EP1001966A1

EP1001966A1 - A senescence gene and its use in the treatment of cancer and other diseases

Info

Publication number: EP1001966A1
Application number: EP98933073A
Authority: EP
Inventors: Olivia M. Pereira-Smith; Yi Ning; Michael J. Bertram; Nathalie Berube; Xin Swanson; James K. Leung; Qitao Ran
Original assignee: Baylor College of Medicine
Current assignee: Baylor College of Medicine
Priority date: 1997-07-03
Filing date: 1998-07-02
Publication date: 2000-05-24
Also published as: WO1999001465A1; AU8282498A; EP1001966A4

Abstract

The gene sequences that comprise genetic determinants that control the capacity of cells to proliferate and methods for isolating same are provided. The sequences can be used in a gene therapy for cancer and viral infection, particularly AIDS.

Description

A SENESCENCE GENE AND ITS USE IN THE TREATMENT OF CANCER AND OTHER DISEASES

The work herein was supported by grants from the United States Government. The United States Government may have certain rights in this invention.

Field of the Invention

The present invention is in the field of recombinant DNA technology. This invention is directed to a gene sequence that controls the capacity of cells to proliferate. The invention is directed further to the use of this gene sequence in the diagnosis and treatment of cancer and other diseases.

Background of the Invention I. Cellular Control of Proliferation

Normal human diploid cells have a finite potential for proliferative growth. Thus, as the aging process occurs, the capacity of cells to proliferate gradually diminishes. Under controlled conditions, in vitro cultured human cells can proliferate maximally only to about 80 cumulative population doublings. The proliferative potential of such cells has been found to be a function of the number of cumulative population doublings which the cell has undergone, and to be inversely proportional to the in vivo age of the cell donor.

Such loss of cellular proliferative capacity is termed "senescence," and is the in vitro analog of aging (Hayflick, L. et al, Exper. Cell Res. 37: 614-636 (1965); Norwood, T.H. et al, In: Handbook of the Biology of Aging (2nd ed.), Finch, C.E. et al. (eds.) Van Nostrand, New York pp. 291-

311 (1985); Goldstein, S., Science 249:1129-1133 (1990); Smith, J.R., Monogr. Devel. Biol. 17:193-208 (1984); Smith, J.R. et al, Exper. Gerontol. 24:377-381 (1989), all herein incorporated by reference).

Experimental evidence suggests that the age-dependent loss of proliferative potential may be the function of a genetic program. Indeed, the onset of senescence and aging are accompanied by significant changes in the profile of genes that are expressed. Through an analysis of such changes, researchers have identified unique mRNAs that are amplified in senescent cells in vitro, thus suggesting that cellular senescence is mediated by an inhibitor of DNA synthesis (Spiering, A.I. et al., Exper.

Cell Res. 179:159-167 (1988); and Pereira-Smith, O.M., et al, Exper. Cell Res. 160:297-306 (1985)). The recognition of such changes has prompted efforts to clone the genes that encode the factors that control cellular senescence and proliferative capacity. Smith, J.R. (PCT Patent Appln. Publication No. WO 93/12251) describes senescent cell-derived inhibitors of DNA synthesis.

Although the proliferative capacity of a cell is believed to be regulated carefully, cells can, through mutation or viral infection, lose their ability to respond to such regulatory factors and thereby re-acquire a capacity to proliferate. Thus, cells cultured in vitro can, at low frequency, escape senescence and thereby become immortalized. In vivo, such uncontrolled cellular proliferation is a defining characteristic of cancer.

Insight into the control of cellular proliferation has been gained from studies in which normal and immortal cells have been fused to form synkaryons. Such studies have demonstrated that the quiescent phenotype of a normal cell is dominant over the proliferative phenotype of an immortalized carcinoma cell (Bunn, CL. et al, Exper. Cell Res. 127:385-396 (1985); Pereira-Smith, O.M., et al, Somat. Cell Genet. 7:411- 421 (1981); Pereira-Smith, O.M., et al, Science 221:964-966 (1983);

Muggleton-Harris, A., et al, Somat. Cell Genet. 6:689-698 (1980)).

Pereira-Smith, O.M. et al. demonstrated that pair-wise fusions between different immortalized cells occasionally resulted in hybrids that had lost their capacity to proliferate (Pereira-Smith, O.M., et al, Proc. Natl Acad. Sci. (U.S. A) 85:6042-6046 (1988)). By systematically conducting a pair-wise analysis, four complementation groups were identified (A, B, C, and D). The fusion of cells having the same complementation group created hybrids that maintained the immortalized character of the parental cells. In contrast, when immortalized cells of different complementation groups were fused, the normal genes of one parent "complemented" the deficient mutant genes of the other, and the resulting hybrids became senescent. This discovery suggested the possibility that a small number of undefined and unidentified genes or pathways might control the ability of a cell to proliferate.

π. AIDS and HIV Infection AIDS (Acquired Immunodeficiency Syndrome) is believed to be caused by a human immunodeficiency virus (HIV). By the end of the 20th century, 40 million people are expected to suffer from AIDS. At least two primary strains of HIV, designated HIV-1 and HIV-2, have been associated with the onset of AIDS. HIV is a member of the retrovirus family. As such, it replicates through an RNA intermediate. Unlike other families of viruses, the propagation of retroviruses occur preferentially, if not exclusively, in proliferating cells. Indeed, the primary drugs used for treating AIDS patients are nucleotide analogues (such as azidothymidine (AZT) and deoxynucleotides other than AZT such as 2',3'-dideoxyadenosine (ddA), dideoxyinosine (ddl), and dideoxycytidine (ddC)) that are directed against the HIV reverse transcriptase gene and viral replication. Administration of these nucleotide analogues to AIDS patients has had some success in slowing the rate of intercellular spreading of the virus in a small number of cases. The available drugs and therapies have, however, been unable to halt the fatal progress of the disease. Moreover, undesired side effects and high cost have limited the applicability and availability of such agents. At present, no long term successful therapy for AIDS exists.

In sum, an ability to arrest the proliferative capacity of cells would provide a means for attenuating aberrant cellular proliferation, and thus would provide an effective therapy for cancer. Similarly, because retroviruses preferentially replicate in proliferating cells, an ability to arrest temporarily cellular proliferation would provide a means for combatting retroviral infection, and in particular, would comprise a therapy for AIDS. In fact, arresting or inhibiting cellular proliferation would prove useful in treating any condition characterized by undesirable cellular growth. The present invention provides molecules and methods capable of mediating such effects.

All references cited herein are incorporated by reference.

Summary of the Invention

The present invention concerns, in part, the recognition of a gene sequence that is capable of inhibiting or arresting the proliferative capacity of a cell. In one embodiment, such a gene sequence, when combined with a suitable genetic therapy comprises a therapy for cancer and diseases, such as AIDS, that involve proliferative cells.

In a second embodiment, the identification of the gene sequence provides a unique means for diagnosing the extent and/or severity of malignancy in a biopsy or other tissue sample. Such diagnostic utility reflects the expectation that the gene sequence is expressed in non- proliferating cells, and thus, the presence of RNA corresponding to the gene sequence, or of the protein encoded by the gene sequence is usually indicative of a normal, non-proliferative state. Hence, the failure to identify such RNA or protein in a histological sample provides a means for identifying or characterizing tumors and neoplastic tissue. The diagnostic utility is further reflected in the use of other diagnostic modes such as detection by polymerase chain reaction (PCR) or hybridization. PCR can be used to detect the presence of the senescence sequence in cells. The absence of the sequence or its presence at low levels is indicative of proliferating cell types. Furthermore, PCR can also be used to determine the existence of mutations in the senescence sequence. Similarly the absence of hybridization is evidence of the absence of the senescence sequence or represents mutational changes in the material tested. Either of these methods can be used to identify and characterize the senescence state of the target cells.

In detail, the invention provides a nucleic acid molecule that is capable of inhibiting undesired or uncontrolled cellular proliferation, such as the proliferation of hyperproliferative cells. Hyperproliferative cells include cells such as cancerous cells, or virally-infected cells of a human patient.

The invention is particularly concerned with the embodiments wherein the cell is a cancerous cell belonging to complementation group B.

The invention is concerned additionally with the embodiments wherein the nucleic acid molecule is capable of hybridizing to a nucleic acid sequence identical to, or substantially similar to one of the following nucleic acid molecules: a fragment of human chromosome 4, a fragment of human chromosome 4 located^' at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4. The invention further provides embodiments wherein the nucleic acid molecule contains its own promoter, or a tumor specific promoter operably linked to the chromosomal fragment and is capable of mediating the preferential expression of the chromosomal fragment in a tumor cell, and/or wherein the nucleic acid molecule is a viral vector, the vector being encapsulated in a viral coat. The invention further provides a protein, substantially free of its natural contaminants, wherein the protein is encoded by the nucleic acid molecule of the present invention and is capable of inhibiting undesired or uncontrolled cellular proliferation, such as the proliferation of hyperproliferative cell, such as a cancerous or virally-infected cell of an animal.

The invention also provides a method for treating cancer in an individual which comprises providing to a cancerous cell of the individual an effective amount of a nucleic acid molecule having a sequence identical to, or substantially similar to one of the following nucleic acid molecules: a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or

MORF 4, that is capable of inhibiting the proliferation of the cell.

The invention also provides a protein, substantially free of its natural contaminants, where the protein sequence is substantially identical to SEQ ID NOS. 2, 4, or 6 and where that protein is capable of inhibiting the proliferation of a cell of a patient. The invention is particularly drawn to inhibiting the proliferation of a cancerous cell, wherein said cancerous cell belongs to a group B complementation group.

A further object of the present invention is to provide a nucleic acid molecule comprising a sequence substantially identical to a fragment of human chromosome 4, which is capable of inhibiting undesired or uncontrolled proliferation of a cell of an animal. Another object of the present invention is to provide the nucleic acid molecule fragment of human chromosome 4 located at 4q 33-34.1.

Another object of the present invention is to provide the nucleic acid molecule fragment of human chromosome 4 with the sequence in

SEQ ID NO. 1, or a fragment or fragments thereof.

One object of the present invention is to provide a nucleic acid molecule which is MORF 4 or a fragment or fragments thereof.

Another object of the invention is to target the invention against various cell-types such as a hyperproliferative cell. Further objects of the invention target virally-infected cells or cancer cells as the hyperproliferative cells. Another object of the invention is to target virally-infected cells infected with human immunodeficiency virus. Still another object of the present invention is to target cancerous cells belonging to a group B complementation group.

Another object of the present invention provides a nucleic acid molecule additionally containing a tumor specific promoter which is operably linked to the chromosomal fragment, and where the promoter is capable of mediating the preferential expression of the chromosomal fragment in a tumor cell. Another object of the present invention provides a nucleic acid molecule additionally containing a native promoter which is operably linked to the chromosomal fragment, and where the promoter is capable of mediating the preferential expression of the chromosomal fragment in a cell. One object of the present invention is to provide a nucleic acid molecule inserted into a non-viral vector.

Yet another object of the present invention is to provide a nucleic acid molecule which has been inserted into a viral vector, where the viral vector has been encapsulated in a viral coat. A further object is to provide the nucleic acid molecule where the viral vector is an adenovirus, and the viral coat is an adenoviral coat.

Another object of the present invention is to provide a protein, substantially free of its natural contaminants, wherein the protein is encoded by a nucleic acid molecule having a sequence substantially identical to a fragment from human chromosome 4, and wherein the protein is capable of inhibiting the proliferation of a cell of a patient. Other objects of the present invention are to target cells such as cancerous cells or virally-infected cells. Still another object is to target cancerous cells belonging to a group B complementation group. Another object of the present invention targets virally-infected cells infected with human immunodeficiency virus.

One object of the present invention is to provide a method for treating cancer in an individual which comprises providing to a cancerous cell of said individual an effective amount of a nucleic acid molecule having a sequence substantially identical to a fragment from human chromosome 4, wherein the nucleic acid molecule is capable of inhibiting the proliferation of said cell. Another object is to target cancerous cells belonging to a group complementation group B.

Another object of the present invention is to provide a method for treating viral infection in an individual which comprises providing to a virally-infected cell of said individual an effective amount of a nucleic acid molecule having a sequence substantially identical to a fragment of human chromosome 4, wherein said nucleic acid molecule is capable of inhibiting the proliferation of virus in the virally-infected cell. A further object of the present invention is to target virally-infected cells infected with human immunodeficiency virus.

