σ, S , ς, " /L, θ, λ) (Coussens, et al.,1986; Knopf, et al., 1986; Ono, et al.. 1986, 1987, 1988; Parker, et al., 1986; Housey, et al., 1987; Ohno, et al., 1988; Backer, et al., 1991; Osada, et al., 1990; Nishizuka, 1992). The retention of different PKC isoforms during evolution, the subtle differences in enzymatic properties and substrate specificities of the PKC isoforms and the different tissue distribution of the PKC isoforms suggests a potential role for specific PKC isoenzymes in regulating defined biological functions (Ohno, et al., 1991; Nishizuka, 1992). Although extensively investigated, the specific functions of the various PKC subtypes still remain to be established (Ohno, et al.,1991; Nishizuka, 1988, 1992). One approach to study the effects of the various PKC isoforms on cellular physiology, although perhaps somewhat artificial because of the high levels of expression, is to over- express specific enzyme subtypes in target cells and analyze their effect on cellular phenotype (Housey, et al., 1988; Persons, et al., 1988., Krauss, et al., 1989; Megidish & Mazurek, 1989; Borner et al., 1991, 1992 a,b; Su,et al., 1991, 1992; Watanabe, et al., 1992; Mischak, et al. , 1993) . Over-expression of the PKC β 1 gene in specific immortal cell lines, such as Rat 6, CREF and C3H10T1/2, can induce morphological, biological and biochemical changes often observed in oncogene transformed cells (Housey, et al., 1988; Krauss, et al., 1989, Su, et al. , 1992). In addition, cells over-expressing PKC B-, are more sensitive to transformation by Ha-ras (Hsiao, et al. , 1989) and Ad5 Su, et al., 1991). In the present study, applicants have determined if the PKC β x gene could cooperate with Ad5 transforming genes in Ad5 transformed RE cells resulting in an induction of the progression phenotype. Such an interaction was observed and TPA further enhanced anchorage independence in PKC &-_ expressing Ad5- transformed RE cells. As was previously found with spontaneous and oncogene-mediated progression (Babiss, et al., 1985; Duigou, et al., 1989; 1991), a single 24 hour exposure to the demethylating agent 5-azacytidine (AZA) resulted in revertant clones which displayed a stable extinction of the transformation progression phenotype. In PKC β : expressing Ell cells, AZA treatment was associated with a decrease in PKC β : RNA transcriptional rates and steady state mRNA and a reduction in PKC enzymatic activity [ 3 H]-PDBu binding to cell surface phorbol ester receptors and anchorage independence. These observations indicate that the state of progression in Ad5-transformed RE cells, resulting spontaneously or by increased expression of Ha-ras or PKC β lf is mediated by a cellular progression-suppressor-gene(s) whose expression may be regulated by DNA methylation (Babiss, et al., 1985; Reddy, et al., 1993) Biιmwnrγ of the Invention This invention provides a method of identifying a progression suppressor gene which comprises: a) introducing DNA containing a progression gene into a population of transformed cells; b) treating the introduced transformed cells from step (a) so as to induce the expression of at least one progression suppressor gene and cause a subpopulation of the cells to revert to a characteristic phenotype; c) selecting cells which express the progression suppressor gene and exhibit the characteristic phenotype; d) isolating mRNA from the cells from step (c) ; e) comparing the mRNA so obtained with mRNA obtained from the uninduced transformed cells as to identify mRNA expressed only by the cells from step (c) ; and f) isolating the gene encoding such mRNA so as to thereby identify the progression suppressor gene. This invention further provides a method of identifying a progression suppressor gene which suppresses expression of long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C βj ^ gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor gene and cause a subpopulation of the cell to selectively suppress the expression of the long terminal repeat regulated protein kinase C & x gene; and c) selecting cells which express the progression suppressor gene; d) isolating mRNA from the cells from step (c) ; e) comparing the mRNA so obtained with mRNA obtained from the uninduced transformed cells so as to identify mRNA only expressed by the cells from step (c) ; and f) isolating the gene encoding such mRNa so as to thereby identify the progression suppressor gene. This invention provides a method of selecting a molecule capable of inhibiting the function of a long terminal repeat of a retrovirus comprising: a) introducing DNA containing the long terminal repeat regulated protein kinase C β : gene into a population of transformed cells; b) selecting cells from step a) which expresses protein kinase C 5 λ gene; c) treating the selected cells with an amount of the molecule effective to inhibit the function of the long terminal repeat; and c) determining the level of expression of the protein kinase C β : gene, the decrease in the level of expression indicating that the molecule is capable of inhibiting the function of the long terminal repeat. This invention provides a method of selecting a molecule capable of activating the function of a long terminal repeat of a retrovirus comprising: a) introducing DNA containing the long terminal repeat regulated protein kinase C β χ gene into a population of transformed cells; b) selecting cells which expresses the protein kinase C β : gene; c) contacting the selected cells from step b) with an amount of the molecule effective to activate the function of the long terminal repeat; c) determining the level of expression of the protein kinase C &■, gene, the increase in the level of expression indicating that the molecule is capable of activating the ftmction of the long terminal repeat. This invention provides a method of identifying a gene which inhibits the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C & x gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor genes and suppress expression of the long terminal repeat regulated protein kinase C &-. gene; and c) isolating the gene which inhibits the function of the long terminal repeat. This invention provides a method of identifying a protein factor capable of inhibiting the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C β : gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor gene and to suppress expression of the long terminal repeat regulated protein kinase C β x gene; c) isolating and lysing the nuclei from the cell from step (b) to produce an extract; d) contacting the extract with the long terminal repeat; and e) isolating the protein factor which binds with the terminal repeat, thereby isolating the factor capable of inhibiting the function of the long terminal repeat. This invention provides a method of identifying a gene which activates the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C β x gene into a transformed cell; b) treating the cells of step (a) so as to induce the expression of at least one progression suppressor gene and suppress expression of the long terminal repeat regulated protein kinase C &■, gene; c) treating the induced cell from step b) with an amount of protein kinase C activating compound or an inhibitor of serine or threonine specific protein phosphatase effective to activate the long terminal repeat; and d) isolating a gene which activates the long terminal repeat. This invention provides a method of identifying a protein factor capable of activating the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C B l gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor gene and suppress expression of the long terminal repeat regulated protein kinase C β x gene; c) treating the induced cell from step b) with an amount of a protein kinase C activating compound or an inhibitor of serine or threonine specific protein phosphatase effective to activate the long terminal repeat; d) isolating and lysing the nuclei from the cell from step c to produce an extract; e) contacting the extract with DNA containing the long terminal repeat; and f) isolating the protein factor which binds with the terminal repeat, thereby identifying the factor capable of activating the function of the long terminal repeat. Brief Description of the Figures Figure 1 Analysis of steady state levels of PKC B l t neomycin resistance (NEO) and GAPDH mRNA expression in Ell and Ell subclones. Northern analysis using 15μg of total cytoplasmic mRNA from logarithmically growing cells was performed as previously described (Babiss, et al., 1983; Su & Fisher, 1992) . Cell lines include Ell, Ell-NMT (nude mouse tumor-derived Ell subclone) , the PKC β x expressing Ell subclone Bl/PKC, single-cell subclones of Bl/PKC (Bl/PKC-cll, B1/PKC-C12, B1/PKC-C17 and Bl/PKC-cllO) and single-cell subclones of Bl/PKC cells treated for 24 hours prior to isolation with lOμM AZA (Bl/PKC-AZAIIb, Bl/PKC-AZAIIe , Bl/PKC-AZAIIg, Bl/PKC/AZAIVa and Bl/PKC-AZAVIIa) . Fiσure 2 Analysis of steady state levels of Ad5, E1A, Ad5 E1B and GAPDH mRNA expression in Ell and Ell subclones. Northern analysis using 15μg of total cytoplasmic mRNA from logarithmically growing cells was performed as previously described (Babiss, et al., 1983; Su & Fisher, 1992). Cell line description can be found in the legend to Figure 1. Fjqur? ? Effect of TPA on expression of PKC β 1# PKCe, c-jun and GAPDH in Bl/PKC-AZAIIg cells. Cells were treated with 100 ng ml " 1 of TPA for 15, 30, 60 or 120 minutes prior to RNA isolation and analysis by Northern hybridization. For comparison, mRNA samples from untreated Ell-NMT, Ell, Bl/PKC, Bl/PKC-cll, Bl/PKC-cllO and Bl/PKC-AZAIIg were analyzed. Fiσure 4 Effect of continuous and transient TPA treatment on expression of PKC B-, and GAPDH mRNA in Ell and Ell subclones. RNAs were isolated from untreated Ell and Ell subclones or subclones grown for 24 hours in the presence of 100 ng/ml "1 of TPA (+ TPA) , or subclones grown for 24 hours in the presence of 100 ng ml "1 of TPA followed by growth for one week in the absence of TPA (± TPA) . Figure 5 Analysis of gene transcription rates in Ell, Ell-NMT, Bl/PKC and Bl/PKC-AZAIIg cells grown in the absence or presence of TPA. Nuclei from 2 x IO 6 cells were isolated from each cell line shown (either untreated or treated with 100 ng ml "1 of TPA for one hour) and nuclear RNA was labeled in vitro and