Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-21
2001-06-12
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024330, C536S024500, C536S025300, C536S027400
Reexamination Certificate
active
06245515
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to diagnostic and therapeutic methods for detecting and alleviating cancers. Specifically it relates to the identification of DNA sequences specifically bound by tumor suppressor p53.
BACKGROUND OF THE INVENTION
The gene for the nuclear phosphoprotein p53 is the most commonly mutated gene yet identified in human cancers (Vogelstein, B.,
Nature,
348:681 (1990)). Missense mutations occur in tumors of the colon, lung, breast, ovary, bladder, and several other organs (S. J. Baker, et al.,
Science,
244:217 (1989); J. M. Nigro, et al.,
Nature,
342:705 (1989); T. Takahashi, et al.,
Science,
246:491 (1989); Romano, et al.,
Oncogene,
4:1483 (1989), Menon,
Proc. Natl Acad. Sci. USA,
87:5435 (1990); Iggo, et al.,
Lancet ii,
675 (1990); T. Takahashi, et al.,
J. Clin. Invest.
86:363 (1990); Mulligan,
Proc. Natl Acad. Sci. USA,
87:5863 (1990); Bartek, et al.,
Oncogene,
5:893 (1990); Stratton et al.,
Oncogene,
5:1297 (1990)). One of the important challenges of current cancer research is the elucidation of the biochemical properties of the p53 gene product and the way in which mutations of the p53 gene affect these properties.
Although some biological characteristics of p53 have been defined, such as its ability to suppress the growth of in vitro transformed murine cells (Eliyahu, et al.,
Proc. Natl Acad. Sci. USA
86:8763 (1989); Finlay, et al.,
Cell,
57:1083 (1989)) or human cancer cells (Baker, et al.,
Science,
249:912 (1990); Mercer, et al.,
Proc. Natl Acad. Sci, USA,
87:6166 (1990); Diller et al.,
Mol. Cell Biol.
10:5772 (1990)), the biochemical basis of this suppression remains largely unknown. As a step towards understanding such properties, we have attempted to determine whether p53 binds to specific DNA sequences within the human genome.
SUMMARY OF THE INVENTION
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 another object of the invention to provide a method for providing the physiological effect of wild-type p53 protein to a cell.
It is yet another object of the invention to provide a double-stranded DNA fragment which contains a p53-specific DNA binding site.
It is yet another object of the invention to provide a single-stranded oligonucleotide or oligonucleotide containing nucleotide analogs which can specifically complex to a p53-specific DNA binding site.
It is still another object of the invention to provide methods for identifying compounds which specifically bind to p53-specific DNA binding sequences.
It is still another object of the invention to provide methods for identifying compounds which restore the ability of mutant p53 proteins to bind to specific DNA binding sequences.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment a method is provided for detecting the presence of wild-type p53 protein in a cell, comprising the steps of: contacting a p53-specific-binding DNA fragment with a cell lysate from a tissue of a human to bind the DNA fragment to wild-type p53 present in the cell lysate; and detecting the binding of the p53-specific-binding DNA fragment to wild-type p53.
In another embodiment of the invention a method is disclosed for providing the physiological effect of wild-type p53 protein to cell, comprising the steps of: providing to a cell a compound which is able to specifically complex with a p53-specific binding site.
In yet another embodiment a double-stranded DNA fragment is provided which comprises a p53-specific-DNA binding site, wherein the fragment comprises more than one monomer repeat of the sequence 5′-RRRCWWGYYY-3′ (SEQ ID NO:3) and wherein the fragment is covalently attached to an insoluble polymeric support.
In another embodiment of the invention a single-stranded oligonucleotide containing natural nucleotides and/or nucleotide analogs is provided which is able to complex specifically with a p53-specific binding site, said binding site comprising more than one monomer of the sequence 5′-RRRCWWGYYY-3′ (SEQ ID NO:3).
In yet another embodiment of the invention a method is provided for identifying compounds which specifically bind to p53-specific DNA binding sequences, comprising the steps of: contacting a p53-specific DNA binding fragment immobilized on a solid support with a test compound to bind the test compound to the DNA fragment; and determining the amount of test compound which is bound to the DNA fragment.
In even another embodiment of the invention a method is provided for identifying compounds which specifically bind to p53-specific-DNA binding sequences, comprising the steps of: contacting a p53-binding DNA fragment immobilized on a solid support with both a test compound and wild-type p53 protein to bind the wild-type p53 protein to the DNA fragment; determining the amount of wild-type p53 protein which is bound to the DNA fragment, inhibition of binding of wild-type p53 protein by the test compound suggesting binding of the test compound to the p53-specific DNA binding sequences.
In still another embodiment a method of prescreening agents for use in cancer therapy is provided comprising: measuring the amount of binding of a p53 protein which is encoded by a mutant gene found in cancer cells of a patient to a DNA molecule which comprises more than one monomer of RRRCWWGYYY (SEQ ID NO: 28); measuring the amount of binding of said p53 protein to said DNA molecule in the presence of a test substance; and comparing the amount of binding of the p53 protein in the presence of said test substance to the amount of binding of the p53 protein in the absence of said test substance, a test substance which increases the amount of binding being a candidate for use in cancer therapy.
In another embodiment of the invention a method is provided for prescreening agents for use in cancer therapy comprising: contacting a transfected cell with a test substance, said transfected cell containing a p53 protein which is encoded by a mutant gene found in cancer cells of a patient and a reporter gene construct comprising a reporter gene which encodes an assayable product and a sequence which conforms to the p53 consensus binding site having more than one monomer of RRRCWWGYYY (SEQ ID NO: 3), wherein said sequence is upstream from and adjacent to said reporter gene; and determining whether the amount of expression of said reporter gene is altered by the test substance, a test substance which alters the amount of expression of said reporter gene being a candidate for use in cancer therapy.
In still another embodiment a method of prescreening agents for use in cancer therapy is provided comprising: adding RNA polymerase and ribonucleotides to a transcription construct, said transcription construct comprising a reporter gene which encodes an assayable product and a sequence which conforms to the p53 consensus binding site having more than one monomer of RRRCWWGYYY (SEQ ID NO: 3), said sequence being upstream from and adjacent to said reporter gene, said step of adding being effected in the presence and absence of a test substance; determining whether the amount of transcription of said reporter gene is altered by the presence of said test substance, a test substance which alters the amount of transcription of said reporter gene being a candidate for use in cancer therapy.
In a further embodiment a DNA construct is provided comprising: a reporter gene which encodes an assayable product; and a sequence which conforms to the p53 consensus binding site having more than one monomer of RRRCWWGYYY (SEQ ID NO: 3) upstream from and adjacent to said reporter gene; wherein said DNA construct is selected from the group consisting of a recombinant plasmid, a viral vector or an isolated molecule of DNA.
In another embodiment of the invention a method is provided of diagnosing tumor-inducing or hyperplastia-inducing strains of human papilloma virus (HPV) comprising: contacting cells or cell extracts of patients suspected of being
Kinzler Kenneth W.
Sherman Michael I.
Vogelstein Bert
Banner & Witcoff , Ltd.
Chakrabarti Arun
Jones W. Gary
The Johns Hopkins University
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