One object of the present invention is to provide a method for treating cancer in an individual which comprises providing to a cancerous cell in said individual an effective amount of a protein, substantially free of its natural contaminants, wherein said protein is encoded by a nucleic acid molecule having a sequence substantially identical to a fragment from human chromosome 4, and wherein said protein is capable of inhibiting the proliferation in the cancerous cell. A further object of the invention is to target cancerous cells belonging to complementation group B.

Still another object of the present invention is to provide a method for treating viral infection in an individual which comprises providing to a virally-infected cell in said individual an effective amount of a protein, substantially free of its natural contaminants, wherein the protein is encoded by a nucleic acid molecule having a sequence substantially identical to a fragment from human chromosome 4, and wherein said protein is capable of inhibiting the proliferation of virus in the virally- infected cell. A further object of the present invention is to target virally- infected cells infected with human immunodeficiency virus.

Another object of the invention is to provide antibodies directed against the protein products of the senescence inducing gene sequences. These proteins can be derived from the chromosomal fragments, or from the specific genes themselves. This object of the invention includes antibodies that specifically bind the protein products of nucleic acid molecules capable of inducing senescence in a proliferating cell. A further object includes binding to a protein encoded by one a sequence identical to, or substantially similar to one of the following nucleic acid molecules: a fragment of human chromosome 4, a fragment of human chromosome

4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4. Another object is to provide antibodies to protein products that are the same or substantially similar to SEQ ID NOS. 2, 4, or 6.

Another object of the invention is to provide a method of detecting the amount of protein in a cell identical or substantially similar to a protein encoded by a sequence selected from a group including a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4 by exposing the cellular contents to an antibody directed against a member of the group and measuring the amount of antibody binding.

Another object of the present invention provides a method of pre- screening nucleotide analogs for use in inhibiting cell proliferation, comprising measuring the amount of binding of a protein encoded by a nucleic acid molecule selected from a group including a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1,

SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4 to ATP or GTP; and measuring the amount of binding of said protein to said protein molecule in the presence of a nucleotide analog; then comparing the amount of binding with and without said nucleotide analog. A nucleotide analog which increases the amount of binding being a suitable candidate for use in inhibition of cellular proliferation. A further object of the invention is such an assay in which the protein sequence is identical or substantially similar to SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 6.

Another object of the present invention provides a method of pre- screening test substances for use in inhibiting cell proliferation, comprising: measuring the amount of protein-protein binding at a leucine zipper site of a protein encoded by a nucleic acid molecule selected from the a group comprising a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4 on cellular proliferation; and measuring the amount of binding at said leucine zipper site in the presence of said test substance; and comparing the amount of binding with and without said test substance. A test substance which increases the amount of binding being a suitable candidate for use in inhibition of cellular proliferation. A further object of the invention provides an assay in which the protein sequence is identical or substantially similar to SEQ ID NO.

2, SEQ ID NO. 4, or SEQ ID NO. 6.

Brief Description of the Drawings Figure 1 shows results of the BAC approach to isolation of candidate genes. Figure 2 is a map of a BAC contig corresponding to cytogenetic band 4g33-33.

Figure 3 shows morphology changes and Sen β-Gal staining in two HeLa + MORF 4 clones.

Figure 4 shows morphology of re-induced senescence in group B cells.

Figure 5 shows results of Zoo Southern blots. Figure 6 shows FLAG-tagged MORF 4 staining. Figure 7 shows a comparison of the DNA sequences of the cDNA 386h22 which is on chromosome 15 and contains introns, and MRG 1 (MORF related gene on chromosome 1) and MORF 4.

Figure 8 shows a comparison of predicted proteins from cDNA 386h22, MRG 1 and MORF 4 genomic sequences.

Figure 9 shows the genomic structure of MORF 4. Detailed Description of the Invention

It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

I. The Determinants of Cellular Proliferation

The present invention derives in part from the identification of gene sequences that are determinants of cellular proliferation.

As used herein, an "antibody" is an immunoglobulin protein that has a specific affinity for a given molecule called an antigen. As used herein, a "gene sequence" is a nucleic acid molecule.

As used herein, a "determinant of cellular proliferation" is a gene sequence that, when introduced into a recipient cell, alters the proliferative capacity of that cell. Any cell may serve as the recipient cell, however, the invention is particularly directed to the treatment of "hyperproliferative" cells. Such cells are characterized by undesired or uncontrolled growth, and include cancerous cells, virally infected cells, cells of the immune system, epidermal cells, etc.

As used herein, a molecule comprising "a sequence of a human chromosomal fragment" may be either RNA or DNA, and denotes that the nucleic acid molecule contains a region whose nucleotide sequence is

"substantially identical" to the nucleotide sequence that is naturally found in a chromosome of a human. Two sequences are substantially identical if each will hybridize to the complement of the other under stringent hybridization conditions. The determinants of the present invention thus comprise human chromosomal fragments or genes that are capable of inducing quiescence or senescence in a previously proliferating cell. Such determinants include the natural allelic variants of such sequences (i.e., those that are capable of inducing a proliferative state in previously quiescent or senescent cells as well as those that are capable of inducing quiescence or senescence in a previously proliferating cell). The determinants of the present invention further include mutated gene sequences of such allelic variants.

Methods for isolating, identifying and using such determinants are described below. The present invention provides a means for isolating the determinants of cellular proliferation that are present on human chromosomes, and in particular on human chromosome 4. In a preferred embodiment for isolating such determinants, microcell-mediated chromosomal transfer is used to identify the chromosome that possesses a particular determinant. Microcell-mediated chromosome transfer is a technique by which one can introduce single, intact chromosomes derived from one cell into another. The method has proven to be a valuable tool for genetic manipulation.

For such purpose, microcell donor cells are plated onto plastic bullets cut from tissue culture flasks and incubated in medium (such as

Eagle's minimal essential medium with Earles salts + 10% fetal bovine serum. Following attachment, colcemid (approximately 0.1 μg/ml) is added, resulting in micronucleation of 80-90% of the cells. The bullets are then centrifuged at 28,000g for approximately 50 minutes, and filtered (5 μm filter membrane) to increase the proportion of single chromosome microcells. Hybrid cells are formed preferably by fusing recipient cells with the microcells through a 60 second exposure to 50% polyethylene glycol 1500, followed by washes in serum-free medium.

Preferably, choice of the microcell donor and the recipient cell is such that the recombinant hybrid can be selected directly. Recipient cells, in general, comprise both stable hybrids and unstable hybrids (in which the human chromosome(s) transferred are unstable and are lost or segregated upon further cell growth). The presence of such instability is apparent after several population doublings. Thus, the cells are extensively cultured, and after approximately 100 population doublings, are evaluated to determine whether they contain the introduced human chromosome by DNA polymorphism analysis which allows the distinction of the introduced chromosome from the native genetic material of the recipient cell.

Once a quiescent hybrid cell line has been isolated, and, using the above-described methods, the identity of the human chromosome of its microcell donor has been ascertained, the isolation of a determinant of cellular proliferation is undertaken. Any of a variety of cytogenetic methods may be used for this purpose. Thus karyotype analysis can be performed. Alternatively, human chromosome-specific primers are used to detect human chromosome-specific amplification. A preferred method is to employ fluorescently labelled chromosomal markers such as are widely available, and to assess whether a particular clonal line contains a human chromosome that is capable of binding a particular chromosome- specific marker. Thus, in a preferred embodiment, the donor cell is formed by fusing such a murine cell with a normal human cell that carries a neomycin or hygromycin-resistance determinant on one of its chromosomes. Murine A9 cells are a preferred mouse cell for this purpose. A preferred recipient human cell is a tumor-derived fibroblast cell or epithelial-derived cell that has been previously assigned to one of the four complementation groups.

Such assignation is obtained by fusing the recipient cell with a cell of each of the four groups, and determining which cell from a complementation group is capable of forming a continuously proliferating hybrid cell.

In such manner, hybrid cells are prepared that contain any one of the human chromosomes. Such cells are then evaluated to determine the frequency with which their progeny exhibit a return to a quiescent or senescent state. The recognition of such a state is determined by any of a variety of means, such as by microscopic evaluation, turbidity, confluence, colony morphology, incorporation of radiolabelled nucleotides, etc.

Because human chromosomes are hundreds of thousands of kilobases in length, classical restriction endonuclease cleavage and cloning into plasmid vectors is unwieldy, and unlikely to be successful.

For this reason, the subcloning of the desired determinant of cellular proliferation is performed by conducting the above-described microcell fusion, and then evaluating the resultant quiescent clones to determine whether they contain an entire chromosome, or only a fragment of a chromosome. Clones containing such fragments arise spontaneously, via genetic rearrangement and translocation between the human and murine chromosomes of the microcell donor. Clones containing fragments of chromosomes are then employed to subclone the human chromosomal fragment. Such subcloning is optional, and where desired may be omitted in favor of conducting a marker analysis in the manner described below.

The bacterial artificial cloning (BAC) system is most preferably exploited for such optional sub-cloning. The BAC system employs a bacterial minichromosomal vector that is capable of containing between

50-1,000 kilobases. Thus, by fragmenting the relevant human chromosomes into approximately 300-500 parts, it is possible to obtain a library of chromosomal fragments that may then be screened to identity the fragment that contains the desired cellular proliferation determinant. The screening of such a library, however, is exceedingly laborious and complex.

The original hybrids, or the cloned subchromosomal fragments are preferably evaluated further to determine whether they contain or lack chromosome-specific markers. A senescent clone that was formed from, for example, a chromosome 4 microcell donor, but that lacks a particular chromosome 4-specific marker, indicates that the region adjacent to the marker site is not involved in regulating the quiescent state. Similarly, the identification of a particular chromosome 4-specific marker indicates that the region adjacent to the marker site is linked to the desired determinant of cellular proliferation. Suitable markers are well known in the art. Once one or more marker sites that flank a desired determinant of cellular proliferation have been identified, the determinant is cloned through the use of in vitro amplification procedures and then subcloned into cosmids or into viral vectors. The resulting constructs can be introduced into immortalized cells that, when transformed by a vector containing a determinant, exhibit a quiescent or senescent state.

As indicated, an evaluation of the capacity of normal cells to complement the abnormal proliferation of immortalized cells has identified four complementation groups as relevant to the control of quiescence and senescence. These four genes or gene pathways are likely part of a genetic program, the end point of which includes the overexpression of negative growth regulatory genes (such as the Cdk inhibitors p21 and pl6) which have been found to be overexpressed in senescent cells. The determinant of group B is on chromosome 4. The gene sequence that comprises this determinant is isolated using the above-described methods.

It has been previously shown that the phenotype of cell senescence is dominant over that of immortality, in that hybrids from normal- immortal fusions have limited proliferation potential (regain growth control). By fusing different immortal human cell lines with each other four complementation groups for indefinite division have been identified, indicating that there are four senescence genes or gene pathways.

Using microcell mediated chromosome transfer, it was demonstrated that a normal human chromosome 4 induced senescence specifically in multiple immortal cell lines assigned to Group B and had no effect on cell lines assigned to the other groups. This result provided evidence for a cell senescence gene on human chromosome 4.

A fragment of human chromosome 4 was obtained that was less than a megabase, in a mouse background, that could induce senescence when introduced into group B cell lines. Using AZu-PCR products specific to this piece of human DNA, high density BAC filters were probed and a contig of BACs to this region was obtained (Figures 1 and 2). The BACs were then used to directly screen high density cDNA filters and cDNAs identified were obtained. The BACs were also used to select cDNAs from a brain cDNA library and similar cDNAs were obtained. The cDNAs were sequenced to determine sequence homology and motifs indicative of function, and used as probes for Northern and Southern blots. They were also cloned into an expression vector under control of the CMV promoter for transfection studies. Various genomic regions that spanned the region of the cDNAs were cloned into a plasmid without a promoter, for use in transfection experiments in which the gene would be expressed from its own promoter.

Seven cDNAs have been partially to fully characterized to date. One cDNA 386h22 has homology to Seq. No. 5 (GenBank D14812) in the data base. The latter was cloned in a search for genes expressed during differentiation of a myeloid leukocyte cell line (Nomura, N. et al, DNA Res. 1:27-45 (1994)).