subsequently hybridized to denatured DNA probes on nylon membranes. For each hybridization reaction, an equal number of total counts representing a similar number of cell nuclei was used, so that the comparative rates of transcription could be obtained. The gene probes used are shown in the order presented in the box. Figure 6 Effect of TPA on binding of nuclear proteins from Ell, Bl/PKC and Bl/PKC- AZAIIg to AP-1 and MECA DNA sequences. Gel-shift analysis of complexes formed between an AP-1 oligonucleotide probe (5'- CCAAACAGGATATATGAGTCATGCAGTTC-3') (Seq. ID No. 1) (a) or a MECA oligonucleotide probe (5 , CCAAACAGGATATCTGTGGTAAGCAGTTCC-3 ' ) (Seq. ID No. 2) (b) and nuclear extracts prepared from untreated or TPA (24 hours) treated cells. DNA lane refers to the oligonucleotide probe without the addition of nuclear extracts. Addition of a 100- fold excess of the respective competitor DNA sequence in combination with nuclear extracts eliminated binding to the AP-1 and MECA oligonucleotide probes (data not shown) . Figure 7 Effect of TPA on steady state levels of PKC β lf Ad5 E1A, Ad5 E1B and GAPDH mRNA in Bl/PKC and B1/PKC-PB5 cells. Cells were treated with 50 or 100 ng ml "1 for 1 or 4 hours prior to RNA isolation and analysis by Northern hybridization. Bl/PKC is a PKC Bι expressing subclone. B1/PKC-PB5 is a single-cell subclone of Bl/PKC cells treated for 4 days with 4mM phenylbutyrate and then grown for two weeks in medium lacking PB prior to isolation. Detailed Description of the Invention Throughout this application, the following standard abbreviations are used to indicate specific nucleotides: C = cytosine A = adenosine T = thymidine G = guanosine This invention provides a method of identifying a progression suppressor gene which comprises: a) introducing DNA containing a progression gene into a population of transformed cells; b) treating the introduced transformed cells from step (a) so as to induce the expression of at least one progression suppressor gene and cause a subpopulation of the cells to revert to a characteristic phenotype; c) selecting cells which express the progression suppressor gene and exhibit the characteristic phenotype; d) isolating mRNA from the cells from step (c) ; e) comparing the mRNA so obtained with mRNA obtained from the uninduced transformed cells as to identify mRNA expressed only by the cells from step (c) ; and f) isolating the gene encoding such mRNA so as to thereby identify the progression suppressor gene. This invention provides a method of identifying a progression suppressor gene which suppresses the progression phenotype of a transformed cell comprising: a) introducing a progression gene into a transformed cell; b) selectively suppressing the progression phenotype by inducing expression of at least one progression suppressor gene; and c) isolating the progression suppressor gene. The progression suppressor gene may be identified by subtractive hybridization or differential display. Subtractive hybridization techniques are well-known in the art. Differential display for differential expression cloning was also known (Sager et al. 1993; Liang & Pardee, 1992; Sun, et al. , 1994). The progression phenotype may be selectively suppressed by chemicals such as azacytidine or phenyl butyrate. Such treatment will induce expression of at least one progression suppressor gene and as a consequence, the progression phenotype is suppressed. This invention provides the progression suppressor gene identified by the above described method. This identified gene would be useful in further study of regulation of the suppression and progression of the transformed phenotype. The progression suppressor gene could prove useful for diagnostic applications in tumor staging and for therapeutic uses. The progression suppressor gene described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptide by a variety of recombinant techniques. The gene is useful for generating new cloning and expression vectors, transformed and transfected procaryotic and eucaryotic host cells, and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products. This invention further provides the progression suppressor gene operatively linked to a promoter of RNA transcription. This invention provides a vector which comprises the identifed progression suppressor gene. Suitable vectors comprise, but are not limited to, a plasmid or a virus. These vectors may be transformed into suitable host cell to form a host cell vector system for the production of a polypeptide of the identified progression suppressor gene. This invention provides a virus comprising the identified progression suppressor gene. This invention provides a polypeptide encoded by the identified progression suppressor gene. This invention further provides a method of identifying a progression suppressor gene which suppresses expression of long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C B-, gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor gene and cause a subpopulation of the cell to selectively suppress the expression of the long terminal repeat regulated protein kinase C B-. gene; and c) selecting cells which express the progression suppressor gene; d) isolating mRNA from the cells from step (c) ; e) comparing the mRNA so obtained with mRNA obtained from the uninduced transformed eels so as to identify mRNA only expressed by the cells from step (c) ; and f) isolating the gene encoding such mRNA so as to thereby identify the progression suppressor gene. This invention provides a method of identifying a progression suppressor gene which suppresses expression of long terminal repeat of a retrovirus comprising: a) introducing a long terminal repeat regulated protein kinase C B-. gene into a transformed cell; b) selectively suppressing expression of the long terminal repeat regulated protein kinase C B l gene by inducing the expression of at least one progression suppressor gene; and c) isolating the progression suppressor gene. In an embodiment, the retrovirus is a Moloney leukemia virus long terminal repeat. In another embodiment, the retrovirus is a human immunodeficiency virus. In one embodiment of this invention, the selective suppression of expression of the long terminal repeat regulated protein kinase C β : gene by inducing the expression of at least one progression suppressor gene was achieved by treatment with 5-azacytidine or phenyl butyrate. In another embodiment, the selective suppression is achieved by treatment with a DNA demethylating agent. DNA demethlating agents are well- known in the art. This invention further provides that the progression suppressor gene may be isolated by subtractive hybridization or differential display. This invention also provides the progression suppressor gene identified by the above-described method. This invention provides the progression suppression gene operatively linked to a promoter of RNA transcription. This invention provides a vector which comprises the identified progression suppressor gene. This invention also provides a virus comprising the identified progression suppressor gene. This invention further provides a polypeptide encoded by the identified progression suppressor gene. This invention provides a method of selecting a molecule capable of inhibiting the function of a long terminal repeat of a retrovirus comprising: a) introducing DNA containing the long terminal repeat regulated protein kinase C βi gene into a population of transformed cells; b) selecting cells from step a) which expresses protein kinase C β : gene; c) treating the selected cells with an amount of the molecule effective to inhibit the function of the long terminal repeat; and c) determining the level of expression of the protein kinase C &-, gene, the decrease in the level of expression indicating that the molecule is capable of inhibiting the function of the long terminal repeat. This invention also provides a method of selecting a molecule capable of inhibiting the function of a long terminal repeat of a retrovirus comprising: a) introducing the long terminal repeat regulated protein kinase C β x gene into a transformed cell; b) contacting the cell from step a) with an amount of the molecule effective to inhibit the function of the long terminal repeat; and c) determining the level of expression of the protein kinase C β : gene, the decrease in the level of expression indicating that the molecule is capable of inhibiting the function of the long terminal repeat. In an embodiment of the above method, the retrovirus is Moloney leukemia virus. In another embodiment, the retrovirus is human immunodeficiency virus. This invention provides a method of selecting a molecule capable of activating the function of a long terminal repeat of a retrovirus comprising: a)introducing the long terminal repeat regulated protein kinase C β : gene into a transformed cell; b) contacting the cell from step a) with an amount of the molecule effective to activate the function of the long terminal repeat; c) determining the level of expression of the protein kinase C B-, gene, the increase in the level of expression indicating that the molecule is capable of activating the function of the long terminal repeat. This invention provides a method of identifying a gene which inhibits the function of the long terminal repeat of a retrovirus comprising:a)introducing a long terminal repeat regulated protein kinase C β : gene into a transformed cell;b)selectively suppressing expression of the long terminal repeat regulated protein kinase C B-. gene in the introduced cell of step a) by inducing the expression of at least one progression suppressor gene; and c) isolating the gene which inhibits the function of the long terminal repeat. In an embodiment, the gene is isolated by subtractive hybridization or differential display. This invention further provides the gene identified by the above-described method. This invention also provides the identified gene operatively linked to a promoter of RNA transcription. This invention further provides a vector which comprises the above gene. This invention provides a virus comprising the above gene. This invention also provides a polypeptide encoded by the above gene. This invention provides a method of identifying a protein factor capable of inhibiting the function of the long terminal repeat of a retrovirus