Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-02
2001-04-03
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S023500, C536S024310
Reexamination Certificate
active
06210887
ABSTRACT:
BACKGROUND OF THE INVENTION
Colorectal cancer is a significant cancer burden to the general population of many developed countries. In the United States alone, there are over 130,000 new cases of colorectal cancer per year, and over 65,000 deaths per year resulting from colorectal cancer. Colorectal cancer it is second only to lung cancer in cancer morbidity in the United States.
The progression of colorectal cancer, or colorectal tumorigenesis, is a multi-step process involving the loss of function of so-called tumor suppressor genes, as well as the activation of oncogenes. Fearon et al.,
Cell
61: 759-67 (1990); Paraskeva et al.,
Anticancer Research
10: 1189-200 (1990). It is also marked by several phenotypically distinct stages during progression. These include normal, hyperplastic, benign, carcinoma and metastatic stages. These distinct stages make colorectal cancer an exceptionally useful paradigm for the studying the molecular genetic basis of cancer in general.
Among the classical oncogenes implicated in cancer, the ras and myc genes have been found to be activated and/or show elevated expression in colorectal tumors. About half of large adenomas and at least half of carcinomas contain activated K-ras genes. Forrester et al.,
Nature
327: 298-303 (1987); Bos et al.,
Nature
327: 293-97 (1987); Burmer et al.,
Proc. Nat'l Acad. Sci. USA
86: 2403-07 (1989). C-myc over expression and occasional gene amplification have also been demonstrated in colorectal tumors. Erisman et al.,
Mol. Cell. Biol.
5: 1969-76 (1985); Imaseki et al.,
Cancer
64: 704-09; Finley et al.,
Oncogene
4: 963-71 (1989). Furthermore, deregulated c-myc expression can be suppressed by microcell-mediated transfer of chromosome 5, which is the locus for the putative tumor-suppressor genes, APC (for adenomatous polyposis coli) and MCC (for mutated in colorectal carcinoma) discussed below. Rodriguez-Alfageme et al.,
Proc. Nat'l Acad. Sci. USA
89: 1482-86 (1992). Although the importance of oncogenes in cancer development can not be ignored, it is the presently the tumor suppressor genes which have drawn the most interest for study of cancer development.
Several tumor suppressor genes have been implicated in colorectal tumor progression. One of the more noteworthy tumor suppressor genes is p53. This gene has a locus at chromosome band 17p13 and is lost in a large majority of colon carcinomas (though not as much in adenomas). Often the lesion, which refers to genetic mutations, consists of a deletion of one allele and a point mutation at one of several hotspots in the remaining allele. Baker et al.,
Science
244: 217-21 (1989), Nigro et al.,
Nature
342: 705-07 (1989). Importantly, it has been shown that transfection of a wild-type p53 gene into colon cancer cell lines in vitro results in a suppression of cell growth, thereby demonstrating that the p53 gene product, a tumor suppressor, has a direct effect on one major cancer characteristic. Baker et al.,
Science
249: 912-15 (1990).
Genes APC and MCC identified above have been mapped to a locus at chromosome band 5q21. Groden et al.,
Cell
66: 589-600 (1991); Kinzler et al.,
Science
253: 661-64 (1991); Kinzler et al.,
Science
251: 1366-70 (1991). This is the site which is linked to the inherited disorder adenomatous polyposis coli, which is a disorder marked by multiple polyposis and a very high incidence of colon carcinoma at an early age. Both genes contain mutations and/or deletions in colon carcinoma, however, MCC mutations are not common among tumors, whereas the APC lesions are more common and found in the germ line genomic DNA of APC patients. Kinzler et al.,
Science
251: 1366-70 (1991); Nishisho et al.,
Science
253: 665-69. It is notable that transfer of chromosome 5 to colon cancer cells lacking a normal APC gene suppresses tumorigenicity. Goyette et al.,
Mol. Cell. Biol.
