Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1994-04-14
2004-02-10
Zitomer, Stephanie (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S320100, C435S194000, C435S325000, C536S023100, C536S024330
Reexamination Certificate
active
06689561
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention.
The invention relates to the detection of genetic abnormalities that confer. susceptibility to certain cancers in humans. More specifically, the invention relates to methods for detecting deletions of, or polymorphisms in, a newly discovered gene which encodes a tumor suppressor.
2. History of the Prior Art.
In recent years, a growing body of evidence has developed which supports the theory that the development of certain tumors is suppressed by gene products (“tumor suppressors”) which inhibit cellular proliferation (see, e.g., the review in Marx,
Science,
263:319-320, 1994). Conversely, if the tumor suppressors which would ordinarily be present in a cell are either absent (due, for example, to a gene deletion) or less active (due, for example, to a gene mutation), tumor growth which would otherwise be inhibited may go unchecked. However, although the growth of certain tumors has been positively demonstrated to relate to the deletion of a tumor suppressor expressing gene, it has not yet been shown that mutations in the same genes will allow abnormal cellular proliferation to occur.
The growth cycle of eukaryotic cells is regulated by a family of protein kinases known as the cyclin-dependent kinases (“CDK's”). As shown in
FIG. 1
, the cyclins and their associated CDK's move cells through the three phases of the growth cycle (G
1
, S and G
2
, respectively) leading to division in the mitosis phase (M). The cyclin/CDK complexes whose role in cellular proliferation has been most clearly defined to date are the cyclin D/CDK enzymes, which are believed to assist in the progression of the G
1
growth cycle phase. Of these enzymes, cyclin D1 is believed to be an oncogene, whose overexpression stimulates excessive cell division through the continuous production of kinase, thus contributing to the development of cancers of, for example, the breast and esophagus. Cyclin D1 is specifically bound by CDK4 as part of a multi-protein complex that also consists of a protein known as p21 and cell nuclear antigen.
Known inhibitors of such cyclin/CDK overexpression include the tumor suppressor protein p53 and the protein product of the retinoblastoma (Rb) gene. Recently, another putative inhibitor (p16) was isolated and a cDNA for the inhibitor was partially sequenced by Serrano, et al.,
Nature,
366:704-710, 1993. The authors demonstrated that p16 binds CDK4 to inhibit the activity of the CDK4/cyclin D enzymes. Based on data indicating that p16 prevented phosphorylation by CDK/cyclin D of certain Rb growth cycle proteins, the authors proposed that p16 acts in vivo upstream and downstream of Rb to form a negative feedback loop to regulate cellular proliferation. However, no connection between p16 and the occurrence or inhibition of particular cancers was suggested, nor has any information been published concerning the genomic structure of the gene encoding p16.
SUMMARY OF THE INVENTION
Prior to the publication of the Serrano, et al., article referred to above, the inventors discovered a tumor suppressor gene (hereafter, “CDK4I”) and identified its genomic structure (see, SEQ ID NO's: 1-2). In non-malignant cells, CDK41 maps to chromosome 9p21 and is physically adjacent to the gene for methylthioadenosine phosphorylase (MTAse) (see, FIG.
4
(
b
)). MTAse deficiencies resulting from deletions of, or mutations in, the gene for MTAse have been shown to be directly related to the onset of certain cancers (see, Nobori, etal.,
Cancer Res.
53:1098-1101, 1993, the disclosure of which are incorporated herein for reference regarding the role of MTAse in cancer development, and SEQ ID NO: 14, the nucleotide sequence of genomic MTAse).
Approximately one-half of all tumor cells which have been identified to date as either lacking CDK4I or containing mutations or rearrangements (collectively, “polymorphisms”) of the CDK4I gene also lack MTAse. The inventors have also identified mutations in the CDK4I gene which are present in the tumor cells of patients with certain cancers. The invention is therefore directed to methods to detect (a) deletions of the CDK4I gene in cells, and (b) polymorphisms, which deletions and polymorphisms are indicative of susceptibility to certain cancers.
More specifically, in one aspect, the invention comprises methods for detecting point mutations in, or deletions of, the CDK4I gene. Such methods include polymerase chain reaction (PCR) based assays, gel electrophoresis of single-strand conformation polymorphisms, direct sequencing, and restriction endonuclease digestion. Detection of a deletion of the CDK4I gene will preferably be performed by a unique competitive PCR technique.