comprising: a)introducing a long terminal repeat regulated protein kinase C β : gene into a transformed cell; b) selectively suppressing expression of the long terminal repeat regulated protein kinase C B-, gene in the introduced cell of step a) by inducing the expression of at least one progression suppressor gene; c) isolating and lysing the nuclei from the cell from step b to produce an extract; d) contacting the extract with the long terminal repeat; and e) isolating the protein factor which binds with the terminal repeat, thereby isolating the factor capable of inhibiting the function of the long terminal repeat. This invention provides the protein factor identified by the above-described method. This invention provides a method of identifying a gene which activates the function of the long terminal repeat of a retrovirus comprising: a) introducing a long terminal repeat regulated protein kinase C β : gene into a transformed cell; b) selectively suppressing expression of the long terminal repeat regulated protein kinase C β x gene by inducing the expression of at least one progression suppressor gene; c) treating the induced cell from step b) with a protein kinase C activating compound or an inhibitor of serine or threonine specific protein phosphatase to activate the long terminal repeat; and d) isolating a gene which activates the long terminal repeat. In an embodiment, the gene is isolated by subtractive hybridization or differential display. In an embodiment, the protein kinase C activating compound is a tumor promoting diterpene phorbol ester. In an another embodiment, the protein kinase C activating compound is a synthetic activator of protein kinase C. An example of this compound is ADMB and another example is DHL In another embodiment, an inhibitor of serine or threonine specific protein phosphatase is used to activate the protein kinase C β x . An example of this serine or threonine specific protein phosphatase is calyculin. Another example is okadaic acid. This invention further provides the gene identified by the above-described method. This invention also provides the identified gene operatively linked to a promoter of RNA transcription. This invention further provides a vector which comprises the above gene. This invention provides a virus comprising the above gene. This invention also provides a polypeptide encoded by the above gene. This invention provides a method of selecting a molecule capable of activating the function of a long terminal repeat of a retrovirus comprising: a) introducing DNA containing the long terminal repeat regulated protein kinase C β x gene into a population of transformed cells; b) selecting cells which expresses the protein kinase C B l gene; c) contacting the selected cells from step b) with an amount of the molecule effective to activate the function of the long terminal repeat; c) determining the level of expression of the protein kinase C B-. gene, the increase in the level of expression indicating that the molecule is capable of activating the function of the long terminal repeat. This invention provides a method of identifying a gene which inhibits the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C B-, gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor genes and suppress expression of the long terminal repeat regulated protein kinase C β x gene; and c) isolating the gene which inhibits the function of the long terminal repeat. This invention provides a method of identifying a protein factor capable of inhibiting the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C β x gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor gene and to suppress expression of the long terminal repeat regulated protein kinase C B-, gene; c) isolating and lysing the nuclei from the cell from step (b) to produce an extract; d) contacting the extract with the long terminal repeat; and e) isolating the protein factor which binds with the terminal repeat, thereby isolating the factor capable of inhibiting the function of the long terminal repeat. This invention provides a method of identifying a gene which activates the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C B-, gene into a transformed cell; b) treating the cells of step (a) so as to induce the expression of at least one progression suppressor gene and suppress expression of the long terminal repeat regulated protein kinase C β x gene; c) treating the induced cell from step b) with an amount of protein kinase C activating compound or an inhibitor of serine or threonine specific protein phosphatase effective to activate the long terminal repeat; and d) isolating a gene which activates the long terminal repeat. This invention provides a method of identifying a protein factor capable of activating the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C B-. gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor gene and suppress expression of the long terminal repeat regulated protein kinase C β : gene; c) treating the induced cell from step b) with an amount of a protein kinase C activating compound or an inhibitor of serine or threonine specific protein phosphatase effective to activate the long terminal repeat; d) isolating and lysing the nuclei from the cell from step c to produce an extract; e) contacting the extract with DNA containing the long terminal repeat; and f) isolating the protein factor which binds with the terminal repeat, thereby identifying the factor capable of activating the function of the long terminal repeat. This invention also provides a method of identifying a protein factor capable of activating the function of the long terminal repeat of a retrovirus comprising: a) introducing a long terminal repeat regulated protein kinase C β x gene into a transformed cell; b) selectively suppressing expression of the long terminal repeat regulated protein kinase C B-. gene in the introduced cell of step a) by inducing the expression of at least one progression suppressor gene; c) treating the induced cell from step b) with a protein kinase C activating compound or an inhibitor of serine or threonine specific protein phosphatase to activate the long terminal repeat; d) isolating and lysing the nuclei from the cell from step c) to produce an extract; e) contacting the extract with the long terminal repeat; and f) isolating the protein factor which binds with the terminal repeat, thereby identifying the factor capable of activating the function of the long terminal repeat. Finally this invention provides the identifed protein factor described by the above method. This invention provides a powerful tool for identifying critical transcriptional regulators, both activators and inhibitors, controlling expression of LTRs of retroviruses. Once appropriate DNA-binding proteins are identified, they can serve as models for designing drugs which can be used to specifically inhibit replication of viruses controlled by LTRs, i.e., such as HIV which is the mediator of AIDS. In addition, replicating retroviruses containing dominant acting genes which encode proteins functioning as transcriptional suppressors of LTRs could be transferred into uninfected T-cells preventing subsequent production of HIV after infection. Another approach could be to use the genes encoding mutated activators of LTR expression as dominant-negative mutants to infect uninfected T-cells preventing subsequent production of HIV following infection. The approaches briefly outlined above represent novel strategies for: designing new classes of compounds to inhibit HIV replication; constructing viruses (which can function as potential vaccines) preventing HIV-infected T-cells from producing new viruses; and generating retroviruses which can prevent uninfected T-cells from producing new progeny HIV and releasing virus capable of infecting additional target T- cells. These approaches used alone or in various combinations could result in the effective treatment of HIV-infected individuals and a means of curtailing HIV pathogenesis. This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter. EXPERIMENTAL DETAILS MATERIALS AND METH0D8 Cell ςμJ ure? Ell is a single-cell clone of H5tsl25-transformed Sprague-Dawley secondary RE cells (Fisher et al., 1978). Ell-NMT is a subclone of cells derived from a nude mouse tumor (Babiss et al. , 1985). Ell/pMV7-J3 and Ell/pMV7-K2 are two independent neomycin (neo) resistant Ell cultures obtained following transfection with pMV7 (Perkins, et al., 1983) and selection for growth in G418. Al/PKC, Bl/PKC, CI/PKC and DI/PKC are four independent neo resistant Ell cultures obtained following transfection with a full-length cDNA sequence of RP58 (Housey, et al., 1987) , which encodes PKCβ^ subcloned into pMV7 (pMV7-PKC) (Housey, et al., 1988) and selection for G418 resistance. Bl/PKC subclones, Bl/PKC-cll, Bl/PKC-cl2, Bl/PKC-cl7 and Bl/PKC-cllO, were obtained by plating Bl/PKC cells at low density and isolating independent single-cell derived clones using a metal cloning cylinder (Fisher, et al., 1978). AZA treated subclones of Ell, Ell-NMT, Al/PKC, Bl/PKC, Cl/PKC and Dl/PKC were obtained by plating cells at low density, adding lOμm AZA for 24 hours, removing AZA and growing cells in AZA-free culture medium for three weeks and isolating single-cell derived clones (Babiss, et al., 1985). All cultures were grown in Dulbecco's modified Eagle's medium supplemented with 5% FES (DMEM-5) at 37°C in a humidified 5% C0 2 /95% air incubator. DNA transfections Ell cells were seeded at 1 x IO 6 cells per 10 cm plate; 24 hours later cultures were transfected using the calcium phosphate precipitation method with 10 μg of pMV7 or pMV7-PKC as described previously with slight modifications (Babiss, et al., 1984; Su, et al., 1990). Approximately 48 hours after transfection, cultures were seeded at 1 x 10 s and 5 x 10 s per 6 cm plate in G418 (lOOOμg ml "1 ) containing medium and the medium with G418 was changed every three or four days until neo resistant colonies developed (approximately two weeks) . G 18- resistant Ell subclones were isolated and maintained as separate clonal populations in media containing 250μg ml "1 G418. Anchorage-independent growth The ability of the various cell lines to grow when suspended in soft agar was determined as described previously (Fisher, et al., 1979c). Cells were initially seeded in