Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-02
2001-02-06
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S255100, C536S023100
Reexamination Certificate
active
06183964
ABSTRACT:
BACKGROUND OF THE INVENTION
More than half of all human cancers are associated with one or more alterations in the tumor suppressor gene TP53 (1-4). Many premalignant lesions, a subset of malignant clones, and germlines of families prone to cancer are characterized by the presence of one wild-type and one mutant allele of 2P53 (5-9) (See SEQ ID NO:21-32). In this situation the mutant p53 protein may act in a dominant-negative fashion, ultimately leading to loss of heterozygosity and thus a further growth advantage for the malignant cells. Alternatively, the mutant p53 protein may have acquired a new tumor promoting act which is independent of wild-type p53. These hypotheses are based on the analysis of only a few TP53 mutations usually in the setting of over-expression of the mutant protein, and their relevance to TP53 mutations in general has not been proven (8, 10-13).
Absent or significantly reduced activity of the tumor suppressor protein p53 can be due to the presence of abnormally high levels of host proteins, i.e. mdm-2 or viral proteins, i.e. high-risk human papilloma virus E6 (8, 11, 35). However, in the majority of cancers p53 inactivation is caused by missense mutations in one TP53 allele with concomitant loss-of-heterozygosity (8, 10-12, 36. The missense mutations can be further classified into those affecting codons important for contacting the DNA binding sites and structural mutants affecting codons which stabilize the hydrophobic p53 core domain (31-33, 37). The unusually high frequency of TP53 missense mutations in human cancers can be explained by their dominant-negative effect. Interference with the initially still present wild-type p53 allele leads to increased genetic instability, loss-of-heterozygosity and thus complete abrogation of p53 function (10, 11, 38, 39, 40). In addition, there is evidence that at least some of the same missense mutations confer a gain-of-function (35, 40).
Reconstitution of wild-type p53 activity in these cancers could be of large therapeutic benefit (41-47). The anti-tumor effect of reconstituted p53 activity could be further enhanced by utilization of conventional anti-cancer therapies (43, 44, 46). There is a need in the art for means of correcting the abnormalities found in p53 in human cancers.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a human p53 cDNA which suppresses the phenotype of p53 mutations found in human cancers.
It is another object of the invention to provide human cells which carry both dominant-negative and suppressor type p53 mutations.
It is an object of the invention to provide cells which carry human p53 CDNA which suppresses the phenotype of p53 mutations found in human cancers.
It is an object of the invention to provide methods for treating cancer cells.
It is an object of the invention to provide methods for identifying mutations in a human p53 gene which suppress the phenotype of dominant-negative p53 mutations.
It is an object of the invention to provide a yeast cell useful for selecting and studying mutations in human p53.
It is another object of the invention to provide a method of identifying compensatory mutations in TP53 which suppress dominant-negative TP53 mutant phenotypes.
It is still another object of the invention to provide a method of identifying potential therapeutic agents.
It is yet another object of the invention to provide a method of screening putative carcinogens.
It is yet another object of the invention to provide a method for identifying cellular proteins which interact with p53 and abrogate its activity.
It is an object of the invention to provide a kit for isolating mutations in p53.
It is another object of the invention to provide a gene fusion useful for isolating and studying p53 mutations.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention a nucleic acid encoding human p53 is provided which carries at least one intragenic suppressor mutation, wherein said mutation suppresses a dominant-negative mutation of p53, and at least one dominant negative mutation of p53.
In another embodiment of the invention a nucleic acid is provided which encodes human p53. The nucleic acid comprises at least one intragenic suppressor mutation, wherein said intragenic suppressor mutation suppresses a dominant-negative mutation of p53, wherein the at least one intragenic suppressor mutation is selected from the group consisting of: T123P, N268D, N239Y, S240N, and T123A+H168R.
In another embodiment of the invention a human cell is provided which comprises:
a human p53 DNA which comprises a dominant-negative mutation; and
a nucleic acid encoding human p53 which carries at least one intragenic suppressor mutation, wherein said mutation suppresses a dominant-negative mutation of p53 in trans.
In still another embodiment of the invention a method of treating cancer cells is provided. The method comprises:
introducing into the cancer cells a nucleic acid which carries at least one intragenic suppressor mutation, wherein said mutation suppresses a dominant-negative mutation of p53, whereby a neoplastic phenotype of the cancer cells is suppressed or apoptosis is induced.
In yet another embodiment of the invention a method is provided for identifying mutations in a human p53 gene which suppress the phenotype of a dominant-negative p53 mutation. The method comprises the steps of:
introducing a p53 nucleic acid which has been mutagenized into a cell which carries a dominant-negative mutation in a p53 DNA;
testing the phenotype of the cell to determine whether the cell behaves like a cell carrying a wild-type p53 or a cell carrying a dominant-negative p53 mutation; wherein a cell which behaves like a cell carrying a wild-type p53 is a cell carrying a p53 suppressor mutation on the mutagenized p53 nucleic acid.
In another embodiment of the invention a method is provided for identifying mutations in a human p53 gene which suppress the phenotype of dominant-negative p53 mutations. The method comprises the step of:
testing the phenotype of a cell which comprises a p53 nucleic acid which carries a dominant-negative mutation and which has been mutagenized, to determine whether the cell behaves like a cell carrying a wild-type p53 or a cell carrying a dominant-negative p53 mutation; wherein a cell which behaves like a cell carrying a wild-type p53 is a cell carrying a p53 suppressor mutation on the mutagenized p53 nucleic acid.
In one embodiment of the invention a yeast cell is provided. The cell comprises a first reporter gene which is selectable or counterselectable. The reporter gene is operably linked to a DNA sequence to which human p53 specifically binds. The cell also comprises a first fusion gene which expresses a human p53 in the cell. The fusion gene comprises a yeast promoter operably linked to a human p53 coding sequence.
In another embodiment of the invention a method of identifying compensatory mutations in TP53 which suppress dominant-negative 7P53 mutant phenotypes is provided. The method involves providing a cell which comprises:
a reporter gene which is selectable, wherein the reporter gene is operably linked to a DNA sequence to which human p53 specifically binds; and
a first fusion gene which expresses a dominant-negative allele of human p53 in the cell, the fusion gene comprising a promoter operably linked to a human p53 coding sequence.
Then a population of DNA molecules comprising a second fusion gene is introduced into the cell. The second fusion gene comprises a promoter operably linked to a mutagenized human p53 coding sequence. Phenotypic revertants of the dominant-negative allele of human TP53 are selected using the selectable phenotype of the reporter gene.
According to another embodiment of the invention a method of identifying potential therapeutic agents is provided. A cell is provided which comprises:
a reporter gene which is selectable, wherein the reporter gene is operably inked to a DNA sequence to which human p53 specifically binds; and
a fusion gene which-e
Boeke Jef D.
Brachmann Rainer K.
Arthur Lisa B.
Banner & Witcoff , Ltd.
Enewold Jeanine
The Johns Hopkins University
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