Genomic fragments that contain the regions similar to cDNA 386h22 were subcloned into plasmid vectors. These genomic fragments were transfected into group B cell lines. The genomic fragment with similarity to cDNA 386h22 induced senescence in group B cell lines, the others did not. cDNA 386h22 is not encoded by the genomic fragment from chromosome 4, but a gene with extensive similarity is encoded by the genomic fragment. The cDNA 386h22 corresponds to a gene on chromosome 15 as described below. A frame shift mutation introduced into the genomic fragment of 386h22 at the start codon abolished the senescence inducing activity of this molecule. These data indicated that the genomic DNA from chromosome 4 having similarity to cDNA 386h22 encodes the cellular senescence gene. The sequences are shown as Seq. I.D. Nos. 1, 3, and 5. The proteins encoded are also shown as Seq. I.D. Nos. 2, 4 and 6. Homology searches indicate potential phosphorylation, myristylation sites and a leucine zipper motif. The MORF 4 gene has interesting features predicted by computer analysis, such as a helix-loop-helix domain, one ATP/GTP binding domain and a leucine zipper motif that are shared by MRG 15 (386h22) which has a chromodomain plus another ATP/GTP binding domain, characteristic of transcription factors See eg. Busch, S.J., & Sassone-Corsi, P., Trends Genet. 6:36-40 (1990); Kadesch, T., Immunol Today 13:31-36 (1992);

Kageyama, R. et al, Crit. Rev. Neurobiol 9:177-188 (1995), though the DNA binding motif has low similarity to other proteins that bind to DNA. Thus, they can act in transcriptional control by binding to other proteins, including each other. However, they also have three separate amino acid motifs found in the telomere binding protein Eup 51kd, from Euplotes

Crassus (Wang, W. et al, Nucleic Acids Res. 24:6621-6629 (1992)) that is conserved in the S. cerevisiae and S. pombe homologs of MORF. This is in the helix-loop-helix domain and may indicate telomere binding capacity.

There are no known equivalents of the technology. The technology is novel in that there are no correlation between tumor type and complementation group assignment and this is a novel manner of categorizing tumors. In addition, it could be useful in controlling other hyper proliferative disorders and use of the anti-sense in inducing proliferation. The limited proliferative potential of normal human cells in culture is well documented and accepted as a model for aging at the cellular level. Through a genetic analysis aimed at understanding the basic mechanics of this phenomenon of cell aging, we have found that fusion of normal with immortal human cells yields hybrids with limited division potential, indicating that cellular senescence results from dominant genetic events.

By fusing different immortal cell lines with each other we have identified four complementation groups for indefinite division, suggesting that at least four genes or gene pathways are involved in senescence. By microcell mediated chromosome transfer we have determined that two of these genes are on human chromosomes 1 and 4 respectively. Our current hypothesis is that these four genes or gene pathways are part of a genetic program, the end point of which is the increased expression of negative growth regulatory genes, such as the Cdk inhibitors p21 and pl6, which we have found are overexpressed in senescent cells.

The sequence known as MORF 4 was cloned from the human genome region originally covered by the fragment of chromosome four which causes senescence in group B immortalized cell lines. The procedure was to isolate Bacterial Artificial Chromosomes (BACs) using amplified probes specific to the human chromosome 4 fragment. These probes identified BAC clones that could be aligned into a contiguous sequence of clones that spanned most of the chromosome 4 fragment of interest. Individual BACs were then used as probes to isolate fragments of candidate genes as cDNA copies of RNAs. The cDNA clone designated as 386h22 was isolated and confirmed to hybridize strongly to a fragment of BAC (designated 526E7). A fragment of the BAC hybridizing to the cDNA clone was subcloned into a vector and function tested in the cellular senescence assay. The clone caused the group B cells to senesce. Sequencing has revealed that the genomic clone was indeed highly similar to the cDNA clone but sufficient differences between the sequences indicate that the cDNA 386h22 is not encoded by the genomic DNA of BAC 526E7. The cDNA clone is transcribed from a separate gene that is located on human chromosome 15. The resulting proteins of the two genes are nearly identical and may have related function. MORF 4 encodes a protein lacking the first 88 amino acids found in MRG 15.

The relationship between the protein conceptually translated from the BAC derived gene, the protein conceptually translated from the cloned cDNA number 386h22 and the GenBank entry protein (D14812) is demonstrated by an alignment of their sequence (Table 1). BAC S26E7 protein cDHΛ 38es22 protein -MAPKQDPKPKFQEG ERVLCFHGPIΛYEAK CVKVAIKD QVKYFI

GB /D14812 protein MSSRKQGSQPRGQQS AEEENFKKPTRSNMQ RSKMRGAS SG

BAC 52SE7 protein M 89 cDNA 38 GH22 protein HYSGWNKNWDEWVPE SRV KTVOTNLQKQR ELQΪQU3QEQYAEGKM 89 GB SD14S12 protein KTAGPQ QK NLEPALPG — 57

EΛC 526Ξ7 protein RWAAPGKKTSGLQQK NIEVKTKKNKQKTPG NGDGGSTSETPQPPR cOHA 38GB22 psotein RGAAPGKKTSGJ QQK NVEVKTKKNKQKTPG NGDGGSTSE PQPPR GB §014812 protein RWGG RSJUCNPPS GSVRKTRKN QKTPG NGDGGSTSEΛPQPPR

EΛC S26E7 protein KKRAQVDPTVENEET FWNRVEVKVKIPEEL KPTOCVDDWDLITRQ 179 cDNA 386H22 protein KKRARVDPTVENEET FMNRVEVKVKIPEEL KPWLVDDVroi.ITRQK 179 GB {D14812 protein KKRJUUV0PTVESEEΛ FKNRKEVKVKTPEEL KPWLVEDWDLVTRQK 144

BAC 526E7 protein QLFYLPAEK GDSIF EDYANYKKSRGOTDN KEYAVNEVVAGIKGY corø, 38GB22 protein - QLFYLPAKKNVDSIL EDYANYKKSRGNTDN KEYAVNEWAGIKEY GB *D14812 protein QLFQLPAKKNVOAII, EEYANCKKSQGNVDN KEYAVNEWAGIKEY

BAC 52GE7 protein FNLMLGTQVLNKFER PQYAEILADCPDAPM SQVYGVPHLLRLSVQ 269 cDNA 38GH22 protein FNVMLGTQLLYKTER PQYAEIIADHPDAPM SQVYGAPH LRLFVR 269 GB *D14812 prot-.in FNVMLGTQI .YKFER PQYΛEH.IAHPDAEM SQ.VYG&PHT.T.RI,FVR 234

BAC S2GE7 protein IGAMLAYTPLNEKSΪ, ALLLNY HDFLKYIA KNSATLFSASDYEVA cDHA 386H22 protein IGAM AYTPLDEKSL AUJLNYLHDFLKYIΛ. KNSATLFSASDYEVa. GB *D14812 protein IGA IAYXtl-DEKSI. AL1.LGYI.HDFLKΪ1A KNSASLFTASDYKVA AT.T tTOCjHDmKYIA KNSΛΪIi

BAC 526E7 protein I.PEYHRKA.V 323 cDNA 386H22 protein SAEYHRKAL 288

GB *D14812 orotein gPEYHRKRV 323

The amino acids LALLLNYLHDFLKYLAK SATL indicate the region that apparently contains a protein interaction domain (also known as a leucine zipper). Observation of this conserved motif strongly suggests that these related proteins interact specifically with other proteins by contacts to this portion of the protein. These interactions can be critical to the function of the protein or its localization in the cell.

II. Therapeutic Utility

The molecules of the present invention may be used to control or suppress undesired or uncontrolled cellular proliferation. As used herein, cellular proliferation is said to be "undesired" when it comprises growth that for medical or cosmetic reasons is not wanted by a patient. Examples of such undesired growth include warts, moles, psoriasis lesions, etc. The regeneration of tissue incident to injury or trauma can comprise yet another example of undesired growth. For example, in patients who are to receive multiple surgical operations, the formation of "scar" tissue incident to the first such operation may encumber subsequent operations. Similarly, where only limited medical facilities such that the complete treatment of the injury will be delayed, the immediate regeneration of damaged tissue may not be desired. Growth is said to be uncontrolled where it leads to a medically significant proliferation of cells. Cancer, warts, molluscum contagiosum, etc. are examples of uncontrolled proliferation. As is evident, growth may be both uncontrolled and undesired.

As indicated above, the primary drugs presently used for treating AIDS patients are nucleotide analogues that affect the capacity of virally infected cells to replicate DNA. Similarly, nearly all chemotherapeutic anti-cancer agents currently in use interfere with DNA synthesis, or with the cellular ability to produce the precursors needed for DNA or RNA synthesis. Although the available anti-AIDS chemotherapies may slow the onset of the disease, such therapies have been unable to halt the fatal progress of the disease.

Similarly, chemotherapeutic antineoplastic agents have had only limited success in treating cancer. In many cancerous tumors, only a fractional sub-population of the tumor cells is actively dividing (Boyd,

M.R., In: Current Therapy in Oncology, (Niederhuber, J.E., Ed.), B.C. Decker, pp. 11-22 (1993)). The size of this subpopulation is inversely proportional to the size of the tumor. Hence, for large tumors, chemotherapeutic measures may have only very limited effectiveness. Many tissues and organs (such as bone marrow, precursors of gametes, hair follicles, and the epithelium lining the intestinal tract and the skin) have growth rates that exceed those of tumors. Hence, such tissue is adversely affected by conventional chemotherapy, and limits the dosage of the chemotherapeutic agent, and extent to which chemotherapy may be applied. Therefore, temporarily forcing these normal dividing cells into a reversible quiescent state may protect them from chemotherapeutic agents.

The genetic determinants of the present invention have therapeutic utility in the treatment of diseases such as AIDS and cancer. As a preferred initial step in such treatment, a cancerous, or virally infected cell of a patient is evaluated to determine which of the (four complementation group) determinants of cellular proliferation is capable of inhibiting the proliferation of the cell, or its capacity to support viral infection. In the case of T-cells and B-cells, any of the four determinants potentially work, however all of three B-cell types and two T-cell types tested were assignable to complementation group D, suggesting that a lesion in complementation group D may be preferentially responsible for abnormal T-cell or B-cell development. For cancer cells, such evaluation is accomplished by monitoring any of the characteristic features of attributes of such cells: cell surface antigens, immortality, absence of contact inhibition, anti-sense repression, hyperploidy, the capacity of the cells to exhibit the characteristics of aging cells, etc. For virally-infected cells, such evaluation can additionally include monitoring the capacity of the cell for its capacity to replicate the virus. After the particular complementation group has been established, then the molecules of the present invention may be administered to comprise a genetic therapy. The general principles of gene therapy have been discussed by Oldham, R.K., Principles of Biotherapy , Raven Press, NY, (1987); Boggs, S.S. Int. J. Cell Clon. 8:80-96 (1990); and Karson, E.M. Biol Reprod. 42:39-49 (1990); all of which references are incorporated by reference herein. Such gene therapy is provided to a recipient in order to treat (i.e. suppress, or attenuate) an existing condition, or to provide a prophylactic therapy to individuals who, due to inherited genetic mutations, somatic cell mutation, or behavioral or environmental factors are at enhanced risk. In a preferred embodiment, such therapy comprises providing an effective amount of either a single-stranded or a double-stranded nucleic acid molecule (DNA or RNA) to an individual. By controlling the concentration and/or expression of such gene sequences, it is possible to direct selectively either a transient alteration or a permanent alteration of the proliferative state of the recipient cell. Thus, the gene sequences may be incorporated into a viral, retroviral or plasmid vector, which may either be capable of autonomous propagation within the recipient cell, or incapable of such propagation (as by being replication deficient). In yet another embodiment, the nucleic acid sequences (either alone or incorporated into a vector) may be designed to integrate into chromosome of the genome of recipient cell, thereby permitting its "passive" maintenance in the progeny of that cell.