agar at different cell densities ranging from a high of 5 x 10* to a low of 2 x 10 3 . After 21 days of growth in DMEM containing 7.5% FBS and 0.4% noble agar with routine feeding every four days, colonies > 0.1mm in diameter were counted with the aid of an Olympus tissue culture microscope and a calibrated grid. For cultures receiving TPA, 100 ng ml "1 of TPA was incorporated in the 0.8% noble agar/DMEM-7.5 base layer, the 0.4% noble agar/DMEM-7.5 overlay layer and the 0.4% noble agar/DMEM- 7.5 overlay feeder layers. PKC-enzvmatic activity Levels of PKC enzymatic activity were determined using DEAE-fractionated cell lysates (O'Brian, et al., 1989). Lysates were prepared by harvesting cells in the logarithmic phase of growth with Buffer A (20 mM Tris-HCl pH 7.5, 5mM EDTA, 5mM EGTA, 15mM 2-mercaptoethanol, lOμg ml "1 leupeptin, 0.25 mM PMSF, 25μg ml "1 soybean trypsin inhibitor) containing 0.1% Triton X-100, stirring the cell suspensions for 1 hour, and then centrifuging them at 13,800 G for 15 min. Supernatants were loaded onto 0.5 ml DEAE-Sepharose columns equilibrated in Buffer A. Columns were washed with 3 ml of Buffer A, and 2 ml of Buffer A containing 0.2 M NaCl (pH 7.5) was used to elute PKC activity (O'Brian et al., 1989). PKC enzymatic activity was assayed as previously described (Ward & O'Brian, 1992) , by subtracting the phosphotransferase activity between [γ 32 P]ATP and histone III-S observed in the presence of ImM Ca 2+ from the activity observed in the presence of ImM Ca 2+ plus 30 μg PS/ml. Cellular ( 3 H -PDBu binding PDBu binding assays were performed as described by Housey et al. (1988) and Su et al. (1992). Cells were seeded at 2 x 10 3 /35 mm tissue culture plate, DMEM-5 was added 24 hour post-plating and cells were assayed 20 to 24 hours later. Cultures were washed 2 x with DMEM:PBS:BSA (2:1 vol/vol, DMEM:PBS, containing 1.0 mg ml "1 bovine εerum fraction V (Sigma) and incubated at 37°C in the presence of 1.0 ml of DMEM:PBS:BSA containing 50 or 25 nM [ 3 H]-PDBu (New England Nuclear; 8.3 Ci mmol "1 ) . Four replicate plates were incubated with [ 3 H]-PDBu while a fifth plate was incubated with [ 3 H]-PDBu in the presence of 50 nM unlabeled PDBu to determine nonspecific binding. Two additional plates were re-suspended by brief trypsin/versene treatment and counted using a model Z. Coulter Counter (Hialeah, FLA) . Plates receiving PDBu were washed 3 x in 3 ml of ice-cold DMEM:PBS:BSA and solubilized for 2 hours at 37°C using 1.2 ml of 2.25% trypsin, 0.02% EDTA, 1.0% Triton X-100. One ml of lysate was counted by liquid scintillation. Nucleic acid analysis High molecular weight DNA was isolated from the various cells as previously described (Dorsch-Hasler, et al., 1908; Fisher, et al., 1982; Babiss, et al., 1984). Cellular DNAs (lOμg per sample) were digested with the restriction endonuclease EcoRI, size-fractionated through 0.6% agarose gels, transferred to nitrocellulose filters and probed using a 32 P-labeled PKC B-_ DNA probe as described previously (Fisher, et al. , 1982) . Steady state levels of PKC 6-. Ad5 E1A, neo, Ad5 ElB, c-jun, PKCe and GAPDH mRNAs were determined by Northern analysis of total cytoplasmic RNA using appropriate multiprimed 32 P- labeled cloned DNA probes as described previously (Babiss, et al., 1983; Su & Fisher, 1992). Northern blots were washed in a 0.1% SDS, 1 x SSC buffer at room temperature for 30 min followed by washing at 42°C for an additional 30 min in the same buffer. In vitro transcription within isolated nuclei was performed as previously described (Friedman, et al., 1986; Duigou, et al., 1990; Su, et al. , 1993) with modifications (Jiang, et al., 1993). Nuclei from approximately 2 x 10 s cells were isolated, and RNA transcripts previously initiated by RNA polymerase II were allowed to elongate in the presence of [ 32 P]UTP. The [ 32 P]-labeled RNA was extracted with phenol/chloroform and unicorporated nucleotides were removed by passing the probe through a G-50 Sephadex column. Nylon membranes containing 2μg of the appropriate denatured plasmid DNA gene inserts were hybridized with the [ 32 P]-labeled RNA. Nylon membranes contained PKCα, PKC β 1# PKCγ, PKCe, c-jun, jun-B, c-fos, Ad5 E1A, Ad5 ElB, GAPDH, 6-actin and pBR322 DNA probes. Following hybridization, the nylon membranes were washed and exposed for autoradiography. Gel-retardation assays Oligonucleotide probes were synthesized which contain the AP-1 binding sequence (5'CCAAACAGGATATATGAGTCATGCAGTTC- 3') (Seq. ID No.l) (Angel, et al., 1988; Mitchell & Tijian, 1989) or the MECA binding sequence (5'- CCAAACAGGATATCTGTGGTAAGCAGTTCC-3') (Seq. ID No. 2) (Halazonetis, 1992) . Oligonucleotides were end-labeled with [ 32 P-γ]-ATP using T » DNA kinase and then allowed to react with nuclear extracts at room temperature for 30 min. Reaction mixtures consisted of 32 P-labeled oligonucleotide (5000 c.p.m.), 2μg of poly (dl-dC) and lOμg of nuclear protein extract in lOmM HEPES (pH 7.5), 50 mM KC1, 5 mM MgCl 2 , 0.5 mM EDTA, ImM DTT and 12.5% glycerol. After incubation for 30 min at room temperature the reaction mixtures were separated on a 5% polyacrylamide gel with circulating Tris-borate buffer (0.375 x TBE, 160V), the gels were dried and autoradiographed. Nuclear extracts were also incubated with a 100-fold excess of unlabeled competitor DNA and the 32 P-labeled oligonucleotide probes. EXPERIMENTAL RESULTS Induction of the transformation progression phenotype by the PKC B gene in Ad5-transfoπned RE cells In Ad5-transformed Sprague-Dawley RE cells, the ability to form colonies in agar with enhanced efficiency correlates directly with increased tumorigenic potential in nude mice, i.e., the progression phenotype (Fisher, et al., 1979a, b, c; Babiss, et al., 1985; Duigou, et al., 1989, 1991). The Ad5-transformed RE clone, Ell, grows with a lower efficiency in agar and displays a longer latency time for tumor formation in nude mice than its progressed nude mouse tumor-derived subclone, Ell-NMT (Babiss, et al., 1985) (Table 1 and data not shown). Transfection of Ell cells with a cloned PKC β : gene construct which also contains a neomycin (neo) resistance gene (Housey, et al., 1988) results in G-418 resistant clones that display enhanced anchorage independence in comparison with parental Ell cells (Table 1) . In contrast, G418-resistant Ell cells transfected with the pMV7 plasmid construct lacking the PKC β x expressing Ell clones also display a reduction in tumor latency time in nude mice (data not shown) . As observed previously with Ell and Ell-NMT cells, TPA increased the efficiency of agar growth in PKC B λ transfected Ell cells (Table 1) . These results indicate that the PKC β x gene can function as a progression-inducing gene in Ell cells and this process can be further modulated by the phorbol ester tumor promoter, TPA. Table l Effect of TPA on anchorage-independent growth of Ad5- transformed rat embryo cells (Ell) , nude mouse tumor-derived Ell cells (Ell-NMT) , G418-resistant pMV7-transformed Ell cells and Ell cells expressing the B x isoform of PKC Cloning efficiency in agar (%) b Cell type* -TPA +TPA Phenotype" Ell 4.2 ± 0.8 6.1 ± 0.6 P- (1.5) Ell-NMT 32.7 ± 2.9 53.4 ± 4.5 P+ (1.6) Ell/pMV7-J3 2.8 ± 0.4 3.5 ± 0.4 P- (1.3) Ell/pMV7-K2 4.4 ± 0.7 5.9 ± 0.3 P- (1.3) Al/PKC 12.4 ± 1.1 20.1 ± 0.9 P+ (1.6) Bl/PKC 29.1 ± 1.3 56.8 ± 3.5 P+ (2.0) Cl/PKC 10.1 ± 0.7 17.1 ± 1.2 P+ (1.7) Dl/PKC 22.6 ± 2.2 49.5 ± 2.8 P+ (2.2) 'Cell lines include the parental Ell cell line, a nude mouse tumor-derived Ell clone (Ell-NMT) . G418-resistant Ell cells obtained following transfection with the pMV7 vector (Ell/pMV7-J3 and Ell/pMV7-K2) , and PKC β x expressing Ell clones (Al/PKC, Bl/PKC) . Cl/PKC and Dl/PKC, b Agar cloning effeciency, in the absence or presence of 100 ng ml "1 of TPA, was ~ determined as described in Materials and Methods and previously (Fisher, et al., 1979c). Results presented are the average percent agar cloning efficiency ± SD of 4 replicate plates initially seeded with 5 x 10* cells. Values presented in brackets refer to fold-increase in agar cloning in the presence of TPA "Progression phenotype (P+) and absence of the progression phenotype (P-) . Progression phenotype is indicated by enhanced growth in agar in comparison with parental Ell cells with a further increase in anchorage-independent growth after exposure to TPA. Reversal of the transformation progression phenotype in Ell-PKC B. cells bv AZA The progression phenotype of Ell-NMT cells can be stably reversed by a single-application of the demethylating agent AZA (Babiss, et al., 1985). Growth of Ell-NMT and PKC Bi-transfected Ell cells at clonal cell densities (100 and 200 cells/6 cm plate) for 24 hours in the presence of lOμM AZA, followed by continued cultivation for two weeks in the absence of AZA resulted in a series of AZA subc * lones displaying a stable reversion in their anchorage-independence to that of parental Ell cells (Table 2) . In contrast, AZA-treatment did not modify the low anchorage-independence of Ell cells or of neo resistant Ell cells transformed with the pMV7 construct lacking the PKC β x gene (Table 2 and data not shown) . These observations indicate that AZA can also reverse the progression phenotype in PKC β x expressing Ell cells as observed previously in spontaneously progressed and Ha¬ ras-progressed Ell cells treated with AZA (Babiss, et al., 1985; Duigou, et al., 1989). Table 2 Effect of AZA on anchorage-independent growth of Ell and Ell subclones AZA Cloning efficiency Cell type* treatment! in agar (%) I Phenotype* Ell 3.5 ± 0.5 P- Ell-AZA 1 + 4.6 ± 0.4 P- Ell-AZA 2 + 6.1 ± 0.5 P- Ell-NMT - 35.3 ± 2.6 P+ Ell-NMT-AZA 1 + 4.3 ± 0.6 P- Ell-NMT-AZA 2 + 2.7 ± 0.3 P- Al/PKC - 11.8 ± 0.9 P+ Al/PKC-AZAIa + 1.6 ± 0.2 P- Al/PKC-AZAIIi + 4.1 ± 0.4 P- Bl/PKC - 27.5 ± 1.8 P+ BP/PKC-AZAIIg + 4.3 ± 0.5 P- Bl/PKC-AZAIVe + 1.8 ± 0.1 P- Cl/PKC - 12.2 ± 1.0 P+ Cl/PKC-AZAIIId + 4.9 ± 0.4 P- Cl/PKC-AZAVa + 7.1 ± 0.8 P- Dl/PKC - 24.8 ± 1.7 P+ Dl/PKC-AZAVIb + 8.1 ± 1.0 P- Dl/PKC-AZAVIIc + 4.3 ± 0.6 P- 'Cell lines have been described in the legend to Table l and in Materials and Methods. fThe indicated cell type was treated at clonal densities with lOμM AZA for 24 h. , cultures were grown for two weeks in the absence of AZA and independent colonies were isolated and maintained as separate clones (termed AZA clones) . AZA treatment = +; no AZA