12: 1387-95 (1992). This reinforces the concept that APC and/or MCC are tumor suppressor genes. Another gene, the DCC gene (for deleted in colorectal carcinoma), is located at chromosome band 18q21 and also is lost in a large majority of colon carcinomas and about fifty percent of late adenomas. A portion of the DCC gene bears a homology to the neural cell adhesion molecule (N-CAM). Fearon et al.,
Science
247: 49-56 (1990). This suggests that the DCC gene product may play a role in cell-to-cell contacts. A specific role in colorectal tumor progression, however, has not been ascertained.
The identification of such genes, the absence or impairment of which is linked to cancer, yields insights into the initiation and progression of cancer and other abnormalities. Additionally, the existence of such genes raises the possibility that other tumor suppressor genes may exist.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to identify a gene that is down-regulated in colon adenomas and adenocarcinomas.
Another object of this invention is to provide a gene that is down-regulated early in tumorigenesis.
It is another object of this invention to provide a cDNA sequence that corresponds to the aforementioned down-regulated gene.
Still another object of this invention to provide a nucleotide probe that hybridizes to the aforementioned down-regulated gene.
Still another object is to provide for a down-regulated gene herein referred to as “DRA” (for down regulated in adenoma).
It is yet another object of this invention is to provide a method for identifying and isolating candidate tumor suppressor genes.
It is still another object of this invention to provide an assay and method to diagnose and/or identify colon tissue abnormalities by measuring the presence or absence of the mRNA or protein product of a down-regulated gene.
In accomplishing the foregoing objects, there has been provided, in accordance with one aspect of the present invention, a cDNA encoding a polypeptide having a molecular weight of about 84,500 daltons. The mRNA encoding this polypeptide has been found to be down-regulated in adenocarcinomas and adenomas of the colon.
There is also provided a method for evaluating colon tissue comprising the steps of:
obtaining a colon tissue test sample;
evaluating the amount of DRA mRNA expression in, said colon tissue sample by hybridizing the mRNA of said tissue sample with a nucleotide probe derived from the DRA nucleotide sequence;
comparing said amount of DRA mRNA expression in said colon tissue sample to a control to determine relative DRA mRNA expression.
Other objects, features and advantages of the present invention will become apparent from the following detailed description, sequence data and tables.
REFERENCES:
patent: 5650281 (1997-07-01), Vogelstein
Hamilton, S.R. Journal of Cellular Biochemistry, Supplement 16G:41-46, 1992.*
Goelz et al., “Hypomethylation of DNA from Benign and Malignant Human Colon Neoplasms,”Science, vol. 228, pp. 187-190 (Apr. 1985).
Erisman et al., “Deregulation of c-myc Gene Expression in Human Colon Carcinoma Is Not Accompanied by Ampification or Rearrangement of the Gene,”Mol. Cell. Biol., vol. 5, No. 8, pp. 1969-1976 (Aug. 1985).
Forrester et al., “Detection of High Incidence of K-ras Oncogenes during Human Colon Tumorigenesis,”Nature, vol. 327, pp. 298-303 (May 1987).
Feinberg et al., “Reduced Genomic 5-Methylcytosine Content in Human Colonic Neoplasia,”Cancer Research, vol. 48, pp. 1159-1161 (Mar. 1988).
Imaseki et al., “Expression of c-myc Oncogene in Colorectal Polyps as a Biological Marker for Monitoring Malignant Potential,”Cancer, vol. 64, pp. 704-709 (1989).
Finley et al., “Expression of myc Gene Family in Different Stages of Human Colorectal Cancer,”Oncogene, vol. 4, pp. 963-971 (1989).
Burmer et al., “Mutations in the KRAS2 Oncogene during Progressive Stages of Human Colon Carcinoma,”Proc. Natl. Acad. Sci USA, vol. 86, pp. 2403-2407 (Apr. 1989).
Baker et al., “Chromosome 17 Deletions and p53 Gene Mutation in Colorectal Carcinomas,”Science, vol. 244, pp. 217-221 (Apr. 1989).
Nigro et al., “Mutations in the p53 Gene Occur in Diverse Human Tumour Types,”Nature, vol. 342, pp. 705-70
Papas Takis S.
Schweinfest Clifford W.
Fish & Richardson P.C.
Johannsen Diana
Myers Carla J.
The United States of America as represented by the Department of
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