Typically, suitable plasmid vectors are designed to include a prokaryotic replicon and selectable marker, such that the propagation of the vector in bacterial cells is readily accomplished. Plasmid vectors using papovirus replicons ultimately kill their host cells, and thus are most suitable therapies involving transient expression. SV40-based vectors that may be used include pMSG (Pharmacia), pSVT7, pMT2 (Kaufman, R.J., Genetic Engineering: Principles and Methods, Vol. 9.

In contrast, plasmid vectors that employ the replicons of Epstein- Barr or bovine papilloma viruses do not generally cause cell death, and are thus suitable for long term propagation. Examples of such vectors include the BPV-1, pBV-lMTHA, pHEBo, p205 indicated above.

The gene therapy of the present invention can be accomplished using viral or retroviral vectors. Examples of suitable vectors are discussed by Fletcher, FA. et al, J. Exper. Med. 174:837-845 (1991);

Makela, T.P. et al, Gene 118:293-294 (1992); Porgador, A. et al, Cane. Res. 52:3679-3686 (1992); Yoshimura, K. et al, Nucl. Acids Res. 20:3233- 3240 (1992); Li , B. et al, Proc. Natl Acad. Sci. (U.S.A.) 86:8892-8896 (1989); Ohi, S. et al, Gene 89:279-282 (1990); and Russel, S.J. et al, J. Virol 66:2821-2828 (1992).

Adenoviruses, especially modified adenoviruses, are a preferred viral vector for delivering the therapeutic gene sequences of the present invention in order to treat cancer. The human adenoviruses, particularly types 2, 5, and 12, have been characterized most extensively, and these viruses have served as valuable tools in the study of the molecular biology of DNA replication, transcription, RNA processing, and protein synthesis in mammalian cells. The biology of adenoviruses is reviewed by Graham, F.L. et al, Methods in Molecular Biology: Gene Transfer and Expression Protocols (1991), Vol. 7, Chap. 11, pp. 109-128, incorporated by reference herein. Adenoviruses have several salient advantages over other gene therapy vectors. The viral particle is relatively stable, and, in the case of serotypes commonly used as vectors to date, the viral genome does not undergo rearrangement at a high rate. Insertions of foreign genes are generally maintained without change through successive rounds of viral replication. The adenovirus genome is also relatively easy to manipulate by recombinant DNA techniques, and the virus replicates efficiently in permissive 293 host cells. Unlike retroviral vectors, adenoviral vectors do not require host cell replication in order to achieve high-level expression. Thus, they are particularly suitable for prophylactic gene therapy. In a preferred sub-embodiment, the adenoviral vector is modified so as to render it incapable of replicating, such as by deleting a critical gene in the El region of the viral genome. Such vectors can only be propagated in cell lines such as the permissive 293 host cell line, which provides the necessary Ela and Elb gene products in trans (See Graham, F.L. et al. above). Whereas wild-type Ad5 (containing El genes, and competent for viral replication in cells) can be cytopathic within 6 to 48 hours, the deletion of the El genes precludes cytopathy. Replication- deficient adenovirus have a finite lifespan (several weeks or more) before being degraded by host nucleases. Thus, such vectors are used to accomplish transient therapy.

The replication-deficient adenoviral vectors currently utilized for in vivo gene transfer are derived largely from adenovirus serotype 5 (Ad5). Replication-deficit adenoviral vectors have been used to mediate in vivo gene transfer into bronchial epithelium (See Rosenfeld, MA. et al, Cell 68:143-155 (1992)) and skeletal muscle (See Quantin, B. et al, Proc. Natl. Acad. Sci. (U.SA.) 89:2581-2584 (1992)).

The nucleic acids of the present invention can be introduced into target cells by various techniques known in the art: fusion of recipient cells with bacterial spheroblasts, liposomes, erythrocyte-membrane vesicles, whole-cell fusion, through uptake of DNA complexed with non- histone nuclear proteins or with poly lysine-carrying receptor ligands, by microinjection or targeting with microprojectiles, or by the use of transducing viruses.

In a preferred embodiment for treating cancer, the introduced molecules are preferentially expressed in tumor cells. In one embodiment, the vector contains appropriate transcriptional or translational regulatory elements, such that the proliferation determinants are preferentially expressed in tumor cells. Thus, whereas any suitable mammalian promoter may be employed to mediate expression, it is preferable in the treatment of cancer to employ tumor-specific promoters (i.e. promoters that are more active in tumor cells than in non-tumor cells). A promoter is said to be operably linked to a gene sequence if it controls or mediates the transcription or translation of the gene sequence or a subsequence thereof.

Preferred examples of such promoters include the native promoter naturally associated with a gene of a complementation group, the α- fetoprotein promoter, the amylase promoter (especially, the murine amylase promoter), the cathepsin E promoter, the Ml muscarinic receptor promoter, the γ-glutamyl transferase promoter, etc., and especially, the CMV promoter. Suitable α-fetoprotein promoter sequences are present in the vectors PSVA F0.4 CAT^A and PAF 5.1 02-CAT) (Watanabe et al., J.

Biol Chem. 262:4812-4818 (1987)). The PSVA F0.4 CAT^A vector contains 5 kb of flanking DNA with a deletion of approximately 2 kb between -1.0 and -3.0. The PAF 5.1 (Θ2-CAT) vector encompasses approximately 400 base pairs of the a-fetoprotein 5' flanking sequence which lies between - 3.7 kb and -3.3 kb, coupled to the SV40 promoter in the PSC1 CAT vector. Suitable amylase promoters, especially murine amylase promoter sequences are described by Wu et al, Molec. Cell. Biol. 11:4423-4430 (1991). Suitable cathepsin E promoter sequences are described by Azuma et al, J. Biol Chem., 267:1609-1614 (1992). Suitable Ml muscarinic receptor promoter sequences are described by Fraser et al, Molec. Pharmacol 36:840-847 (1989) and by Bonner, Trends Neurosci. 12:148-

151 (1989). Vectors containing suitable γ-glutamyl transferase promoter sequences are described by Rajagopalan, S. et al, J. Biol. Chem. 265:11721-11725 (1990). Suitable CMV promoter sequences are obtained from the CMV-promoted β-galactosidase expression vector, CMBβ (See MacGregor, G.R. et al, Nucleic Acids Res. 17:2365 (1989)).

In a second embodiment, the vector is engineered to array receptors or ligands for an antigen present on the tumor cell that is the recipient of the vector. Such markers are discussed by Drebin, J.A. et al, Current Therapy in Oncology, pp. 58-61 (1993). As a consequence of such array, the vectors preferentially adsorb to tumor cells, and thereby impart selectively their therapeutic value.

For the treatment of AIDS, lymphotrophic viral vectors are preferred. In one embodiment, such vectors are produced by modifying an existing lymphotrophic virus (HIV, SIV, EB, etc.). Alternatively, such vectors can comprise non-lymphotrophic viruses that have been modified to permit them to adsorb to and infect CD4+ cells. For example, synthetic viral vectors are formed that array the HIV gp 120 protein that is capable of binding to the CD4 receptor.

For the treatment of psoriasis, warts, moles, and other skin conditions of undesired or uncontrolled cellular proliferation, dermatrophic viral vectors, such as herpes viral vectors, etc., are employed to deliver the therapeutic gene sequences of the present invention.

As indicated above, a preferred embodiment of the invention comprises vectors that are capable of expressing the incorporated proliferation determinant. In an alternative embodiment, the desired gene therapy is mediated in the absence of expression, most preferably via recombination. As is appreciated, the recipient cells possess a mutated allele of the introduced determinant, and indeed, it is the presence of such a mutated allele that is responsible for the disease that is to be treated. The introduction of a normal allele of the determinant into such cells permits the introduced gene sequence to recombine with the chromosomal allele to thereby accomplish the "repair" or "replacement" of the mutated sequence. Since such repair or replacement converts a proliferating cell into a quiescent cell, such therapy will have a cumulative effect, even if it occurs at low frequency. Indeed, if such methods were used in concert with conventional methods, even very low frequency recombinational events could have significant therapeutic benefit. For example, anaplastic cancer of the thyroid is essentially incurable; such cells fail to take up the radioactive iodine isotopes that comprise a therapy for other forms of thyroid cancer. Even if the therapeutic molecules of the present invention provide only a very low level recombinational repair of the proliferative state of such cells, any incorporation of iodine by such cells would lead to the death of adjacent, non-iodine-incorporating cells.

m. Diagnostic Utility Apart from their therapeutic utility, the molecules of the present invention are used to diagnose the predisposition of an individual to cancer, and to determine which of the four complementation group pathways has been altered. Such information is correlated against the accumulated data of amenability of such tumors, or their refractiveness, with respect to a particular chemotherapeutic agent or regime. Moreover, the identification of the gene sequences of the determinants of cellular proliferation permits the development of determinant-specific probes that are used (in conjunction with an amplification procedure, such as PCR) to assess whether an individual carries a mutation in one of the determinants. The capacity to evaluate the presence of such mutations provides an extremely sensitive method for diagnosing cancer. As such, the method is employed at an extremely early stage, and thereby provide the physician with greater flexibility in treating the cancer. The diagnostic utility is further reflected in the use of other diagnostic modes such as detection or characterization of cellular senescence by polymerase chain reaction (PCR) or hybridization. PCR can be used to detect the presence of the senescence sequence in cells. The absence of the sequence or its presence at low levels is indicative of proliferating cell types. Furthermore, PCR can also be used to determine the existence of mutations in the senescence sequence. Similarly the absence of hybridization is evidence of the absence of the senescence sequence or represents mutational changes in the material tested. Either of these methods can be used to identify and characterize the senescence state of the target cells.

IV. The Analogs of the Molecules of the Present Invention and Their Uses

The present invention contemplates the use of any of a variety of chemical agents to either inhibit or enable DNA synthesis. Such agents may be: (1) a nucleic acid molecule, (2) a protein, or (3) a compound whose structure mimics that of either a nucleic acid molecule or a protein (i.e. a

"peptidomimetic" agent).

As indicated above, in the most preferred embodiment of the present invention, the agents of the present invention comprises nucleic acid molecules. In particular, such molecules include the naturally occurring gene sequences of the determinants of cellular proliferation that have been purified from their natural contaminants. In a preferred sub- embodiment, the sequence of the molecules are selected so as to be superior to such natural gene sequences. To obtain such superior sequences, the naturally occurring sequences are mutagenized, either by random mutagenic means, or by site-directed mutagenic protocols. The mutated species of molecules are then introduced into transformed, immortal or cancer cells, and the kinetics of growth inhibition is determined. Molecules that exhibit increased velocity or efficiency in inhibiting cellular proliferation are identified and recovered. Such molecules provide a non-naturally occurring therapeutic gene sequence.

In an alternative embodiment, the gene sequences of the present invention are used to express encoded gene products that can then be delivered to cancerous or transformed cells via electroporation, liposome mediated fusion, pseudoviral incorporation, etc. The gene products produced by the above-described non-naturally occurring therapeutic gene sequences can alternatively be used for this purpose.

In yet a third embodiment, the invention uses a compound whose structure mimics that of either a nucleic acid molecule or a protein (i.e. a "peptidomimetic" agent). Such structural similarity is determined via binding studies, by crystallographic means, etc.

In addition to use in expressing proteins and polypeptides and in defining desirable analogs, the nucleic acid molecules of the present invention are used to produce antisense nucleic acid molecules capable of binding to an endogenous sequence and inhibiting its activity, etc. A particularly preferred such agent is an antisense oligonucleotide.

In general, an "antisense oligonucleotide" is a nucleic acid (either DNA or RNA) whose sequence is complementary to the sequence of a target mRNA molecule (or its corresponding gene) such that it is capable of binding to, or hybridizing with, the mRNA molecule (or the gene), and thereby impairing (i.e. attenuating or preventing) the translation of the mRNA molecule into a gene product. To act as an antisense oligonucleotide, the nucleic acid molecule must be capable of binding to or hybridizing with that portion of target mRNA. Antisense oligonucleotides are disclosed in European Patent Application Publication Nos. 263,740; 335,451; and 329,882, and in PCT Publication No. WO90/00624, all of which references are incorporated by reference herein. Preferably, the antisense oligonucleotide is about 10-30 nucleotides in length, most preferably, about 15-24 nucleotides in length.

Any means known in the art to synthesize the antisense oligonucleotides of the present invention may be used. Automated nucleic acid synthesizers may be employed for this purpose. In addition, desired nucleotides of any sequence are obtained from any commercial supplier of such custom molecules.