treatment = -. i Results presented are the average percent agar cloning efficiency ± SD of four replicate plates initially seeded with 5 x 10* cells. Further details in Materials and Methods. ^Progression phenotype (P+) and absence of the progression phenotype (P-) . Progression phenotype is indicated by enhanced growth in agar in comparison with parental Ell cells. Molecular and biochemical properties of Bl/PKC cells and AZA-revertant Bl/PKC cells To investigate the mechanism by which the PKC β x gene induces the progression state and AZA reverts progression in PKC β : expressing cells applicants utilized the PKC Bi/neo-construct transfected G418-resistant Ell subclone, Bl/PKC (Tables 1 and 2) . Southern hybridization analysis of EcoRI digested DNA isolated from Bl/PKC cells and its single-cell derived-untreated-subclones, Bl/PKC-cll, B1/PKC-C12, B1/PKC-C17 and Bl/PKC-cllO, and its single- cell derived-AZA-treated subclones, Bl/PKC-AZAIIb, Bl/PKC-AZAIIe, Bl/PKC-AZAIIg, Bl/PKC-AZAIVa and Bl/PKC- AZAVIIa, indicated the presence of a 2.4kb fragment corresponding to the newly inserted PKC β x gene (data not shown) . This DNA fragment was not present in Ell or Ell- NMT cells, whereas additional DNA fragments of approximately 11, 6.8, 4.1 3.1 and 1.5kb that correspond to the endogenous PKC B-, gene were present in all of the Ell-derived cell lines (data not shown) . As anticipated based on their ability to grow in G418, all of the Bl/PKC and Bl/PKC-AZA subclones expressed the neo resistance gene, whereas this mRNA was not detected in Ell or Ell-NMT cells (Figure 1) . In addition, all of the Bl/PKC-AZA subclones produced more neo resistance RNA then parental Bl/PKC and many of the untreated Bl/PKC subclones. Probing of Northern blots with a PKC β x gene demonstrated the presence of multiple hybridizing mRNAs from Bl/PKC and its untreated subclones, whereas the majority of AZA-treated Bl/PKC subclones contained reduced levels of these mRNAs (Figure 1) . The multiple hybridizing RNAs seen in Bl/PKC cells and its subclones presumably represent unspliced transcripts or variably spliced transcripts coming from the retroviral promoter in the PKC β : vector PMV7-PKCB ! . The major transcript is a 6.6kb RNA species, which corresponds to the predicted size for an mRNA transcript that initiates in the 5' LTR and terminates in the 3' LTR of pMV7-PKCβi construct. In contrast, probing similar Northern blots with the Ad5-E1A and ElB transforming genes indicated no consistent differences in expression of these genes in the various cell types (Figure 2). These observations indicate that treatment with AZA results in: (a) a selective suppression in expression of the transfected PKC B l gene, without a similar suppression in expression of the neo resistance gene that is present on the same transfected plasmid construct; and (b) no change in expression of the Ad5 E1A or ElB genes as a function of expression or AZA- induced suppression of the transformation progression phenotype. The biological and biochemical properties of Bl/PKC cells and its untreated and AZA-treated subclones are shown in Table 3. Parental Bl/PKC cells grew with increased efficiency in agar vs. Ell parental cells (Table 1) . An increase in agar cloning efficiency was also apparent in all of the Bl/PKC-derived untreated subclones (Table 3) . Ell, Ell-NMT, Bl/PKC and untreated Bl/PKC subclones displayed a further enhancement in anchorage-independence of 1.4 to 2.3-fold when grown in the continuous presence of TPA (Table 3). Bl/PKC and Bl/PKC untreated subclones displayed increased levels of [ 3 H]-PDBu binding to cell surface receptors in comparison with Ell and Ell-NMT cells (Table 3) . In parental Bl/PKC cells and specific untreated Bl/PKC subclones, the increase in binding of phorbol esters to cell surface receptors correlates with an increase in PKC enzymatic activity (Table 3) . This was not always the case, however, as indicated by Bl/PKC- cllO cells that displayed elevated [ 3 H]-PDBu binding to cell svirface receptors without a significant increase in PKC enzymatic activity. Compared with the dramatic changes observed in phorbol ester binding levels, alterations in PKC enzymatic activity levels were relatively modest. This most likely reflects efficiencies of active enzyme recovery from cell lysates (phorbol ester binding assays are done in intact cells) and/or incomplete post-translational modification of PKC, resulting in a catalytically inactive form of the enzyme (Borner, et al., 1988). Table 3 Growth in agar, binding of phorbol-dibutyratβ (PDBu) and protein kinase C (PKC) activity in Ell cells and Ell cells expressing the B-, isoform of PKC Growth in Agar (%)* PDBU binding Cell line -TPA +TPA (C, .p.m./lO* cells)t Ell 2.5 3.5 (1.4) 707 Ell-NMT 39.9 58.4 (1.5) 771 Bl/PKC 28.7 57.9 (2.0) 13,363 Bl/PKC-cll 28.9 46.1 (1.6) 13,758 B1/PKC-C12 16.6 38.2 (2.3) 14,658 B1/PKC-C17 10.9 19.9 (1.8) 16,211 Bl/PKC-cllO 20.3 38.6 (1*9) 13,989 Bl/PKC-AZAIIb 7.6 7.2 (0.9) 651 Bl/PKC-AZAIIe 6.1 7.4 (1.2) 696 Bl/PKC-AZAIIg 5.2 4.7 (0.9) 1,133 Bl/PKC-AZAVIIa 26.6 40.7 (1.5) 39,942 PKC activity Cell line (mmol/min/mg)+ Phenotype++ Ell N.D. P- Ell-NMT N.D. P+ Bl/PKC 1.83 ± 0.46 P+ Bl/PKC-cll 1.07 ± 0.34 P+ B1/PKC-C12 1.61 ± 0.13 P+ B1/PKC-C17 2.33 ± 0.27 P+ Bl/PKC-cllO 0.37 ± 0.10 P+ Bl/PKC-AZAIIb 1.04 ± 0.54 P- Bl/PKC-AZAIIe 1.62 ± 1.14 P- Bl/PKC-AZAIIg 0.48 ± 0.53 P- Bl/PKC/AZAVIla 3.36 ± 1.26 P+ Table 3 - continued *E11, Ell-NMT, PKC β : expressing Ell (Bl/PKC), single-cell derived Bl/PKC subclones (Bl/PKC-cll, Bl/PKC-cl2, Bl/KC-cl7 and Bl/PKC-cllO and single-cell derived Bl/PKC subclones treate with lOμM AZA for 24 h (Bl/PKC-AZAIIb, Bl/PKC-AZAIIe, Bl/PKC- AZAIIg, and Bl/PKC-AZAVIIa) were analyzed for anchorage- independent growth in the presence and absence of 100 ng ml "1 of TPA as described previously (Fisher et al., 1979c). Results presented are the average percent agar cloning efficiency of 4 replicate plates. Replicate samples varied by <10%. Values presented in brackets refer to fold-increase in agar cloning in the presence of TPA. fPDBu binding assays were performed as described in Materials and Methodε. Results presented are the average specific binding (corrected for non-specific binding) of four replicate plates. Replicate samples varied by <15%. +PKC enzymatic activity was determined as described in Materials and methods. Results are the average of triplicate samples ± standard deviation of the mean. ++Progression phenotype (P+) and absence of the progression phenotype (P-) . Progression phenotype is indicated by enhanced growth in agar in comparison with parental Ell cells with a further increase in anchorage-independent growth after exposure to TPA. With the exception of Bl/PKC-AZAVIIa, all of the AZA- treated Bl/PKC subclones exhibited a reduction in anchorage-independence in comparison with parental Bl/PKC cells. Similarly, except for Bl/PKC-AZAVIIa cells, which behaved in a similar manner as Ell, Ell-NMT, Bl/PKC and Bl/PKC subclones not treated with AZA, TPA did not result in an increase in anchorage-independence in Bl/PKC-AZA cells (Table 3). All of the Bl/PKC-AZA clones, with the exception of Bl/PKC-AZAVIIa, also displayed a reduction in [ 3 H]-PDBu binding to cell surface receptors and PKC enzymatic activity (Table 3) . It should be noted that Bl/PKC-AZAVIIa cells retained similar properties as Bl/PKC cells, including the retention of β x PKC mRNA expression suggesting that this clone had not been reverted in its transformation progression phenotype by AZA (Table 3 and Figure 1) . These results indicate that increased expression of PKC β : in Ell cells is often associated with increased PKC enzymatic activity and enhanced binding of phorbol dibutyrate to PKC-cell surface receptors. In addition, whereas TPA can increase anchorage-independence in Ell, Ell-NMT and Bl/PKC cells, it cannot induce this effect in Bl/AZA clones displaying a loss of the progression phenotype. Effect of TPA on σene expression in AZA-reverted Bl/PKC cells TPA is a potent activator of PKC in diverse target cells (reviewed in Ohno, et al., 1991; Nishizuka, 1992). To determine if TPA could alter gene expression in Bl/PKC- AZAIIg cells, cultures were incubated with 50ng ml "1 of TPA for 15, 30, 60 and 120 minutes (Figure 3). TPA resulted in a time-dependent induction of PKC β x expression in Bl/PKC-AZAIIg cells that was first apparent by 30 minutes and maximum by 60 minutes. TPA also increased c-jun mRNA levels in Bl/PKC-AZAIIg cells with similar kinetics as PKC B-. induction. In contrast, TPA did not alter expression of the endogenous PKCe gene or the GAPDH gene in Bl/PKC-AZAIIg cells (Figure 3). TPA also did not significantly alter PKC β : mRNA levels in Bl/PKC cells already displaying high levels of PKC B-, mRNA, i.e., B1/PKC-C12 and Bl/PKC-AZAVIIa cells (Figure 4). In Bl/PKC-AZAIId cells, which display a higher level of de novo expression of PKC β : mRNA than Bl/PKC-AZAIIg cells, TPA further enhanced PKC β χ expression (Figure 4) . Preliminary studies indicate that additional agents can induce PKC β x mRNA expression in Bl/PKC-AZAIIg cells, including calyculin and okadaic acid (inhibitors of serine/threonine-specific protein phosphatase 1 and 2A and 2B at higher concentrations) and rationally designed protein kinase C activators ADMB (3-(N-acetylamino)-5-(N- decyl-N-decyl-N-methylamino) benzyl alcohol) and DHI (6- (N-decylamino)-4-hydroxymethylindole) (unpublisheddata) . To determine if the effect of TPA on β x PKC expression in Bl/PKC-AZA treated subclones was reversible or irreversible, cells were grown for 24 hours in TPA, cultures were washed and incubated for an additional 7 days in the absence of TPA (Figure 4) . A reduction in PKC B- . mRNA level was observed in Bl/PKC-AZAIIg, Bl/PKC- AZAIVe and Bl/PKC-AZAIId cells grown for one week in the absence of TPA. This experiment indicates that the TPA effect on PKC B-. expression depends on the level of PKC β x initially expressed by the Bl/PKC subclones and the TPA effect is reversible following removal of this tumor promoting agent. The ability of TPA to induce a rapid induction of PKC B l RNA in Bl/PKC-AZAIIg cells suggested that TPA might be transcriptionally activating this gene. To investigate this possibility, applicants used nuclear run-on assays to measure RNA transcription rates in untreated and TPA- treated Bl/PKC-AZAIIg