EP1001966A1 - Ein senescene-gen und seine verwendung zur behandlung von krebs und anderen krankheiten - Google Patents

Ein senescene-gen und seine verwendung zur behandlung von krebs und anderen krankheiten

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Publication number
EP1001966A1
EP1001966A1 EP98933073A EP98933073A EP1001966A1 EP 1001966 A1 EP1001966 A1 EP 1001966A1 EP 98933073 A EP98933073 A EP 98933073A EP 98933073 A EP98933073 A EP 98933073A EP 1001966 A1 EP1001966 A1 EP 1001966A1
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EP
European Patent Office
Prior art keywords
cell
nucleic acid
fragment
acid molecule
seq
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EP98933073A
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English (en)
French (fr)
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EP1001966A4 (de
Inventor
Olivia M. Pereira-Smith
Yi Ning
Michael J. Bertram
Nathalie Berube
Xin Swanson
James K. Leung
Qitao Ran
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Baylor College of Medicine
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Baylor College of Medicine
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Publication of EP1001966A1 publication Critical patent/EP1001966A1/de
Publication of EP1001966A4 publication Critical patent/EP1001966A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • 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.
  • 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.
  • HIV human immunodeficiency virus
  • 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.
  • nucleotide analogues such as azidothymidine (AZT) and deoxynucleotides other than AZT such as 2',3'-dideoxyadenosine (ddA), dideoxyinosine (ddl), and dideoxycytidine (ddC)
  • AZT azidothymidine
  • ddA dideoxyinosine
  • ddC dideoxycytidine
  • 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.
  • undesired side effects and high cost have limited the applicability and availability of such agents.
  • 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.
  • an ability to arrest temporarily cellular proliferation would provide a means for combatting retroviral infection, and in particular, would comprise a therapy for AIDS.
  • 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.
  • 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.
  • a gene sequence when combined with a suitable genetic therapy comprises a therapy for cancer and diseases, such as AIDS, that involve proliferative cells.
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • 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
  • 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.
  • 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 ⁇ -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
  • the present invention derives in part from the identification of gene sequences that are determinants of cellular proliferation.
  • an "antibody” is an immunoglobulin protein that has a specific affinity for a given molecule called an antigen.
  • a “gene sequence” is a nucleic acid molecule.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • microcell donor cells are plated onto plastic bullets cut from tissue culture flasks and incubated in medium (such as
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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);
  • 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.
  • MORF 4 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).
  • BACs Bacterial Artificial Chromosomes
  • 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.
  • 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.
  • the molecules of the present invention may be used to control or suppress undesired or uncontrolled cellular proliferation.
  • cellular proliferation is said to be "undesired” when it comprises growth that for medical or cosmetic reasons is not wanted by a patient.
  • 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.
  • the formation of "scar" tissue incident to the first such operation may encumber subsequent operations.
  • 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.
  • the primary drugs presently used for treating AIDS patients are nucleotide analogues that affect the capacity of virally infected cells to replicate DNA.
  • 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.
  • 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.
  • 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,
  • the genetic determinants of the present invention have therapeutic utility in the treatment of diseases such as AIDS and cancer.
  • 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.
  • 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.
  • 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.
  • cell surface antigens 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.
  • virally-infected cells such evaluation can additionally include monitoring the capacity of the cell for its capacity to replicate the virus.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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);
  • 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.
  • 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.
  • the introduced molecules are preferentially expressed in tumor cells.
  • the vector contains appropriate transcriptional or translational regulatory elements, such that the proliferation determinants are preferentially expressed in tumor cells.
  • 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.
  • promoters include the native promoter naturally associated with a gene of a complementation group, the ⁇ - fetoprotein promoter, the amylase promoter (especially, the murine amylase promoter), the cathepsin E promoter, the Ml muscarinic receptor promoter, the ⁇ -glutamyl transferase promoter, etc., and especially, the CMV promoter.
  • Suitable ⁇ -fetoprotein promoter sequences are present in the vectors PSVA F0.4 CAT A and PAF 5.1 02-CAT) (Watanabe et al., J.
  • 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 ( ⁇ 2-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-