In one embodiment the invention provides a nucleic acid molecule comprising a sequence substantially identical to a fragment of human chromosome 4, wherein the fragment is capable of inhibiting undesired or uncontrolled proliferation of a cell of an animal. In another embodiment the nucleic acid molecule is a fragment of human chromosome 4 located at 4q 33-34.1. In yet another embodiment the nucleic acid molecule is a fragment of human chromosome 4 with the sequence in SEQ ID 1 or is a fragment or fragments thereof.

Another embodiment of the invention utilizes a nucleic acid molecule which is MORF 4 or a fragment or fragments thereof. Other embodiments of the invention are directed against various cell-types such as a hyperproliferative cell. Further embodiments of the invention target virally-infected cells or cancer cells as the hyperproliferative cells. In a further embodiment the virally-infected cell is infected with human immunodeficiency virus. In another embodiment the cancerous cell belongs to a group B complementation group.

Other embodiments of the invention include where the nucleic acid molecule additionally contains a tumor specific promoter which is operably linked to the chromosomal fragment, and the promoter is capable of mediating the preferential expression of the chromosomal fragment in a tumor cell. Another embodiment includes where the nucleic acid molecule additionally contains a native promoter which is operably linked to the chromosomal fragment, and the promoter is capable of mediating the preferential expression of the chromosomal fragment in a cell.

In another embodiment the nucleic acid molecule is inserted into a non-viral vector.

Other embodiments of the invention include where the nucleic acid has been inserted into a viral vector, where the viral vector has been encapsulated in a viral coat. In further embodiments the viral vector is an adenovirus, and the viral coat is an adenoviral coat. Other embodiments of the invention provide a protein, substantially free of its natural contaminants, wherein the protein is encoded by a nucleic acid molecule having a sequence substantially identical to a fragment from human chromosome 4, and wherein the protein is capable of inhibiting the proliferation of a cell of a patient. In further embodiments the cell is a cancerous cell or a virally-infected cell. In one embodiment the cancerous cell belongs to a group B complementation group. In another embodiment the virally-infected cell is infected with human immunodeficiency virus.

One embodiment of the invention provides a method for treating cancer in an individual which comprises providing to a cancerous cell of said individual an effective amount of a nucleic acid molecule having a sequence substantially identical to a fragment from human chromosome 4, wherein the nucleic acid molecule is capable of inhibiting the proliferation of said cell. Other embodiments include where the cancerous cell belongs to a group complementation group B.

Another embodiment of the invention provides a method for treating viral infection in an individual which comprises providing to a virally-infected cell of said individual an effective amount of a nucleic acid molecule having a sequence substantially identical to a fragment of human chromosome 4, wherein said nucleic acid molecule is capable of inhibiting the proliferation of virus in the virally-infected cell. In a further embodiment the virally-infected cell is a cell infected with human immunodeficiency virus.

Another embodiment of the invention provides a method for treating cancer in an individual which comprises providing to a cancerous cell in said individual an effective amount of a protein, substantially free of its natural contaminants, wherein said protein is encoded by a nucleic acid molecule having a sequence substantially identical to a fragment from human chromosome 4, and wherein said protein is capable of inhibiting the proliferation in the cancerous cell. In a further embodiment the cancerous cell belongs to complementation group B.

Still another embodiment provides a method for treating viral infection in an individual which comprises providing to a virally-infected cell in said individual an effective amount of a protein, substantially free of its natural contaminants, wherein the protein is encoded by a nucleic acid molecule having a sequence substantially identical to a fragment from human chromosome 4, and wherein said protein is capable of inhibiting the proliferation of virus in the virally-infected cell. In a further embodiment the virally-infected cell is a cell infected with human immunodeficiency virus. Another embodiment of the invention provides a method of detecting the amount of protein in a cell identical or substantially similar to a protein encoded by a sequence selected from a group including a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4 by exposing the cellular contents to an antibody directed against a member of the group and measuring the amount of antibody binding. Another embodiment of the present invention provides a method of pre-screening nucleotide analogs for use in inhibiting cell proliferation, comprising measuring the amount of binding of a protein encoded by a nucleic acid molecule selected from a group including a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1,

SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4 to ATP or GTP; and measuring the amount of binding of said protein to said protein molecule in the presence of a nucleotide analog; then comparing the amount of binding with and without said nucleotide analog. A nucleotide analog which increases the amount of binding being a suitable candidate for use in inhibition of cellular proliferation. A further embodiment of the invention is such an assay in which the protein sequence is identical or substantially similar to SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 6.

Another embodiment of the present invention provides a method of pre-screening test substances for use in inhibiting cell proliferation, comprising: measuring the amount of protein-protein binding at a leucine zipper site of a protein encoded by a nucleic acid molecule selected from the a group comprising a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4 on cellular proliferation; and measuring the amount of binding at said leucine zipper site in the presence of said test substance; and comparing the amount of binding with and without said test substance. A test substance which increases the amount of binding being a suitable candidate for use in inhibition of cellular proliferation. A further embodiment of the invention provides an assay in which the protein sequence is identical or substantially similar to SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 6.

The following examples are offered by way of illustration and are not intended to limit the invention in any manner.

EXAMPLE 1 Isolation of a Mouse-human Hybrid Cell Line Containing a Fragment of Human Chromosome 4 Carrying the Senescence Inducing Gene(s)

The hybrid cell line was isolated as follows: HT1080, a fibrosarcoma derived immortal human cell line, assigned to group A, was used as a recipient as described in Ning, Y. et al, Proc. Nail. Acad. Sci.

USA 88:5635-5639 (1991). This cell line has a pseudodiploid karyotype which allows for accurate cytogenetic analysis. One of five microcell hybrid clones was found to retain an intact chromosome 4 and did not lose division potential. Of the others, one was found by cytogenetic analysis to retain a fragment of a chromosome. This fragment was confirmed as a fragment of chromosome 4 by fluorescence in situ hybridization (FISH) using probes specific for the centromere of chromosome 4. This hybrid clone was used as a microcell donor to introduce the fragment of chromosome 4 into mouse A9 cells. Since the microcell donor chromosome 4 carried the neomycin (neo) resistance gene, selection with G418 was used and a microcell hybrid was obtained. The fragment of chromosome 4 in this hybrid is capable of inducing senescence when introduced into immortal cell lines assigned to group B, but not cell lines assigned to the other groups. This cell line is referred to as A9+F4. Pulse field gel electrophoresis (PFGE) of DNA isolated from A9+F4 and probed with total human genomic DNA revealed that < 800 kb of human DNA was present.

EXAMPLE 2 Bacterial Artificial Chromosome (BAC) Approach High density BAC filters of a human genomic library derived from white blood cell DNA had been developed and were obtained from Genome Systems Inc. These filters were screened with Alu PCR probes from A9+F4 and a series of BACs were obtained (Fig 1A). Following Southern analysis of BAC DNA digested with a variety of restriction enzymes and screening with Alu PCR probes from several of the BACs, the overlap among them was determined (example Fig IB). Riboprobes made to the ends of the BACs from the promoters T7 & SP6 flanking the insertion site were used to identify contiguous BACs. The final contig covered the fragment of chromosome 4 in A9+F4 as determined by Southern analysis (Fig 2). Both Alu PCR products from A9 + F4 and probes from BACs in the contig were localized to 4q 33-34 by FISH. The region 4q 32-34 had been identified as the potential chromosome 4 senescence gene locus in loss of heterozygosity analyses of normal and matched tumor samples and cell lines derived from head and neck squamous cell carcinomas by Loughran, 0. et al, Oncogene, accepted for publication (1997). Relevant BACs (Fig 2) were used as probes on high density cDNA filters from Genome Systems Inc. to identify the corresponding cDNAs (Fig 1C). The filters were comprised of 24 cDNA libraries from 18 tissues. Direct cDNA selection of PCR-amplified cDNA inserts from a lambda Zap II human brain library from Stratagene was used, using a pool of 12 individual BACs for selection (Fig 2) See eg. Parimoo, S. et al, Nail.

Acad. Sci. USA 88:9623-9627 (1991); Dracopoli, N. et al eds., Current Protocols in Human Genetics. Current Protocols USA (1994). PCR-amplified cDNA inserts were pre-hybridized with human Cot-1 DNA and BAC vector DNA to reduce non-specific hybridization. They were subsequently hybridized in solution to biotinylated BAC DNA. Hybrids were captured on streptavidin-coated beads (Dynabeads M280; Dynal), washed and eluted. The selected cDNA mixture was re-amplified by PCR and processed through a second round of hybridization, capture, wash and elution. The secondary selected cDNAs were amplified by PCR and cloned into a TA vector (Invitrogen). A total of 200 clones were randomly picked in the course of two independent experiments, and the inserts amplified by PCR. Some clones were initially eliminated due to homology to Alu repeats. To determine if any clones were identical, the PCR products were transferred to a membrane and hybridized with PCR probes specific to some of the clones. Many clones were found to have no inserts or vector inserts, thus speeding up the process of elimination. Inserts from the remaining clones were labeled and hybridized to membranes of BAC DNA. Positive clones were sequenced and compared to DNA databases using the BLASTN system. The majority of the BACs used in the case of direct selection were from the region of heaviest overlap in the contig (Fig 2). The same cDNA, 386h22, was identified from this screen as was done from the direct filter screen.

Northern analysis of a multi-tissue blot with the various cDNAs revealed that cDNA 386h22 encodes an expressed gene.

EXAMPLE 3 Transfection Studies with MORF 4 Gene

Genomic DNA was used in transfections because the activity of the cell senescence gene on human chromosome 4 was followed under its own promoter. The genomic region encoding MORF 4 (Mortality Factor on chromosome 4), which is related to cDNA 386h22, had a clear effect in inducing various senescence - like parameters in group B cell lines, as indicated by loss of proliferation, morphology change, sen β-gal activity (Dimri, G.P. et al, Proc. Natl Acad. Sci. USA 92:9363-9367 (1995)) and return of mortalin pattern to that of a normal cell (Figs 3,4). Mortalin staining distinguished normal from immortal human cells by the method of Wadhwa, R. et al, Exp. Cell Res. 216:101-106 (1995) and was found to return to normal in immortal cell microcell hybrid clones. Transfection of the MORF 4 genomic DNA in which a frame shift mutation at the 5' end in the start codon has been introduced, eliminates expression of the gene. Loss of senescence inducing activity in the mutated DNA provides stronger evidence that it is indeed the cell senescence gene on human chromosome 4. A 3' deletion in the MORF 4 genomic DNA was constructed. This is an additional mutated DNA that does not affect proliferation of immortal cells assigned to group B.

EXAMPLE 4 Sequence Analysis and FISH Results with MORF Family Members Six cDNAs hybridized to BAC 526e7 which carries the genomic DNA that encodes MORF 4. The cDNA 386h22 was chosen because it had the longest insert. Sequencing indicated that it lacked the 5' end because there was no start codon and the insert length was not sufficient to encode the transcripts recognized on northerns. 5' RACE was performed on a Marathon Ready brain library (Clontech) and the 5' end cloned and sequenced. When it was used as probe on a Zoo blot it hybridized to multiple bands in human DNA and was present in all species (Fig. 5A). Analysis of the sequence indicated that there were many regions in common with other genes, such as a leucine zipper and an ATP/GTP binding domain. A 5' probe lacking these regions was used. Multiple bands were again observed, but not as many as previously (Fig. 5B). Therefore, MORF 4 is a member of a family.

Consistent with the idea that the MORF family might bind DNA or act as transcription factors is the fact that a Flag-tagged construct of

MORF 4, when expressed in EJ or HeLa cells, produces a protein that is present in the nucleus, as detected by anti-Flag antibodies (Fig. 6).

The gene and the cDNA 386h22 have been fully sequenced and show some differences at the nucleotide and predicted protein level, but have 95% similarity (Figs. 7, 8). The cDNA 386h22 has an additional 105 base pairs at the 5' end. In the MORF 4 gene a frame shift has occurred so that it utilizes the second ATG start site and encodes a smaller protein.