cells (Figure 5) . As appropriate control cell lines, RNA transcription rates in the presence and absence of 1 hour treatment with 50 ng ml "1 TPA were also determined in Ell, Ell-NMT and Bl/PKC cells. Using GAPDH and β-actin as internal control indicators of RNA transcription, the transcription rates of the PKC α, PKC γ, PKC e, Jun-B, Ad5 E1A and Ad5 ElB genes were similar (within two-fold) in all four cell types grown in the presence or absence of TPA (Figure 5) . Transcription of the PKC γ gene in all four cell types and the PKC B x gene in Ell and Ell-NMT was negligible in the presence or absence of TPA. In contrast, the inserted PKC β x gene was transcribed at a high rate in Bl/PKC cells, whereas transcription of this gene was reduced > 10-fold in Bl/PKC-AZAIIg cells (Figure 5) . When treated with 50 ng ml "1 of TPA for l hour, the transcription rate of PKC B-, in Bl/PKC-AZAIIg cells increased five-fold. Smaller increases (two- to three¬ fold) were also observed in the transcription of the c- jun an c-fos genes in TPA-treated Bl/PKC-AZAIIg cells (Figure 5) . TPA can induce transcriptional activation of genes containing the palindromic DNA sequence TGACTCA (Seq. ID No. 3) , referred to as the TPA-responsive element (TRE) (Lee, et al., 1987; Angel, et al., 1988; Mitchell & Tijian, 1989) . The AP-1 multiprotein transcription factor complex, consisting of either c-jun protein homodimers or c-jun/c-fos heterodimers, can bind to the TRE through the leucine zipper dimerization domain resulting in changes in gene expression (Kouzarides & Ziff, 1988; Sasson-Corsi, et al. , 1988). Sequence analysis of the Moloney leukemia virus long terminal repeat (LTR) , which transcriptionally regulates expression of the PKC β x gene in the pMV7 plasmid, indicated the presence of two tandem AP-1 recognition sites. To determine if the ability of TPA to induce transcriptional increases in the PKC β : gene in Bl/PKC- AZAIIg cells involved changes in the level of AP-1 transcription factors applicants isolated nuclear extracts from control and TPA-treated cells and performed DNA-binding (gel retardation) assays. As can be seen in Figure 6, the level of AP-1 activity in Bl/PKC-AZAIIg cells was reduced relative to the levels found in Ell and Bl/PKC cells. Following treatment with TPA, the amount of AP-1 activity increased to that observed in Bl/PKC cells. In contrast, the level of DNA binding proteins recognizing an enhancer core sequence element (MECA) (Halazonetis, 1992) found in the Moloney leukemia virus LTR was similar in Ell, Bl/PKC and Bl/PKC-AZAIIg cells grown in the presence or absence of TPA. Both AP-1 and MECA binding activities were eliminated by using a 100- fold excess of the respective competitor DNA sequence in combination with nuclear extracts (data not shown) . Reversal of the transformation progression phenotype in Ell-PKC B- cells bv ohenylbutyrate (PB) and effect of TPA on gene expression in PB-reverted Bl/PKC cells Experiments were also conducted to determine if the demethylating agent PB could induce a suppression in the progression phenotype in PKC B-. expressing Ell cells. Bl/PKC cells were seeded at clonal cell densities (100, 200 and 400 cells/6 cm plate) treated with 4 mM PB for 4 days and then grown for 2 weeks in medium lacking PB. Colonies were then isolated and grown as independent cell lines in the absence of PB. The Bl/PKC-PB 5 clone displayed a reversion of the progression phenotype, i.e., it grew in agar with an efficiency similar to Ell cells. Analysis of gene expression in Bl/PKC-PB 5 cells indicated that they no longer synthesized PKC B-, or Ad5 ElB mRNA (Figure 7) . As observed with AZA treated Bl/PKC cells, treatment with TPA for 1 hour or 4 hours resulted in the induction of PKC β x mRNA expression. In addition, TPA also induced expression of Ad5 ElB mRNA in Bl/PKC-PB 5 cells. In contrast, no change in Ad5 E1A or GAPDH expression was observed in Bl/PKC or Bl/PKC-PB 5 cells grown in the absence or presence of TPA. These observations indicate that PB, like AZA, can reverse the progression phenotype in PKC B γ expressing Ell cells and progression suppression is associated with a TPA- reversible inhibition in PKC β : and Ad5 ElB expression. EXPERIMENTAL DISCQ8SION The Ad5 transformed rat embryo cell culture system is being used to study the contribution of specific genetic and epigenetic changes in the process of transformation progression (Fisher, 1984; Babiss, et al., 1985; Duigou, et al., 1991; Reddy, et al., 1993). Using this model, it has been demonstrated previously that Ad5 transformation is often a multistep process that is profoundly affected by diverse agents, including growth factors, hormones and tumor promoters (reviewed in Fisher, 1984) . Using a progressed H5tsl25-transformed rat embryo clone, Ell-NMT, it has been shown that transformation progression can be reversed, at an efficiency >90%, by a single treatment for 24 hours with AZA (Babiss, et al. 1985) . Transformation progression can also be stably suppressed in somatic cell hybrids formed between Ell-NMT and non- transformed CREF cells (Duigou, et al., 1990). These results support the hypothesis that transformation progression may be mediated by the expression of an unidentified gene(s) that is regulated by changes in DNA methylation (Babiss, et al., 1985; Duigou, et al., 1991; Reddy, et al., 1993). A basic tenet of applicants' hypothesis is that methylation of the progression suppressor gene(s) results in a loss of expression of this gene and consequently induction of the progression phenotype, whereas demethylation of the putative progression suppressor gene(s) results in gene activation and suppression of the progression phenotype (Babiss, et al., 1985; Duigou, et al., 1991; Reddy, et al., 1993). The PKC β x gene, which when expressed at low levels can enhance transformation of CREF cells by Ad5 or the Ad5 E1A gene (Su, et al. , 1991) , induced a progression phenotype in the H5tsl25-transformed rat embryo clone. Ell. Transformation progression in Ell/PKC cells correlated with an increase in both the transcriptional rate and the steady state levels of PKC λ RNA, an increase in PKC enzymatic activity, an increase in [ 3 H]- PDBu binding to cell surface receptors and an increase in anchorage-independence. A single 24 hour treatment with AZA resulted in a reversion of the progression phenotype and a return in the properties of Bl/PCK cells to that of the untransfected parental Ell cells (Table 3) . As was previously demonstrated in spontaneous progression induced by tumor selection in nude mice (Duigou, et al., 1991) , suppression of PKC progression by AZA in Bl/PKC cells did not result in changes in the levels of expression of the Ad5 E1A or ElB transforming genes. Progression suppression by AZA in Ell/PKC cells also did not extinguish expression of the neo resistance gene or change expression of the PKCe or c-jun genes. These observations indicate that transformation progression in Ell cells can be stably induced by expression of a specific PKC isoform, i.e., PKC β : , and induction of a putative progression suppressor gene by AZA can nullify this phenotype by selectively altering the expression of specific transformation progression-inducing genes. Several studies have focused on the effects of over- expression of specific cPKCs on cellular physiology (Housey, et al., 1988; Persons, et al., 1988; Megidish & Mazurek, 1989; Krauss, et al., 1989; Borner, et al., 1991, 1992a, b; Su, et al., 1991, 1992; Watanabe, et al., 1992) . Over-expression of PKC β x induced profound changes in cellular phenotype that were dependent on the level of PKC B-. expressed and the target cell used (Housey et al., 1988; Krauss, et al., 1989; Choi, et al., 1990; Su, et al., 1991, 1992). When PKC was inserted by retroviral vector into CREF, Rat 6 or C3H10T1/2 cells, sublines developed which expressed high levels of PKC B x mRNA, [ 3 H]-PDBu binding and PKC enzymatic activity (Housey, et al., 1988; Krauss, et al. , 1989; and Su, et al. 1992). High-level PKC Bx-expressing CREF cells exhibited a transformed morphology and grew more rapidly in monolayer culture, formed macroscopic colonies in agar in the absence of TPA and in many independent clones TPA further enhanced anchorage-independent growth (Su, et al., 1992). In contrast, insertion of the same PKC β χ construct into CREF cells by Ca 2+ -mediated DNA transfection resulted in cultures that displayed a normal CREF-like morphology, exhibited only small changes in [ 3 H]-PDBu binding and PKC enzymatic activity and failed to form macroscopic colonies in agar in the absence or presence of TPA (Su, et al., 1991, 1992). In the case of retroviral PKC β x transformed Rat 6 and C3H10T1/2 cells, high-level PKC x expressing clones displayed only marginal changes in morphology in the absence of TPA, whereas TPA resulted in profound morphological changes (Housey, et al., 1988; Krauss, et al., 1989). The growth rate and saturation density of high-level expressing PKC β χ expressing C3H10T1/2 clones was increased, transformed cells grew in agar in the absence or presence of TPA and transformed cells were tumorigenic in nude mice (Housey, et al., 1988) . In contrast, high-level PKC β : expressing C3H10T1/2 clones displayed similar growth rates as control retroviral vector transformed C3H10T1/2 cells and transformed cells did not display anchorage-independent growth and they were not tumorigenic in nude mice (Krauss et al., 1989) . Target cell specificity for the action of cPKCs is further indicated by the observation that over- expression of PKC βj. in the human HT29 colorectal carcinoma cell line results in growth suppression and an inhibition of oncogenic potential in nude mice (Choi et al., 1990). These studies demonstrate both PKC isoform and cell type specific effects of over-expression of various cPKCs in different target cells. Over-expression of PKC B. in Rat 6 cells has been εhown to alter the expression and TPA regulation of endogenous PKC genes, such as PKC£ and PKCe (Borner et al., 1992a). A differential alteration in the regulation of individual PKC isoforms has also been observed in parental Rat 6 (R6) and high-level PKC B t expressing Rat 6 cells (R6- PKC3) transformed with a series of oncogenes, including Ha-ras, src and fos (Borner et al., 1992b). Transformation of R6 or R6-PKC3 cells with an activated