Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-02-22
2004-11-09
Nickol, Gary B. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023500
Reexamination Certificate
active
06815166
ABSTRACT:
TECHNICAL FIELD
The invention relates to cancer, and more particularly to cancer suppressor genes.
BACKGROUND
Breast carcinoma is the second leading cause of cancer-related death in women of the western world. In the United States alone over 175,000 new cases are diagnosed annually. The natural history of breast cancer involves a sequential progression through defined clinical and pathologic stages starting with initially benign then atypical hyperproliferation, progressing into in situ then invasive carcinomas, and culminating in metastatic disease. Ductal carcinoma in situ (DCIS) is the precursor of invasive ductal carcinoma.
SUMMARY
The invention is based on the identification of a human gene that, while highly expressed in normal breast tissue, is not expressed or is poorly expressed in DCIS tumors as well as breast tumors at other stages. This gene has been designated the “HIN-1” (High in Normal 1) gene. The inventors have also identified homologues of the human HIN-1 gene in mice and rats. Human HIN-1 is designated as hHIN-1, mouse HIN-1 as mHIN-1, and rat HIN-1 as rHIN-1. Text that refers to HIN-1 without specifying human, mouse or rat is pertinent to all three forms of HIN-1. The inventors have, in addition, identified two Drosophila genes containing sequences encoding proteins with significant homology to hHIN-1. Thus, the invention features an isolated DNA encoding an HIN-1 polypeptide, purified HIN-1 polypeptides, vectors containing the DNAs, and cells containing the vectors. In addition, the invention features a method of making an HIN-1 polypeptide, in vitro and in vivo methods of inhibiting proliferation of a cancer cell, and methods of diagnosing cancer.
More specifically, the invention features an isolated DNA containing a nucleic acid sequence encoding a polypeptide consisting of SEQ ID NO:1 or SEQ ID NO:22. The DNA can, for example, include the nucleic acid sequence designated SEQ ID NO:3 or SEQ ID NO:23. The invention also includes a vector containing: (a) a nucleic acid sequence that (i) encodes a polypeptide that inhibits proliferation of breast cancer cells, and (ii) hybridizes under highly stringent conditions to a probe consisting of a sequence that is the complement of SEQ ID NO:3; or (b) the complement of the nucleic acid sequence. Vectors of the invention can also contain any of the isolated DNAs of the invention. In the vectors, polypeptide encoding sequences can be operably linked to a transcriptional regulatory element (TRE). Also encompassed by the invention is a cell (e.g., a prokaryotic or a eukaryotic cell) comprising any vector of the invention.
Also featured by the invention is an isolated polypeptide containing: (a) a protein that inhibits proliferation of breast cancer cells and that is encoded by a nucleic acid sequence that hybridizes under highly stringent conditions to a probe that includes or is the sequence that is the complement of SEQ ID NO:3; or (b) the protein, except for one or more conservative amino acid substitutions. The polypeptide can include the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:22. Another polypeptide of the invention is an isolated polypeptide containing (a) a functional fragment of any of the above-described polypeptides; or (b) the functional fragment, except for one or more conservative amino acid substitutions. Also included in the invention is a method of making a polypeptide; the method involves culturing a cell of the invention and extracting the polypeptide from the culture. The invention also features fragments of any of the DNAs of the invention, e.g., fragments of the DNA with SEQ ID NO:3 that include nucleotides 55 and 56 of SEQ ID NO:3. The fragments of the DNAs of the invention will be at least 10 bp, 15 bp, 25 bp, 50 bp, 75 bp, 100 bp, 125 bp, 150 bp, 175 bp, 200 bp, 250 bp, 300 bp, 305 bp, or 309 bp long.
Another aspect of the invention is a method of inhibiting proliferation of a cancer cell. The method involves contacting the cancer cell with any of the polypeptides of the invention. The cancer cell can be, for example, a breast cancer cell. The contacting can be in vitro. Alternatively, the cancer cell can be in a mammal and the contacting in the mammal can involve administering either the polypeptide or a polynucleotide encoding the polypeptide to the mammal. Where the cancer cell is in a mammal, the method can involve: (a) providing a recombinant cell that is the progeny of a cell obtained from the mammal and has been transfected or transformed ex vivo with a nucleic acid encoding the polypeptide; and (b) administering the cell to the mammal.
Another embodiment of the invention is a method of identifying a compound that enhances inhibition of proliferation of cancer cells. The method involves: (a) providing a first and a second plurality of cancer cells; (b) combining a test compound, the first plurality of cancer cells, and any of the polypeptides of the invention; (c) combining the second plurality of cancer cells and; (d) determining the level proliferation of the first plurality of cancer cells. A decreased level of proliferation of the first plurality of cancer cells, as compared to the second plurality of cells, indicates that the test compound enhances inhibition of proliferation of cancer cells by the polypeptide.
Also featured by the invention is a method of diagnosis. The method can involve (a) providing a test cell; and b) measuring the level of expression of a HIN-1 gene in the cell. Lack of expression of the HIN-1 gene or a low level of expression of the HIN-1 gene is an indication that the test cell is a cancer cell. Expression of the HIN-1 gene can be measured as a function of the level of HIN-1 mRNA in the cell or as a function of the level of HIN-1 polypeptide in the cell.
In another aspect, the invention provides a method of diagnosis. The method involves (a) providing a test cell; and (b) determining the degree of methylation of a HIN-1 promoter region in the test cell. A high degree of methylation of the HIN-1 promoter region is an indication that the test cell is a cancer cell. The test cell can be, for example, a breast cell, a prostate cell, a pancreatic cell, or a lung cell.
The invention features an antibody that binds to any of the polypeptides of invention. The antibody can be a monoclonal antibody or a polyclonal antibody.
Also included in the invention is a method of treatment that involves identifying a patient as having cancer cells in which (a) HIN-1 gene expression is low or (b) a HIN-1 promoter region is methylated; and treating the patient with a compound that reduces methylation of the HIN-1 promoter region or with a compound that induces expression of a gene with a methylated promoter region, e.g., the HIN-1 gene.
Yet another aspect of the invention is a method of identifying a compound that replaces the function of HIN-1 in cells that do not express HIN-1. The method involves: (a) providing a first cell that does not express HIN-1; (b) providing a second cell that does express HIN-1; (c) treating the first cell and the second cell with a test compound; and (d) determining whether the test compound decreases proliferation of the first or the second cell. A compound that decreases proliferation of the first cell but not the second cell can potentially replace the function of HIN-1 in cells that do not express HIN -1.
“Polypeptide” and “protein” are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification. The invention also features HIN-1 polypeptides with conservative substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.
As used herein, “full-length HIN-1” is HIN-1 with its native signal sequence.
The term “isolated” polypeptide or peptide fragment as used herein refers to a polypeptide or a peptide fragment which either has no naturally-o
Krop Ian
Polyak Kornelia
Porter Dale
Sgroi Dennis
Dana-Farber Cancer Institute Inc.
Fish & Richardson P.C.
Nickol Gary B.
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