EXAMPLE 5 MORF 4 and Related Genes (MRG 1, 5, 11) are Intronless and Inserted in LINE 1 (LI) Sequences

The MORF 4 gene and related genes (MRG) on chromosome 4 is intronless and contained entirely within LINE 1 (LI) repeat sequences

(Fig. 9). The MRG gene on chromosome 15 has been analyzed by sequencing and has introns and no LI sequence. The sequence contains exons identical to the sequence of cDNA 386h22. It therefore encodes the original gene. Genes within LI repeats have been described recently and are believed to be a mechanism of DNA repair, in which a transcribed cDNA is picked up by LI repeats and placed in the genome to fix double strand breaks (Moran, J.V. et al, Cell 87:917-927 (1996); Feng, Q. et al, Cell 87:905-916 (1996); Moore, J.K. & Haber, J.E., Nature 383:644-647 (1996); Teng, S.-C, et al, Nature 383:641-644 (1996)). The LI repeats can then act as transposons and insert in various additional regions of the genome. Some of such gene members acquire stop codons and point mutations and are not expressed. However, the LI element itself has been found to encode an active promoter as well as have a leucine zipper motif within one of the open reading frames (Britten, R.J., Mol.

Phylogenet. Evol. 5:13-17 (1996); Hattori, M. et al, Nature 321:625-628

(1986); Holmes, S.E. et al, J. Biol Chem. 267:19765-19768 (1992);

Dombroski, BA. et al, Proc. Natl Acad. Sci. USA 90:6513-6517 (1993)).

The observation that a gene picked up by a retrotransposon that is expressed and has negative growth regulatory activity is novel and unique.

It is necessary to determine whether the MORF 4 sequence alone is responsible for the growth inhibitory activity, or whether the active gene product is expressed from the LI promoter. An effect on immortal cell lines assigned to complementation Group B using genomic DNA that does not encompass all of the upstream LI region has been observed. However, the phenotype does not occur as rapidly as that seen when the intact chromosome 4 or the fragment of chromosome 4 is introduced. Following transfection of the genomic MORF 4 DNA, the cells proliferated for 19-40 population doublings (P.D.) before losing division capability. In contrast, when the intact or fragment of the chromosome was introduced, the majority of the hybrids senesced at less than 20 P.D. though some did proliferate through more than 40 P.D. before becoming senescent. LI and its encoded sequences contribute to the growth regulatory activity we observed and either a combined Ll+MORF 4 transcript produces a protein with a more rapid senescence inducing effect or LI enhancer elements contribute to the activity. This could well explain differences in immortalization rates between different species, as human cells appear to have acquired multiple copies of this senescence related gene.

The EST D14812 which was picked up in a database search with our sequence is very similar to the MORFs and diverges primarily at the 5' end. The genomic DNA encoding this EST was cloned and sequenced to ensure that the sequence in the database is correct. These family members are referred to as ORF 4 and ORF X.

EXAMPLE 6 Northern Analysis The Northern data demonstrates two transcripts at 1.8 kb and 1.2 kb detected by a MORF specific DNA probe that distinguishes

MORF/MRG genes.

EXAMPLE 7 Sequencing Since MORF 4 is a member of a family of genes the sequence of all five family members has been obtained. There were base changes/frame shifts in the sequence of the MRG 5 and 11 genes (MORF 4 related genes on chromosomes 5 and 11), indicating they were most likely unprocessed pseudogenes. Indeed analysis using 17 oligomer probes specific for each indicated that though the gene could be amplified from the relevant BAC, it was not expressed in young and senescent normal cells as well as various immortal human cells. Probes specific for MRG 1, the gene on chromosome 1, could not be designed as it is highly homologous to the CDNA corresponding to MRG 15. However there is a stop codon at nucleotide 613 from the Ll/MRG 1 boundary which suggests the gene may be transcribed but a truncated protein will result from the transcript. (Hellman, L. & Pettersson, U., Gene Anal Techn. 4:9-13 (1987); Guillaume, T. et al, J. Immunol. 145:1934-1945 (1990); Clements, J. et al, J. Biol Chem. 265 1077-1081 (1990); Courtay, C. et al, Biochem. J. 297:503-508 (1994); DeMarchi, J.M. et al, Human Mutation 8:116-125 (1996)). In addition, sequencing information allowed the design of PCR primers to analyze only MORF 4 at the DNA level, to determine mutations/deletions in immortal cell lines assigned to Group B, versus the other groups, and in tumor tissue matched to normal material as well as oligonucleotide to be used in RNAse protection assays. The potential chromosome 4 senescence gene locus was identified as 4q 32-34 by loss of heterozygosity analyses in these cells and tissues (Loughran, O. et al, Oncogene accepted for publication (1997)). These studies are extended to other normal and tumor matched tissues. ORF 4 sequence has multiple base changes when compared with ORF X and the Genbank # D14812 and will not be expressed.

EXAMPLE 8 Antibody Production

To further understand the expression of the MORF genes at the protein level, specific polyclonal and monoclonal antibodies are raised.

Synthetic peptides which correspond to unique coding regions of MORF 4 (aa 1-9) and the cDNA 386h22 (aa 54-64) and ORF X (aa 5-16) are synthesized and conjugated to the carrier protein keyhole limpet hemacyanin (KLH) using glutar aldehyde. These are used as antigens in antibody production, according to standard protocols described by Harlow,

E. and Lane, D., ANTIBODIES: A LABORATORY MANUAL (1988). Pre-immune sera from 10 New Zealand white rabbits are screened and 2 rabbits identified each for injection with the individual peptides. 500 μg of antigen mixed with complete Freund's adjuvant is used in the primary inoculation and 250 μg antigen mixed with incomplete Freund's adjuvant is used for three boosts, done at 14, 21 and 49 days. Serum is used for testing at days 35 and 56. Depending on the titre at this time additional boosts are given, or the animals are exsanguinated.

Although synthetic peptides provide an immediate source for antibody generation, one potential problem is that a peptide antibody might not be able to recognize the native protein. Therefore monoclonal antibodies are generated using recombinant protein as the antigen source. MORF 4 protein, cDNA 386h22 protein, and ORF X protein are expressed in bacteria using a T7 bacteriophage expression plasmid (pET vector). These overexpressed proteins are purified by SDS-polyacrylamide gel electrophoresis. In this case, the primary inoculation is protein in polyacrylamide gel slices emulsified with an equal volume of saline. If sufficient proteins are not obtained from the gels, the protein is purified in inclusion bodies using 50 μg of protein in saline emulsified with an equal volume of complete Freund's adjuvant in the primary inoculation.

The standard protocols for injections are followed (Harlow, E. and Lane, D., ANTIBODIES: A LABORATORY MAUNAL (1988)). Boosts are performed with antigens mixed either with saline (polyacrylamide gel slices) or antigens mixed with incomplete Freund's adjuvant (purified proteins). The pre-immune sera from 10 mice is tested. Six female BALB/c mice are injected intraperitoneally. One mouse is selected based on tail bleed titre and cell fusion between the splenocytes of the mouse and myeloma cells are performed three days following the final boost. The regimen of boosting is the same as that for production of polyclonal antibodies, described above. The cells are plated into multiple plates and supernatant from these dishes of fused cells are tested to determine which are producing antibodies. The supernatants are screened by ELISA using an antibody capture assay. 96-wells are coated with purified inclusion bodies prepared from the bacterial overexpression system and positive clones are identified using a horseradish-peroxidase conjugated anti-mouse IgG.

Further screening is performed using a combination of immunoblotting and immunoprecipitation analyses. After a positive supernatant is identified, the cells are subcloned by limiting dilution to isolate stable, single cell cloned hybridomas. Screening is performed, as described above, during the subcloning process and one should obtain 1-6 subcloned cell lines for each antigen. Cloned hybridomas are injected into mice and high-titer monoclonal antibodies collected in the form of ascitic fluid. Several different monoclonal antibodies are generated which specifically recognize MORF 4, 386h22, and ORF X as well as other proteins encoded by the additional family members, if expressed, in order to distinguish the different expression patterns of these proteins.

EXAMPLE 9 Cloning the mouse and rat homologs of MORF 4

The mouse and rat homologs of the MORF 4 gene were identified.

Mouse and rat lambda ZapII brain cDNA libraries from Stratagene were screened by plating the cDNAs and screening with a MORF 4 probe

(Sambrook, J. et al. MOLECULAR CLONING: A LABORATORY MANUAL 2D ED. (1989)). The cDNA clones were sequenced and analyzed for their sequence similarity to the human MORF 4 gene. RACE is used to generate full length cDNAs, if necessary. The MORF family genes have a single EcoRI site within the DNA.

The zoo blot probed contains EcoRI cut genomic DNA. The results with a MORF 4 probe indicated that -there are probably 2 genes in rat and perhaps 3 genes in mouse DNA recognized by this probe (Fig. 5B). It is not known whether these are related genes or actual members of a family, since it could be identifying the rodent homolog of the human EST

D14812 in this screen. If there are two members of a rodent equivalent of the human MORF 4 gene, the isolated cDNA as probe is used on genomic mouse and rat libraries to identify and clone the other family members. Then it is possible to determine whether any are pseudogenes that are not expressed and design oligonucleotide probes that detect true transcripts.

EXAMPLE 10

Studies of the MORF 4 Gene in Cell Senescence and Immortalization,

Cell Cycle and DNA Damage in Human Cells RNA and protein from normal human fibroblasts, adrenal cells and melanocytes at different points in their in vitro lifespan are analyzed using a MORF 4 specific oligonucleotide probe (described above) and antibodies. This allows the determination of the pattern of expression during in vitro senescence. Young and senescent cells made quiescent by removal of serum growth factors and then induced to enter the cell cycle by addition of 10% FBS, are analyzed to determine changes in expression during the cell cycle. The results demonstrate whether patterns of expression are the same or different in these different cell types. RNA is probed from various tissues to determine if there is tissue specific expression of the various members of the MORF family.

RNA and protein from various immortal cell lines are tested to determine whether expression is intact in non-group B cells and whether transcription or translation is impaired in cell lines assigned to group B. It is possible that transcript and an inactive protein continue to be expressed in the group B cells, and that point mutations, identified in

Bunn, CL. and Tarrant, G.M., Exp. Cell. Res. 127:385-396 (1980), are actually responsible for expression of an inactive product.

A hypothesis that cell senescence is triggered by a sensing of DNA damage, such as a severely shortened telomere, has been proposed (Harley, CB. et al, Exp. GerontoL, 27:375-382 (1992)). A senescent like phenotype has been observed in cells treated with H₂0₂ (Chen, Q. & Ames, B.N., Proc. Natl. Acad. Sci. USA 91:4130-4134 (1994)), inhibitors of histone deacetylase (Ogryzko, V.V. et αϋ., Mol Cell Biol, 16:5210-5218 (1996)) and after g-irradiation (Linke, S.P. et al, Can. Res. 57:1171-1179 (1997)). Changes in expression of the MORF 4 gene is a critical event in the path to senescence. It was determined whether DNA damage results in altering expression of this gene in response to DNA damage that results in a senescence-like exit from the cell cycle. Young normal fibroblasts were treated with varying doses of hydrogen peroxide and g-irradiation and expression of the MORF 4 gene at different times following treatment was determined, similar to studies done with the p21^sdlx gene (Johnson, M. et al, Mol. Carcin. 11:59-64 (1994)).

A natural extension of such studies examined normal fibroblasts from donors of different age, individuals with Werner syndrome, progeria and Cockayne syndrome (since the latter are associated with potential defects in a helicase that could have implications for DNA damage repair) and also other differentiated cell types, to give a few examples.

The gene was cloned under the control of a modulatable promoter system such as the MMTV promoter which responds to dexamethasone, or the tetracycline inducible system, and obtain stable transfectants of young normal cells. Modulating expression of the gene at different times in the in vitro lifespan allows further insights into regulation in the path to senescence.

EXAMPLE 11 Mechanism(s) of Regulation of the MORF 4 Gene Based on the results of experiments described above whether the mechanism of regulation of the MORF 4 gene occurs at the RNA level or not can be determined. If change's are observed in RNA levels during cell senescence and immortalization, cell cycle or DNA damage in cultured cells it is determined whether this is occurring as a result of transcriptional changes, focusing initially on cell senescence and immortalization.