In another aspect, the invention comprises methods for detection of CDK4I proteins and biologically active fragments thereof (collectively, “CDK4I”) in a biological cell sample.
In another aspect, the invention comprises screening protocols for susceptibility to particular cancers based on detection of polymorphisms associated with the occurrence of the cancers.
In another aspect, the invention comprises screening protocols for susceptibility to particular cancers based on detection of polymorphisms in, or deletions of, the genes for both CDK4I and MTAse, as well as detection of deficiencies in the products of the genes.
In another aspect, the invention comprises genomic CDK4I, expression products of the CDK4I gene, CDK4I and fragments thereof, as well as antibodies which will specifically bind CDK4I gene expression products, CDK41 and CDK4I fragments.
REFERENCES:
patent: 4681840 (1987-07-01), Stephenson et al.
James, Analytical Chemistry and Chemotherapy 2(4): 191-214, 1991.*
Freeman, Adv. Drug Delivery Reviews, 12: 169-183, 1993.*
Gutierrez et al., The Lancet 339: 715-721, 1992.*
Talmadge Adv. Drug. Deliv. Rev. 10: 247-299, 1993.*
Goldberg et al. Clin. Chem 39: 2360-2374, 1993.*
Schreiber, “Tumor Immunology” in Fundamental Immunology, Ed. William Paul, Raven Press, p. 923-955, 1989.*
Tidd, Anticancer Research 10, 1169-1182, 1990, Cited as of Interest.*
Dorlands Medical Dictionary, W.B.Saunders Co., p. 461.*
A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4 Serrano, et al.,Nature, 366:704-707, Dec. 16, 1993.
Allele-specific enzymatic amplification of &bgr;-globin genomic DNA for diagnosis of sickle cell anemia Wu, et al.,Proc. Natl. Acad. Sci. USA, 86:2757-2760, 4/89.
&dgr;-Aminolevulinate Dehydratase Deficient Porphyria: Identification of the Molecular Lesions in a Severely Affected Homozygote Plewinska, et al.,Am. J. Hum Genet., 49:167-174, 1991.
Detection of polymorphisms of human DNA be gel electrophoresis as single-strand conformation polymorphisms Orita, et al.,Proc. Natl. Acad. Sci USA, 86:2766-2770, 4/89.
How Cells Cycle Toward Cancer Jean Marx,Science, 263:319-321, Jan. 21, 1994.
Absence of Methylthioadenosine Phosphorylase in Human Gliomas Nobori, et al.,Cancer Research, 52:3193-3197, Jun. 15, 1991.
Methylthioadenosine Phosphorylase Deficiency in Human Non-Small Cell Lung Cancers Nobori, et al.,Cancer Research, 53:1098-1101, Mar. 1, 1993.
Selective killing of human malignant cell lines deficient in methylthioadenosine phosphorylase, a purine metabolic enzyme Kamatani, et al.,Proc. Natl. Acad. Sci. USA, No. 2, 78:1219-1223, 2/81.
Methylthioadenosine Phosphorylase Deficiency in Human Leukemias and Solid Tumors Fitchen, et al.,Cancer Research, 46:5409-5412, 10/86.
Deficiency of 5′deixt-5′-methylthioadenosine phosphorylase activity in malignancy Della Ragione, et al.,Biochem. J, 281:533-538, 1992.
Kamb, et al., “A Cell Cycle Regulator Potentially Involved in Genesis of Many Tumor Types,”Science, vol. 264, Apr. 15, 1994, pp. 436-440.
Nobori, et al., “Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers,”Nature, vol. 368, Apr. 21, 1994, pp. 753-756.
Tam, et al., “Differential Expression and Cell Cycle Regulation of the Cyclin-dependent Kinase 4 Inhibitor p16Ink4,”Cancer Research, 54:5816-5820, Nov. 15, 1994.
ATCC Catalogue of Recombinant DNA mat
Carson Dennis A.
Nobori Tsutomu
The Regents of the University of California
Townsend and Townsend / and Crew LLP
Zitomer Stephanie
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