  • Suitable ⁇ -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 ⁇ -galactosidase expression vector, CMB ⁇ (See MacGregor, G.R. et al, Nucleic Acids Res. 17:2365 (1989)).
  • the vector is engineered to array receptors or ligands for an antigen present on the tumor cell that is the recipient of the vector.
  • array receptors or ligands for an antigen present on the tumor cell that is the recipient of the vector.
  • markers are discussed by Drebin, J.A. et al, Current Therapy in Oncology, pp. 58-61 (1993).
  • the vectors preferentially adsorb to tumor cells, and thereby impart selectively their therapeutic value.
  • lymphotrophic viral vectors are preferred.
  • such vectors are produced by modifying an existing lymphotrophic virus (HIV, SIV, EB, etc.).
  • such vectors can comprise non-lymphotrophic viruses that have been modified to permit them to adsorb to and infect CD4+ cells.
  • synthetic viral vectors are formed that array the HIV gp 120 protein that is capable of binding to the CD4 receptor.
  • dermatrophic viral vectors such as herpes viral vectors, etc., are employed to deliver the therapeutic gene sequences of the present invention.
  • a preferred embodiment of the invention comprises vectors that are capable of expressing the incorporated proliferation determinant.
  • the desired gene therapy is mediated in the absence of expression, most preferably via recombination.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • hybridization 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.
  • 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.
  • the present invention contemplates the use of any of a variety of chemical agents to either inhibit or enable DNA synthesis.
  • 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
  • the agents of the present invention comprises nucleic acid molecules.
  • such molecules include the naturally occurring gene sequences of the determinants of cellular proliferation that have been purified from their natural contaminants.
  • the sequence of the molecules are selected so as to be superior to such natural gene 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid molecule is a fragment of human chromosome 4 located at 4q 33-34.1.
  • 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.
  • the virally-infected cell is infected with human immunodeficiency virus.
  • the cancerous cell belongs to a group B complementation group.
  • 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.
  • 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.
  • nucleic acid molecule is inserted into a non-viral vector.
  • nucleic acid has been inserted into a viral vector, where the viral vector has been encapsulated in a viral coat.
  • the viral vector is an adenovirus
  • 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.
  • the cell is a cancerous cell or a virally-infected cell.
  • the cancerous cell belongs to a group B complementation group.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • FISH fluorescence in situ hybridization
  • 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.
  • PFGE Pulse field gel electrophoresis
  • 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.
  • 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.
  • Genomic DNA was used in transfections because the activity of the cell senescence gene on human chromosome 4 was followed under its own promoter.
  • MORF 4 Motality Factor on chromosome 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.
  • 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.
  • MORF 4 is a member of a family.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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 ⁇ g of protein in saline emulsified with an equal volume of complete Freund's adjuvant in the primary inoculation.
  • 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.
  • the mouse and rat homologs of the MORF 4 gene were identified.
  • 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 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.
  • 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.
  • 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.
  • a modulatable promoter system such as the MMTV promoter which responds to dexamethasone, or the tetracycline inducible system
  • 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.
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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)).
  • MORF 4 is examined to see if it is phosphorylated in vivo by analysis of immunoprecipitates from cells metabolically labeled with [ 32 p] orthophosphate by the method of Rosfjord, E. et al, Biochem. Biophys. Res. Commun. 212(3) :847-853 (1995).
  • 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.
  • YORF YORF chromosome
  • 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.
  • 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.
  • 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.
  • the group B cell lines for mutations or deletions in these region can be analyzed.
  • Cells are labeled with 35 S methionine and extracts prepared.
  • 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.
  • 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.
  • 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.
  • 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.
  • yeast two-hybrid system Fields, S. & Song, O., Nature 340:245-
  • 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.
  • a reporter gene such as lacZ
  • MORF 4 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 ⁇ Z., Development 108:47-58
  • 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.
  • 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.

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EP98933073A 1997-07-03 1998-07-02 Ein senescene-gen und seine verwendung zur behandlung von krebs und anderen krankheiten Withdrawn EP1001966A4 (de)

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DATABASE EMBL [Online] 2 April 1993 (1993-04-02) OHARA ET AL.: "Human mRNA for KIAAA0026 gene, complete cds" Database accession no. D14812 XP002206665 *
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