EXAMPLE 12 Transcriptional Regulation / mRNA Stability

If the transcript(s) expressed from MORF 4 is increased in expression in senescent versus young normal cells and immortal cell lines assigned to groups A, C and D, and is not expressed in cell lines assigned to complementation group B, the following is done.

Nuclear run-on assays are used to examine the rate of mRNA transcription in senescent cells (Sambrook, J. et al, MOLECULAR CLONING: A LABORATORY MANUAL (1989); Mitchell, M.T. & Benfield, P.A, J. Biol. Chem. 265:8259-8267 (1990)). Nuclei prepared from these cells is labeled with radioisotope for a period of time and the amount of synthesized RNA of the MORF 4 gene determined by hybridization of the labeled RNA products to the membrane on which the gene is immobilized. Specific binding is determined quantitatively and nonspecific binding is corrected for using unrelated DNAs as internal control. If the data indicates that the activity of the MORF 4 gene is due to a significant increase in transcription rate, the transcriptional regulatory mechanisms are investigated. If the data demonstrate that transcription is unchanged, although mRNA is elevated, message stability is examined by determining the rate of mRNA degradation using RNA synthesis inhibitors such as DRB and actinomycin D (Sambrook, J. et al., MOLECULAR CLONING: A LABORATORY MANUAL (1989); Harris, M.E. et al, Mol. Cell. Biol 11:2416-2424 (1991); Meyer, AS. et al, J. Steroid Biochem. Mol Biol 55:219-228 (1995)).

EXAMPLE 13 Promoter Studies

If MORF 4 gene is not expressed in immortal cell lines assigned to group studies are focused on the promoter region to identify cis- and irans-activating elements. The transcripts that are currently recognized by the MORF 4 gene are 1.8 and 1.2kb. This suggests that the promoter most likely involves elements just 5' of the gene, that have been identified by computer analysis. It is not known that the observed transcripts are indeed expressed from MORF 4. It is possible that the other family members express these mRNAs and that a different, lower abundance

RNA, that uses the L_x promoter is transcribed from MORF 4. RNA analyses using oligonucleotide probes distinguish the two possibilities.

If MORF 4 utilizes promoter elements upstream of the 5' end, a series of different lengths of promoter constructs of this region linked to a reporter gene such as CAT or luciferase are generated. These constructs are used to transiently transfect either normal or non-group B immortal cells to identify the minimal region needed for activity. The element is narrowed by deletion mutagenesis. DNA binding protein is identified by electrophoretic mobility shift assays using nuclear and whole cell extracts to determine whether any specific factors are present. Extracts from immortal cells that assign to group B, in which the gene is not expressed, are used as controls. The transfactor(s) is cloned by either biochemical or genetic approaches. Using biochemical methods the potential transcript factor(s) is purified from crude extracts by chromatography methods such as DNA affinity columns. Once the protein is purified, the sequence is analyzed by peptide mapping and used to further identify the coding genes (Ausubel, F.M. et al, CURRENT PROTOCOLS LN MOLECULAR BIOLOGY (1987)). In a genetic approach, a relevant cDNA expression library is screened with radiolabeled recognition site DNA as probes. Clones encoding proteins that can specifically recognize the target DNA sequence are isolated for further studies (Latchman, D.S., TRANSCRIPTION FACTORS: A PRACTICAL APPROACH (1993)).

If the MORF 4 gene is transcribed from the LI promoter the existing data is utilized to design experiments. Other laboratories have identified cis regulatory elements in the LI open reading frame and found that the YYl transcription factor and other protein complexes bind to these elements (Minakami, R. et al, Nucleic Acids Res. 20:3139-3145 (1992); Becker, KG. et al, Hum. Mol. Genet. 2:1697-1702 (1993); Mathias, S.L. & Scott, A.F., Biochem. Biophys. Res. Commun. 191:625-632 (1993); Kurose, K. et al, Nucleic Acids Res. 23:3704-3709 (1995)).

Whether the same DNA elements are important in regulation of MORF 4 expression is determined by methods described above. Antibodies to YYl are obtained and antibodies to the other putative proteins are used to determine whether these act to super shift in gel shift assays. This indicates whether these DNA sequences and proteins are involved in regulation of MORF 4. EXAMPLE 14 Translational and Post-translational Studies

If regulation is not at the level of transcription and the protein is expressed in all cell types, indicating no translational defects, translation initiation regulation is analyzed. This has been described in the case of the two closely related family members of the elongation factor 1 family

(Lee, S. et al, J. Biol Chem.268:24453-24459 (1993); Lee, S. et al,

CELLULAR AGING AND CELL DEATH 139-151 (1995)). It is not clear precisely what the mechanism is, but when protein of one of the family members is present there is downregulation of expression of protein of the other family member.

If the studies above do not reveal any mechanism of regulation post-translational regulation is determined. Computer analysis of the predicted protein identifies two putative phosphorylation sites on MORF 4. Whether phosphorylation at one or both of these sites is involved in regulating the activity of the protein is determined. To accomplish this, MORF 4 is examined to see if it is phosphorylated in vivo by analysis of immunoprecipitates from cells metabolically labeled with [³²p] orthophosphate by the method of Rosfjord, E. et al, Biochem. Biophys. Res. Commun. 212(3) :847-853 (1995). If the data indicate that MORF 4 is phosphorylated, whether phosphorylation is critical for potential DNA binding or protein binding function (described below) is determined by using CIAP (calf intestinal phosphatases) or acid phosphatase treated cell extracts in these assays by the method of Franklin, C.C et al, Methods Enzymol 254:550-564 (1995). If the results demonstrate that phosphorylation is important for function of the gene, it is confirmed that the putative sites on MORF 4 are indeed phosphorylated by phosphoamino acid analysis. These sites are mutated and thereby demonstrate that phosphorylation is critical for function.

EXAMPLE 15

Mechanism(s) of action of the MORF 4 gene in Yeast While, searching the complete S. cerevisiae genome database a single homolog to MORF was identified on chromosome XVI as a putative open reading frame, YP9367.03c, among a long stretch of genomic sequence. This homolog has been termed YORF. The genetic capabilities of S. cerevisiae are used to get some indication of the function of YORF and thus some inference about the function of MORF.

Homologous recombination in <S. cerevisiae is utilized to create null mutants for YORF (Baudin, A. et al, Nucleic Acids Res. 21:3329-3330 (1993)). This is done by making a stretch of DNA by PCR containing the selectable marker HIS3 flanked by sequence from the 5' and 3' ends of

YORF. This DNA is transformed into a yeast strain that is his3 null by lithium acetate protocol. Colonies that grow on plates in the absence of histidine are analyzed for the replacement of YORF by both PCR and Southern blotting. Confirmed gene knockout clones are analyzed for their ability to grow as a culture and also as individual cell. Wild type mother

S. cerevisiae cells have been found to divide a definite number of times before losing their division potential. Given an assayable phenotype the human MORF gene is put into the nulls and determine whether it can return the cells to wild type. Another possible outcome is that knock out of the gene may be lethal, indicating it is an important survival gene. If knock out is non-lethal and does not affect proliferation, it indicates that compensatory pathways must exist.

Conversely YORF was overexpressed in S. cerevisiae to see what, if any, effects this has on the viability and division potential of the yeast.

This was done by cloning YORF into two different expression vectors and transforming them separately into yeast host strains. Both vectors utilize heterologous promoters to drive the expression of YORF. One vector utilizes the alcohol dehydrogenase (adh) promoter which drives transcription at a constitutively high level. The other vector uses the inducible GAL promoter which gives a high level of expression in the presence of galactose.

Overexpression may result in either growth arrest or senescence of an entire culture or individual mother cells, with or without telomere shortening. If senescence with telomere shortening is observed it will indicate this gene is in the telomere associated senescence pathway. If not, it indicates that an alternative path to senescence exits in yeast cells. A final possibility is that overexpression of the gene results in no phenotype, indicating that the gene is not involved in senescence in yeast cells, or that other genes can compensate for the overexpression. The characterization of null and overexpression phenotypes give some preliminary indications of the role of YORF in yeast.

EXAMPLE 16 Mechanism of Action of the MORF Gene in DNA Binding

Since the MORF 4 gene contains a potential helix-loop-helix domain as well as has similarity to a domain found in the telomere binding protein Eup 51kd from Euplotes Crassus (Wang, W. et al, Nucleic Acids Res. 24:6621-6629 (1992)), the' ability to bind DNA is determined. Gel-shift or footprinting analysis (Andrisani, 0. & Dixon, J.E., J. Biol Chem. 265:3212-3218 (1990)) using the region with homology to the telomere binding sequence as probe is used. Pools of random 12 mer oligonucleotides are made, protein extracts are added and analyzed for gel-shift (Sambrook, J. et al, MOLECULAR CLONING: A LABORATORY MANUAL 2D ED. (1989)). Positive pools are further analyzed and the sequence of the positive oligo determined. Southwestern analysis or UV-crosslinking is also performed to confirm the sequence to which

MORF 4 binds (Antalis, T.M. et al, Genetics 134:201-208 (1993)). In these assays, overexpressed MORF 4 proteins from a baculovirus expression system are used, since they provide an immediate source of large quantities of biologically active proteins. A series of deletion mutants of MORF 4 protein are constructed and used in similar binding assays. The results pinpoint the functional DNA-binding domain of MORF 4 protein and aid in understanding of the mechanism of action in the cell.

Analysis with cell extracts from immortal cell lines assigned to group B may provide indications of either no binding or abnormal pattern of DNA binding. When the important DNA binding region of the protein is identified then the group B cell lines for mutations or deletions in these region can be analyzed.

EXAMPLE 17 Mechanism of Action of the MORF Gene in Protein-protein Interactions Protein-protein interactions are performed by immunoprecipitation with the various antibodies generated as well as use the yeast two-hybrid system.

The common leucine zipper and the motifs of the various MORF family members as well as the EST dl4812, potentially indicate a family of transcription factors interacting proteins. It is logical to assume that homo/hetero dimerization of these molecules can occur to affect transcription. Therefore each possible interactions in both the assays described below is tested, as well as other candidate genes identified in the text.

EXAMPLE 18

Immunoprecipitation Analysis

Cellular extracts are immunoprecipitated with various antibodies against MORF 4 to determine what additional proteins co-precipitate.

Cells are labeled with ³⁵S methionine and extracts prepared. The use of different antibodies which have been raised against various peptides of

MORF 4, reduce the chances of failing to precipitate complexes because of sequestration of MORF 4 epitopes by complex formation, or by disruption of the protein-protein interaction by antibody binding. The immunoprecipitated proteins are denatured and run on reducing SDS polyacrylamide gels. If, on analysis of the gel patterns, it appears that nonspecific binding is a problem, the detergent concentration in the cellular extracts is slowly increased until only specific binding is observed. If a large number of proteins co-precipitate with MORF 4, to decrease these, the cells are sub-fractionated and the nuclear enriched fraction is used to immunoprecipitate proteins. If possible, the identity of the associated protein(s) is deduced from the mobility on SDS polyacrylamide gels. Besides the other MORF family members, obvious candidates that come to mind are tested such as Rb, p53, Id. This test is verified by immunoblotting using the appropriate antibodies. Any candidate protein association with MORF 4 is further confirmed by immunoprecipitation using antibodies to the candidate protein.

Alternatively, if the protein(s) is of a sufficiently high abundance, it is purified from the gel. It is then microsequenced. The protein is also used to raise antibodies and to screen an expression library for cDNAs. This approach has been successfully used by others (Wadhaw, R. et al, J.

Biol. Chem. 268:6615-6621 (1993); Bhattacharyya, T. etal, J. Biol. Chem. 270:1705-1710 (1995)). To determine that the cDNAs isolated do indeed code for the protein of interest, the cDNA is sequenced and compared with this microsequence information. It is also expressed in bacteria to obtain a protein that is potentially unique in the bacterial cell. Immunoblotting of the bacterially produced protein with antibodies specific for the protein of interest confirms that they are the same. Peptide mapping also confirms the identity of the protein.