c-Ha-ras oncogene resulted in an increase in both the mRNA and protein levels of PKCα and PKCS, decreased levels of PKCe mRNA and protein and no change in the expression level of PKCC (Borner, et al., 1992b). The mechanism involved in the enhanced expression of PKC a in c-Ha-ras transformed Rat 6 cells involved an increase in the transcription rate of this PKC gene (Borner et al., 1992b) . In contraεt, no εignificant changes in the levels of expression of PKCα, PKC«S, PKCe or PKCC was found in R6 cells transformed by myc, neu/erb-B2 or mos oncogenes (Borner et al., 1992b). In the present study, applicants have determined the effect of over-expression of PKC B-. and suppression of PKC β x expression by AZA on the transcriptional rates of various cPKC and nPKC genes. No changes occurred in the transcriptional rates of PKCα, PKCγ or PKCe in Bl/PKC or Bl/PKC-AZAIIg cells (Figure 5) . Similarly, no difference was observed in the mRNA levels of PKCe in Ell, Ell-NMT, Bl/PKC or Bl/PKC AZA cells (Figure 3) . These resultε suggest that rat embryo cells transformed by Ad5 behave more like myc, neu/erb-B2 or mos oncogene transformed rat cells, than c-Ha-ras transformed rat embryo cells. The ability of PKC B λ to induce transformation progression in Ell cells correlated with the continued expression of this gene, as reflected by increased PKC β x transcriptional rates and mRNA production, enhanced [ 3 H]- PDBu binding and in many instances an increase in PKC enzymatic activity. Induction of expression of the putative transformation progression suppression gene following AZA treatment in Bl/PKC-AZAIIg cells was associated with a reduction in all of these PKC-related phenomena. An important question that applicants have begun to investigate is the mechanism by which AZA- treatment induced a suppression in transformation progression in Bl/PKC cells. Progression suppression induced by AZA was associated with a decreased level of PKC β x transcription and steady state RNA (Figures 3 and 4) . Since transcription of the PKC β x gene resulted from transcriptional initiation from a Moloney leukemia virus LTR aε oppoεed to its own endogenous promoter (Perkins et al., 1983; Housey, et al., 1988), the above results suggested the possibility that the induction of the putative progression suppressor gene by AZA might be influencing, either directly or indirectly through positive and/or negative acting cis-regulatory elements, transcription from the Moloney leukemia virus LTR. To begin to dissect the region(s) of the Moloney leukemia virus LTR responsive to AZA εuppression studies have been conducted to determine if the binding of transcription factors to sequence specific DNA binding domains are altered following AZA treatment in Bl/PKC-AZA cells. A difference was found in the levels of the AP-1 multiprotein transcription factor complex in Bl/PKC versus Bl/PKC-AZAIIg cells, with the latter cell type displaying less AP-1 activity (Figure 6) . In contrast no consistent changes occurred in AZA treated Bl/PKC with respect to the binding of transcription factors interacting with the enhancer core sequence element, MECA, also found in the Moloney leukemia LTR. The level of AP-1 in Bl/PKC-AZAIIg returned to that of Bl/PKC cells following treatment with TPA. In addition, TPA enhanced transcription and increased steady state RNA levelε of the PKC Bι and the c-jun gene in Bl/PKC-AZAIIg cells. The effect of TPA, however, was transitory and it did not result in a return of the progression phenotype in Bl/PKC-AZAIIg cells. These results indicate that AZA can selectively alter the levels of gene expression and transcription regulatory factors in Bl/PKC cells. Further studies are necessary to determine if selective modulation of the transcriptional machinery of progressed cells is a component of the mechanism by which AZA induces suppresεion of the transformation progression phenotype in Ell cells over-expresεing PKC βj and/or in spontaneously progressed Ell-NMT cells. In summary, the currently described cell culture model system represents a valuable experimental tool for defining the molecular and biochemical events mediating transformation progreεsion and transformation progresεion suppression. On the basis of the apparent selective shut-down of tranεcription from the Moloney leukemia virus LTR following AZA treatment, the present system would also appear amenable for identifying transcriptional regulatory factors that can directly govern expression of genes controlled by LTRs. Further studies designed to address these issues should provide important insightε into the molecular determinantε of multistage carcinogenesis. REFERENCES 1. Angel, P., Allegretto, E.A. , Okino, S.T. , Hattori, K., Boyle, W.J., Hunter, T. & Karin, M. (1988), Nature, 332:166-171. 2. Babiss, L.E., Young, C.S.H., Fisher, P.B. & Ginsberg, H.S. (1983) J. Virol., 46:454-465. 3. Babiss, L.E., Fisher, P.B. & Ginsberg, H.S. (1984). J. Virol., 49:731-740. 4. Babiss, L.E., Zimmer, S.G. & Fisher, P.B. (1985). Science, 228:1090-1101. 5. Bacher, N. , Zisman, Y., Berent, E. & Livneh, E. (1991). Mol. Cell. Biol., 11:126-133. 6. Bishop, M.J. (1978). Science, 235:305-311. 7. Bishop, M.J. (1991). Cell, 64:235-248. 8. Borner, C. , Eppenberger, U. , Wyss, R. & Fabbro, D. (1988). Proc. Natl. Acad. Sci. USA 85:2110-2114. 9. Borner, C, Filipuzzi, L. , Weinεtein, I.B. & Imber, R. (1991) Nature, 353:78-80. 10. Borner, C. , Guadagno, S.N., Fabbro, D. & Weinstein, I.B. (1992a). J. Biol. Chem., 267:12892-12899. 11. Borner, C. , Guadagno, S.N., Hsiao, W.W.-L., Fabbro, D., Barr, M. & Weinstein, I.B. (1992b). J. Biol. Chem., 267:12900-12910. 12. Choi, P.M., Tchou-Wang, K.M. & Weinstein, I.B. (1990) Mol. Cell. Biol., 10:4650-4657. 13. Coussenε, L. , Parker, P., Rhee, L. , Yang-Feng, T.L. , Waterfield, M.D., Francke, U. & Ullrich, A. (1986) Science, 233:859-866. 14. Dorεch-Haεler, K. , Fisher, P.B. , Weinstein., I.B. & Ginsberg, H.S. (1980). J. Virol., 34:305-314. 15. Duigou, G.J. , Babiss, L.E. & Fisher, P.B. (1989) N.Y. Acad. Sci., 567:302-306. 16. Duigou, G.J., Babiss, L.E., Iman, D.S., Shay, J.W. & Fisher, P.B. (1990) Mol. Cell. Biol., 10:2027- 2034. 17. Duigou, G.J. , Su, Z.-z., Babiss, L.E., Driscoll, B., Fung, Y.-K.T. & Fisher, P.B. (1991) Oncogene, 6:1813-1824. 18. Eldar, H. , Zisman, Y., Ullrich, A. & Livneh, E. (1990) J. Biol. Chem., 265:13290-13296. 19. Fisher, P.B. (1984) Mechanisms of Tumor Promotion. Ill, Tumor Promotion and Carcinogenesis in vitro, Slaga, T.J. (ed.). CRC Press:Boca Raton, FL, pp. 57- 123. 20. Fisher, P.B., Weinstein, I.B. Eisenberg, D. & Ginsberg, H.S. (1978) Proc. Natl. Acad. Sci. USA, 75:2311-2314. 21. Fisher, P.B., Bozzone, J.H. & Weinstein, I.B. (1979a) Cell, 18:695-705. 22. Fisher, P.B., Dorsch-Haεler, K. , Weinεtein, I.B. & Ginsberg, H.S. (1979b) Nature, 281:591-594. 23. Fisher, P.B. , Goldstein, N.I. & Weinstein, I.B. (1979b) Cancer Res. 39:3051-3057. 24. Fisher, P.B., Babiss, L.E., Weinstein, I.B. & Ginεberg, H.S., (1982), Proc. Natl. Acad. Sci., U.S.A., 79:3527-3531. 25. Friedman, J.M. , Babiss, L.E. & Darnell, Jr., J.E., (1986), J. Mol. Biol. 6:3791-3797. 26. Halazonetis, T.D., (1992) Anticancer Res., 12:285- 292. 27. Harrington, M.A. , Gonzales, F. & Jones, P.A., (1988) Mol. Cell. Biol., 8:4322-4327. 28. Housey, G.M. , 0'Brian, CA. , Johnson, M.D., Kirschmeier, P. & Weinstein, I.B., (1987), Proc. Natl. Acad. Sci., U.S.A., 84:1065-1069. 29. Housey, G.M. , Johnson, M.D., Hsiao, W.-L.W., O'Brian, CA. , Murphy, J.P., Kirschmeier, P. & Weinstein, I.B., (1988), Cell, 52:343-354. 30. Hsiao, W.-L.W., Housey, G.M. , Johnson, M.D. & Weinstein, I.B., (1989), Mol. Cell. Biol., 9:2641- 2647. 31. Jiang, H. , Waxman, S. & Fisher, P.B., (1993), Mol. Cell. Different., 1(2) :197-214. 32. Jones, P.A. , (1985), Cell, 40:485-486. 33. Knopf, J.L., Lee, M.-H., Sultzman, L.A. , Kriz, R.W., Loomiε, CR. , Hewick, R.M. & Bell, R.M. , (1986), Cell, 46:491-502. 34. Kouzarideε, T. & Ziff, E. (1988), Nature, 336:646- 651. 35. Krauss, R.A. , Housey, G.M., Johnson, M.D. & Weinstein, I.B., (1989), Oncogene, 4:991-998. 36. Lee, W., Mitchell, P. & Tijian, R. , (1987), Cell, 49:741-752. 37. Liang, P. and Pardee, A.B. (1992) Science, 257:967- 971. 38. Marshall, CJ. (1991), Cell, 64:313-326. 39. Megidish, T. , & Mazurek, N. (1989), Nature, 342:807- 811. 40. Mischak, H. , Goodnight, J. , Kolch, W. , Martiny- Baron, G. , Schaechtle, C, Kanietza, M.G., Blumberg, P.M., Pierce, J.H. & Mushinski, J.F. (1993), J. Biol. Chem., 268:6090-6096. 41. Mitchell, P.J. & Tijian, R. , (1989), Science, 245:249-251. 42. Nicholson, G.L., (1987), Cancer Res. 47:1473-1487. 43. Nishizuka, Y. , (1988), Nature, 334:661-665. 44. Nishizuka, Y., (1992), Science, 258:607-614. 45. Nowell, P.C, (1986), Cancer Res. , 46:2203-2207. 46. O'Brian, CA. , Fan, D., Ward, N.E., Seid, C & Fidler, I.J., (1989), FEBS Lett., 246:78-82. 47. Ohno, S., Akita, Y., Konno, Y., Imajoh, S. & Suzuki, K., (1988), Cell, 53:731-741. 48. Ohno, S., Akita, Y. , Hata, A., Osada, S. , Kubo, K. , Konno, Y., Akimoto, K. , Mizuno, K.,Saido, T., Kuroki, T. & Suzuki, K. , (1991), Adv. Enzyme Reg. 31:287-303. 49. Ono, Y., Kurokawa, T. , Kawahara, K. , Nishimura, O Marumoto, R. , Igarashi, K. , Sugiro, Y., Kikkawa, Y., Ogita, K. & Nishizuka, Y., (1986), FEBS Lett., 203:111-115. 50. Ono, Y., Fujii, T., Ogita, K. , Kikkawa, U. , Igarashi, K. , & Nishizuka, Y., (1988), J. Biol. Chem., 263:6927-6932. 51. Osada, S., Mizuno, K. , Saido, T.C, Akita, Y. , Suzuki, K. , Kuroki, T. & Ohno, S., (1990), J. Biol. Chem., 265:22434-22440. 52. Parker, P.J., Coussens, L. , Totty, N. , Rhee, L. , Young, S., Chen, E. , Waterfield, M.D., Francke, U. & Ullrich, A., (1986), Science, 233:853-859. 53. Perkins, A.S., Kirschmeier, P.T., Gattoni-Celli, S. & Weinstein, I.B., (1983), Mol. Cell. Biol., 3:1123- 1132. 54. Persons, D.A. , Wilkison, W.O., Bell, R.M. & Finn, O . J . , ( 1988 ) , Cell , 52 : 447-458 . 55. Reddy, P.G. , Su, Z.-z & Fisher, P.B., (1993), Chromosome and Genetic Analysis. Methods in Molecular Genetics Adolph, D.W. (ed) . Academic Press, Orlando, Florida., Vol. I, pp. 68-102. 56. Sager, R. (1989), Science, 246:1406-1411. 57. Sager, R. Anisowicz, A., Neveu, M. , Liang, P. and Sotiropoulou, G. (1993) FASEB, 7:964-970. 58. Sasεone-Corεi, P., Ransome, L.J. , Lamph, W.W. & Verma, I.M., (1988), Nature, 336:692-695. 59. Scott, R.E., Estervig, D.N., Tzen, C.Y., Minoo, P., Maercklein, P.B. & Hoerl, B.J. , (1989), Proc. Natl. Acad. Sci. U.S.A., 86:1652-1656. 