Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-08-28
2004-05-25
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S377000, C435S320100, C536S023100, C536S024100, C536S024500, C514S04400A
Reexamination Certificate
active
06740523
ABSTRACT:
TECHNICAL FIELD
The invention relates to the fields of diagnosis and therapy of cancers. More particularly, the invention relates to a protein which mediates cell cycle arrest upon damage to cellular DNA.
BACKGROUND OF THE INVENTION
It has long been known that DNA-damaging agents induce a cell cycle arrest, allowing time for repair and thus protecting the organism from the deleterious consequences of mutation. In mammalian cells, these arrests are often dependent on the functionality of the p53 gene product, a transcription factor which is translationally and posttranslationally activated following DNA damage (reviewed in Cox, Levine, and Morgan). Because p53 is mutated in a large fraction of cancers of diverse type, it is thought that the tumorigenic process may be intimately related to the disruption of p53-mediated control of the cell cycle. Accordingly, there has been much effort to define the molecular links between DNA damage, p53 expression, and cell cycle regulation.
Cells treated with ionizing radiation or other DNA-damaging agents arrest in both G1 and G2, with a consequent decrease in the fraction of cells in S phase. In colorectal cancer cells and many other epithelial cell types, 15-40% of the cells arrest in G1 and the remainder arrest in G2/M. The G1 block is in part mediated by p21
WAF1/CIP1/SDI1
, a cyclin dependent kinase inhibitor, which is transcriptionally controlled by p53. Several studies have suggested that the G2/M block following DNA damage is also p53-dependent. However, the basis for this G2/M block, though accounting for the predominant form of arrest induced by radiation in many cell types, is unknown.
Thus, there is a need in the art for elucidation of the pathway by which p53 exerts its cell cycle arresting effects. There is also a need in the art for new diagnostic and therapeutic tools for evaluating and ameliorating human cancers.
SUMMARY OF THE INVENTION
It is an object of the invention to provide DNA molecules useful for diagnosing and treating human tumors.
It is another object of the invention to provide proteins useful for treating human tumors and for raising diagnostically useful antibodies.
It is yet another object of the invention to provide methods of suppressing growth of tumor cells.
It is an object of the invention to provide a method for screening potential therapeutic agents for treating cancer.
It is another object of the invention to provide methods for diagnosing cancer.
It is yet another object of the invention to provide a reporter construct, useful for screening potential antineoplastic agents.
It is an additional object of the invention to provide an antisense construct for inhibiting expression of a tumor suppressor gene.
It is still another object of the invention to provide antisense oligonucleotides for inhibiting expression of a tumor suppressor gene.
It is yet another object of the invention to provide methods for promoting growth of cells in which a tumor suppressor gene's expression is inhibited.
It is another object of the invention to provide a method for assessing susceptibility to cancers.
It is an object of the invention to provide a method for detecting the presence of wild-type p53 protein in a cell.
It is still another object of the invention to provide methods for identifying compounds which specifically bind to p53-specific DNA binding sequences which regulate expression.
It is an object of the invention to provide methods for screening for anti-cancer drugs.
It is another object of the invention to provide cell lines useful for screening for anti-cancer drugs.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention an isolated and purified subchromosomal DNA molecule is provided. The molecule encodes 14-3-3&sgr; protein (a) as shown in SEQ ID NO: 2. The sequence of cDNA encoding &sgr; is shown in SEQ ID NO: 1.
In another embodiment of the invention an isolated and purified 14-3-3&sgr; protein is provided. The protein has a sequence as shown in SEQ ID NO: 2.
In still another embodiment of the invention a method of suppressing growth of tumor cells is provided. The method comprises administration of a 14-3-3&sgr; protein having a sequence as shown in SEQ ID NO: 2 to said cells.
In an additional embodiment of the invention a method of suppressing growth of tumor cells is provided. The method comprises administration to said cells of a DNA molecule which causes said cells to express 14-3-3&sgr;, said DNA molecule having a sequence as shown in SEQ ID NO: 1.
According to another embodiment of the invention a method for screening potential therapeutic agents for the ability to suppress the growth of tumor cells by activating the expression of 14-3-3&sgr; is provided. The method comprises incubation of a potential therapeutic agent with a cell which contains a 14-3-3&sgr; reporter construct, said reporter construct comprising a 14-3-3&sgr; transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable product. Further, the method comprises measurement of the production of the assayable product. A potential therapeutic agent is identified as useful if it increases the production by the cell of the assayable product.
In still another embodiment of the invention a method for diagnosing cancer is provided. The method comprises testing a tissue to determine if the tissue expresses less 14-3-3&sgr; than normal tissue.
In another embodiment of the invention a method for diagnosing cancer is provided. The method comprises testing a tissue to determine if DNA in said tissue contains a mutant 14-3-3&sgr; gene.
In still another embodiment of the invention a 14-3-3&sgr; reporter construct is provided. The reporter construct comprises a 14-3-3&sgr; transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable product.
In another embodiment of the invention an antisense 14-3-3&sgr; construct is provided. The construct comprises: a transcriptional promoter; a transcriptional terminator; and a DNA segment comprising one or more segments of the 14-3-3&sgr; gene, said gene segment located between said promoter and said terminator, said DNA segment being inverted with respect to said promoter and said terminator, whereby RNA produced by transcription of the DNA segment is complementary to a corresponding segment of 14-3-3&sgr; RNA produced by human cells.
According to another embodiment a method of identifying a chromosome is provided. The method comprises the steps of contacting one or more chromosomes with a polynucleotide probe which comprises at least 11 contiguous nucleotides of the sequence shown in SEQ ID NO: 1; and detecting chromosomes which specifically bind to the polynucleotide probe, wherein a chromosome which specifically binds to the probe is identified as containing at least a portion of human chromosome 1.
In another embodiment of the invention a 14-3-3&sgr; antisense oligonucleotide is provided. The oligonucleotide comprises at least ten nucleotides complementary to a sequence present in 14-3-3&sgr; mRNA.
In yet another embodiment of the invention a triplex oligonucleotide is provided. The oligonucleotide comprises at least ten nucleotides complementary to a sequence present in a 14-3-3&sgr; gene.
In still another embodiment of the invention a method is provided for promoting growth of cells. The method comprises: administering a 14-3-3&sgr; antisense or triplex-forming oligonucleotide comprising at least ten nucleotides complementary to 14-3-3&sgr; mRNA or 14-3-3&sgr; gene, respectively, to said cells to inhibit the expression of 14-3-3&sgr;. In an alternative method an antisense 14-3-3&sgr; construct is administered to said cells to inhibit the expression of 14-3-3&sgr;. The construct comprises:
a. a transcriptional promoter;
b. a transcriptional terminator;
c. a DNA segment comprising one or more segments of the 14-3-3&sgr; gene, said gene segment located between said promoter and said terminator, said DNA segment being inverted with re
Hermeking Heiko
Kinzler Kenneth W.
Vogelstein Bert
Banner & Witcoff , Ltd.
McGarry Sean
The Johns Hopkins University
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