EXAMPLE 19 Yeast Two-hybrid System

The yeast two-hybrid system (Fields, S. & Song, O., Nature 340:245-

247 (1989); Chien, C.T. et al, Proc. Natl Acad. Sci. USA 88:9578-9582

(1991); Harper, J.W. et al, Cell 75:805-816 (1993); Li, B. & Fields, S.,

FASEB J. 7:957-963(1993)) is employed to directly clone proteins that interact with MORF 4. A cDNA library from normal human cells has been cloned into a vector which contains the GAL4 activation domain. The MORF 4 cDNA is cloned into the vector that includes the GAL4 DNA-binding domain. The two vectors are then introduced into yeast cells along with a reporter gene, such as lacZ, which contains upstream GAL4 binding sites. When the MORF 4 protein interacts with another protein, the two GAL4 domains are brought together, are transcriptionally active and cause expression of the lacZ gene in the yeast cells, which are then identified as b-gal positive. This allows the identification of cDNAs of proteins that interact with the MORF 4 protein in normal cells. If necessary, libraries in the GAL4 activation domain containing vector are available from Clontech and from a variety of normal tissue sources. Candidates are eliminated/included, genes that immediately come to mind as potential interacting proteins, such as MORF 4 itself and other MORF family members, Rb, Id, and p53, by direct selection with these genes.

EXAMPLE 20 Analysis of MORF 4 Gene" Expression During Development

Expression of MORF 4 during mouse development is analyzed by in situ hybridization and immunostaining of whole mount embryos at various stages of development (Sundin, O.H. et αZ., Development 108:47-58

(1990); Nuovo, G.J. e* αZ., Am. J. Pathol 139(6):1239-1244 (1991); Long, AA. et al, Histochem. 99:151-162 (1993); Millar, M.R. et al, Microsc. Res. Tech. 32:498-503 (1995); Lutz, B. et al, Development 120:25-36 (1994)). It is then determined whether MORF 4 has a role in particular cell lineages during development. The data reveals a role, if any, in development and differentiation of various cell types.

Based on the results obtained here and from other experiments proposed, a decision is made concerning whether it is important and feasible to generate a knockout or over expressing transgenic MORF 4 mouse. EXAMPLE 21 Analysis of MORF 4 Gene Expression During in vivo Aging

Expression of the MORF 4 gene is studied during in vivo aging using various tissues from young, middle aged and old Fisher 344 rats and C57BL/6J mice, both ad lib fed and diet restricted. These tissues are used to prepare RNA and study the expression of the MORF 4 gene. If the antibodies generated cross-react with the rodent protein, they are used to determine changes at the protein level. Sections from paraffin embedded tissues are prepared for in situ hybridization and immunostaining. The results demonstrate a role, if any, of the MORF 4 gene during in vivo aging.

EXAMPLE 22 PCR Analysis of MORF in Senescent and Proliferating Cells

PCR analysis of senescent, proliferating and quiescent cells was performed on Group B cells. MORF 4 was expressed at higher levels in senescent versus proliferating or .quiescent cells. PCR analysis of Group

B immortal cells showed no expression of MORF 4. The results demonstrated a direct correlation between senescence and the level of

MORF 4 expression.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid molecule comprising a sequence substantially identical to a fragment of human chromosome 4, wherein said fragment is capable of inhibiting undesired or uncontrolled proliferation of a cell of an animal.

2. The nucleic acid of claim 1, wherein said fragment of human chromosome 4 is located at 4q 33-34.1.

3. The nucleic acid of claim 1, wherein said fragment of human chromosome 4 is identical or substantially similar to SEQ ID NO. 1 or a fragment or fragments thereof.

4. The nucleic acid of claim 1 wherein said fragment has a sequence identical or substantially similar to MORF 4 or a fragment or fragments thereof.

5. The nucleic acid molecule of claim 1, wherein said cell is a hyperproliferative cell.

6. The nucleic acid molecule of claim 5, wherein said hyperproliferative cell is a virally-infected cell.

7. The nucleic acid molecule of claim 6, wherein said virally- infected cell is infected with human immunodeficiency virus.

8. The nucleic acid molecule of claim 5, wherein said hyperproliferative cell is a cancerous cell.

9. The nucleic acid molecule of claim 8, wherein said cancerous cell belongs to a group B complementation group.

10. The nucleic acid molecule of claim 1, wherein said molecule additionally contains a tumor specific promoter, said promoter being operably linked to said fragment, and capable of mediating the preferential expression of said fragment in a tumor cell.

11. The nucleic acid molecule of claim 1, wherein said molecule additionally contains a native promoter, said promoter being operably linked to said fragment, and capable of mediating the preferential expression of said fragment in a cell.

12. The nucleic acid molecule of claim 1, wherein said molecule has been inserted into a non-viral vector.

13. The nucleic acid molecule of claim 1, wherein said molecule has been inserted into a viral vector, said vector being encapsulated in a viral coat.

14. The nucleic acid molecule of claim 13, wherein said viral vector is an adenovirus, and wherein said viral coat is an adenoviral coat.

15. A nucleic acid molecule comprising a sequence identical or substantially similar to SEQ ID. NO. 3, wherein said fragment is capable of inhibiting undesired or uncontrolled proliferation of a cell of an animal.

16. The nucleic acid molecule of claim 15, wherein said cell is a hyperproliferative cell.

17. The nucleic acid molecule of claim 16, wherein said hyperproliferative cell is a virally-infected cell.

18. The nucleic acid molecule of claim 17, wherein said virally- infected cell is infected with human immunodeficiency virus.

19. The nucleic acid molecule of claim 16, wherein said hyperproliferative cell is a cancerous cell.

20. The nucleic acid molecule of claim 19, wherein said cancerous cell belongs to a group B complementation group.

21. The nucleic acid molecule of claim 15, wherein said molecule additionally contains a tumor specific promoter, said promoter being operably linked to said fragment, and capable of mediating the preferential expression of said fragment in a tumor cell.

22. The nucleic acid molecule of claim 15, wherein said molecule additionally contains a native promoter, said promoter being operably linked to said fragment, and capable of mediating the preferential expression of said fragment in a cell.

23. The nucleic acid molecule of claim 15, wherein said molecule has been inserted into a non-viral vector.

24. The nucleic acid molecule of claim 15, wherein said molecule has been inserted into a viral vector, said vector being encapsulated in a viral coat.

25. The nucleic acid molecule of claim 24, wherein said viral vector is an adenovirus, and wherein said viral coat is an adenoviral coat.

26. A nucleic acid molecule comprising a sequence identical or substantially similar to SEQ ID NO. 5, wherein said fragment is capable of inhibiting undesired or uncontrolled proliferation of a cell of an animal.

27. The nucleic acid molecule of claim 26, wherein said cell is a hyperproliferative cell.

28. The nucleic acid molecule of claim 27, wherein said hyperproliferative cell is a virally-infected cell.

29. The nucleic acid molecule of claim 28, wherein said virally- infected cell is infected with human immunodeficiency virus.

30. The nucleic acid molecule of claim 27, wherein said hyperproliferative cell is a cancerous cell.

31. The nucleic acid molecule of claim 30, wherein said cancerous cell belongs to a group B complementation group.

32. The nucleic acid molecule of claim 26, wherein said molecule additionally contains a tumor specific promoter, said promoter being operably linked to said fragment, and capable of mediating the preferential expression of said fragment in a tumor cell.

33. The nucleic acid molecule of claim 26, wherein said molecule additionally contains a native promoter, said promoter being operably linked to said fragment, and capable of mediating the preferential expression of said fragment in a cell.

34. The nucleic acid molecule of claim 26, wherein said molecule has been inserted into a non-viral vector.

35. The nucleic acid molecule of claim 26, wherein said molecule has been inserted into a viral vector, said vector being encapsulated in a viral coat.

36. The nucleic acid molecule of claim 35, wherein said viral vector is an adenovirus, and wherein said viral coat is an adenoviral coat.

37. A protein, substantially free of its natural contaminants, wherein said protein is encoded by a nucleic acid molecule capable of inhibiting the proliferation of a cell of a patient wherein said nucleic acid molecule sequence is identical or substantially similar to a sequence selected from the following group; a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1,

SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4, and wherein said protein is capable of inhibiting the proliferation of the cell of a patient.

38. The protein of claim 37, wherein said cell is a cancerous cell.

39. The protein of claim 38, wherein said cancerous cell belongs to a group B complementation group.

40. The protein of claim 37, wherein said cell is a virally-infected cell.

41. The protein of claim 40, wherein said virally-infected cell is infected with human immunodeficiency virus.

42. A method for treating cancer in an individual which comprises providing to a cancerous cell of said individual an effective amount of a nucleic acid molecule, wherein said nucleic acid molecule is capable of inhibiting the proliferation of said cell and wherein said nucleic acid molecule sequence is identical or substantially similar to a sequence

-57-

SUBST1TUTE SHEET (RULE26) selected from the following group; a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4.

43. The method of claim 42, wherein said cancerous cell belongs to a group complementation group B.

44. A method for treating viral infection in an individual which comprises providing to a virally-infected cell of said individual an effective amount of a nucleic acid molecule, wherein said nucleic acid molecule is capable of inhibiting the proliferation of virus in said cell and wherein said nucleic acid molecule sequence is identical or substantially similar to a sequence selected from the following group; a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4.

45. The method of claim 44, wherein said virally-infected cell is a cell infected with human immunodeficiency virus.

46. A method for treating cancer in an individual which comprises providing to a cancerous cell in said individual an effective amount of a protein, substantially free of its natural contaminants, wherein said protein is encoded by a nucleic acid molecule having a sequence identical or substantially similar to a sequence selected from the following group; a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4, and wherein said protein is capable of inhibiting the proliferation in said cell.

47. The method of claim 46, wherein said cancerous cell belongs to complementation group B.

48. A method for treating viral infection in an individual which comprises providing to a virally-infected cell in said individual an effective amount of a protein, substantially free of its natural contaminants, wherein said protein is encoded by a nucleic acid molecule having a sequence identical or substantially similar to a sequence selected from the following group; a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4a fragment from human chromosome 4, and wherein said protein is capable of inhibiting the proliferation of virus in said cell.

49. The method of claim 48, wherein said virally-infected cell is a cell infected with human immunodeficiency virus.

50. A method of detecting or differentiating senescent cells from non-senescent cells comprising the steps of: detecting the presence or absence of a sensecence indicator in a cell; and comparing the level of said senescence indicator in said cell to the level of said senescence indicator in a normal cell of similar type.

51. The method of claim 50, wherein said sensecence indicator is a nucleic acid molecule with a sequence identical or substantially similar to a sequence selected from the following group; a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4.

52. The method of claim 50, wherein said sensecence indicator is a protein encoded by a nucleic acid molecule with a sequence identical or substantially similar to a sequence selected from the following group: a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4.

53. Antibodies that specifically bind the protein products of nucleic acid molecules wherein said nucleic acid molecules are capable of inducing sensecence in a proliferating cell.

54. The antibodies of Claim 53, wherein said nucleic acid molecule is obtained from human chromosome 4.

55. The antibodies of Claim 53, wherein said protein product is the same or substantially similar to SEQ ID NOS. 2, 4, or 6.

56. A method of detecting the amount of protein in a cell identical or substantially similar to a protein encoded by a sequence selected from the following group: a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4 comprising: exposing said cellular contents to an antibody directed against a member of said group; and measuring the amount of antibody binding.

57. A method of pre-screening nucleotide analogs for use in inhibiting cell proliferation, comprising: measuring the amount of binding of a protein encoded by a nucleic acid molecule selected from the following group: a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4 to ATP or GTP; measuring the amount of binding of said protein to said protein molecule in the presence of a nucleotide analog; and comparing the amount of binding with and without said nucleotide analog, a nucleotide analog which increases the amount of binding being a suitable candidate for use in inhibition of cellular proliferation.

58. The method of claim 57, wherein said protein sequence is identical or substantially similar to SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 6.

59. A method of pre-screening test substances for use in inhibiting cell proliferation, comprising: measuring the amount of protein- protein binding at a leucine zipper site of a protein encoded by a nucleic acid molecule selected from the following group: a fragment of human chromosome 4, a fragment of human chromosome 4 located at 4q 33-34.1,

SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or MORF 4 on cellular proliferation; and measuring the amount of binding at said leucine zipper site in the presence of said test substance; and comparing the amount of binding with and without said test substance, a test substance which increases the amount of binding being a suitable candidate for use in inhibition of cellular proliferation.

60. The method of claim 59, wherein said protein sequence is identical or substantially similar to SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 6.