60. SU, Z.-Z. & Fisher, P.B., (1992), Poly ADP- Riboεylation Reactions. Poirier, G.L. & Moreau, P. (eds.). Springer-Verlag, New York, pp. 203-210. 61. Su, Z.-z, Zhang, P. & Fisher, P.B., (1990), Mol. Carcinogenesis, 3:309-318. 62. Su, Z.-z., Duigou, G.J. & Fisher, P.B., (1991), Mol Carcinogenesis, 4:328-337. 63. Su, Z.-z., O'Brian, CA. & Fisher, P.B., (1992), Cell. Mol. Biol., 38:27-39. 64. Su, Z.-z., Austin, V.N, Zimmer, S.G. & Fisher, P.B., (1993), Oncogene, 8:1211-1219. 65. Sun, Y., Hegamyer, G., and Colburn, N.H. , (1994), Cancer Res. 54:1139-1144. 66. Taylor, S.M. & Jones, P.A., (1979), Cell, 17:771- 779. 67. Ward, N.E. & O'Brian, C.A., (1992), Biochemistry, 31:5905-5911. 68. Watanabe, T., Ono, Y. , Taniyama, Y., Hazama, K. , Igarashi, K. , Ogita, K. , Kikkawa, U. & Nishizuka, Y., (1992), Proc. Natl. Acad., Sci., U.S.A., 89:10159-10163. 69. Weinberg, R.S., (1985), Science, 230:770-776. 70. Weinεtein, I.B., (1988), Cancer Res., 48:4135-4143. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Fisher, Paul B. Su, Zao-zhong (ii) TITLE OF INVENTION: METHODS OF IDENTIFYING INDUCIBLE INHIBITORSOFTRANSFORMATIONPROGRESSION (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Cooper & Dunham (B) STREET: 30 Rockefeller Plaza (C) CITY: New York (D) STATE: New York (E) COUNTRY: United States of America (F) ZIP: 10112 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: White, John P. (B) REGISTRATION NUMBER: 28,678 (C) REFERENCE/DOCKET NUMBER: 0575/45426 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (212) 977-9550 (B) TELEFAX: (212 664-0525 (C) TELEX: 422523 COOP UI (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CCAAACAGG TATATGAGTC ATGCAGTTC 29 (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CCAAACAGGA TATCTGTGGT AAGCAGTTCC 30 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGACTCA

Claims: What is claimed is: 1. A method of identifying a progression suppressor gene which comprises: a) introducing DNA containing a progression gene into a population of transformed cells; b) treating the introduced transformed cells from step (a) so as to induce the expression of at least one progression suppressor gene and cause a subpopulation of the cells to revert to a characteristic phenotype; c) selecting cells which express the progression suppressor gene and exhibit the characteristic phenotype; d) isolating mRNA from the cells from step (c) ; e) comparing the mRNA so obtained with mRNA obtained from the uninduced transformed cells as to identify mRNA expressed only by the cells from step (c) ; and f) isolating the gene encoding such mRNA so as to thereby identify the progression suppressor gene. 2. A method of claim 1 where in step f) the progression suppressor gene is isolated by subtractive hybridization or differential display. 3. The progression suppressor gene identified by the method of claim 1. 4. The progression suppressor gene of claim 3 operatively linked to a promoter of RNA transcription. 5. A vector which comprises the progression suppressor gene of claim 3. 6. A virus comprising the progression suppressor gene of claim 3. 7. A polypeptide encoded by the progression suppressor gene of claim 3. 8. A method of identifying a progression suppressor gene which suppresses expression of long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C β χ gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor gene and cause a subpopulation of the cell to selectively suppress the expression of the long terminal repeat regulated protein kinase C B 1 gene; and c) selecting cells which express the progression suppressor gene; d) isolating mRNA from the cells from step (c) ; e) comparing the mRNA so obtained with mRNA obtained from the uninduced transformed cells so as to identify mRNA only expressed by the cells from step (c) ; and f) isolating the gene encoding such mRNA so as to thereby identify the progression suppressor gene. 9. A method of claim 8, wherein the retrovirus is a Moloney leukemia virus. 10. A method of claim 8, wherein the retrovirus is a human immunodeficiency virus. 11. A method of claim 8, where in step b) the cells are treated with 5-azacytidine or phenyl butyrate. 12. A method of claim 8, where in step b) the cells are treated with DNA demethylating agents. 13. A method of claim 8, where in step f) the progression suppressor gene is isolated by subtractive hybridization or differential display. 14. The progression suppressor gene identified by the method of claim 8. 15. The progression suppression gene operatively linked to a promoter of RNA transcription. 16. A vector which comprises the progression suppressor gene of claim 14. 17. A virus comprising the progression suppressor gene of claim 14. 18. A polypeptide encoded by the progression suppressor gene of claim 14. 19. A method of selecting a molecule capable of inhibiting the function of a long terminal repeat of a retrovirus comprising: a) introducing DNA containing the long terminal repeat regulated protein kinase C B x gene into a population of transformed cells; b) selecting cells from step a) which expresses protein kinase C β x gene; c) treating the selected cells with an amount of the molecule effective to inhibit the function of the long terminal repeat; and c) determining the level of expression of the protein kinase C β x gene, the decrease in the level of expression indicating that the molecule is capable of inhibiting the function of the long terminal repeat. 20. A method of claim 19, wherein the retrovirus is Moloney leukemia virus. 21. A method of claim 20, wherein the retrovirus is human immunodeficiency virus. 22. A method of selecting a molecule capable of activating the function of a long terminal repeat of a retrovirus comprising: a) introducing DNA containing the long terminal repeat regulated protein kinase C Bι gene into a population of transformed cells; b) selecting cells which expresses the protein kinase C β x gene; c) contacting the selected cells from step b) with an amount of the molecule effective to activate the function of the long terminal repeat; c) determining the level of expression of the protein kinase C β x gene, the increase in the level of expression indicating that the molecule is capable of activating the function of the long terminal repeat. 23. A method of identifying a gene which inhibits the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C β χ gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor genes and suppress expression of the long terminal repeat regulated protein kinase C β x gene; and c) isolating the gene which inhibits the function of the long terminal repeat. 24. A method of claim 23, where in step c) the gene is isolated by subtractive hybridization or differential display. 25. The gene identified by the method of claim 23. 26. The gene operatively linked to a promoter of RNA transcription. 27. A vector which comprises the gene of claim 25. 28. A virus comprising the gene of claim 27. 29. A polypeptide encoded by the gene of claim 25. 30. A method of identifying a protein factor capable of inhibiting the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C B gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor gene and to suppress expression of the long terminal repeat regulated protein kinase C β x gene; c) isolating and lysing the nuclei from the cell from step b to produce an extract; d) contacting the extract with the long terminal repeat; and e) isolating the protein factor which binds with the terminal repeat, thereby isolating the factor capable of inhibiting the function of the long terminal repeat. 31. The protein factor identified by method of claim 30. 32. A method of identifying a gene which activates the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C β x gene into a transformed cell; b) treating the cells of step (a) so as to induce the expression of at least one progression suppressor gene and suppress expression of the long terminal repeat regulated protein kinase C fii gene; c) treating the induced cell from step b) with an amount of protein kinase C activating compound or an inhibitor of serine or threonine specific protein phosphatase effective to activate the long terminal repeat; and d) isolating a gene which activates the long terminal repeat. 33. A method of claim 32 wherein the protein kinase C activating compound is a tumor promoting diterpene phorbol ester. 34. A method of claim 32 wherein the protein kinase C activating compound is a synthetic protein kinase C activator. 35. A method of claim 34, wherein the activator is ADMB or DHL 36. A method of claim 32 wherein the inhibitor is calyculin or okadaic acid. 37. A method of claim 32, where in step d) the gene is isolated by subtractive hybridization or differential display. 38. The gene identified by the method of claim 32. 39. The gene operatively linked to a promoter of RNA transcription. 40. A vector which comprises the gene of claim 38. 41. A virus comprising the gene of claim 38. 42. A polypeptide encoded by the gene of claim 38. 43. A method of identifying a protein factor capable of activating the function of the long terminal repeat of a retrovirus comprising: a) introducing DNA containing a long terminal repeat regulated protein kinase C B x gene into a population of transformed cells; b) treating the cells from step (a) so as to induce the expression of at least one progression suppressor gene and suppress expression of the long terminal repeat regulated protein kinase C β x gene; c) treating the induced cell from step b) with an amount of a protein kinase C activating compound or an inhibitor of serine or threonine specific protein phosphatase effective to activate the long terminal repeat; d) isolating and lysing the nuclei from the cell from step c to produce an extract; e) contacting the extract with DNA containing the long terminal repeat; and f) isolating the protein factor which binds with the terminal repeat, thereby identifying the factor capable of activating the function of the long terminal repeat. 44. The protein factor identified by method of claim 42.

Inventors: FISHER PAUL B, SU ZAO-ZHONG, FISHER, PAUL, B., SU, ZAO-ZHONG
Applicants: UNIV COLUMBIA, THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF
Application year: 1995
Main classification: C12Q1
IPC codes: C12N9, C12N15, C12Q1