Sequence specific DNA binding by p53
AssigneesThe Johns Hopkins University, Genzyme Corporation
InventorsBert Vogelstein, Kenneth W. Kinzler, Michael I. Sherman
Life of this patent
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Abstract
Specific sequences in the human genome are the sites of strong binding of wild-type p53 protein, but not mutant forms of the protein. These sequences are used diagnostically to detect cells in which the amount of wild-type p53 is diminished. The sequences can also be used to screen for agents which correct for loss of wild-type p53 to DNA in cancer cells.
Claims41
A method 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;
- detecting the presence of wild-type p53 protein in the cell by detecting binding of the DNA fragment to wild-type p53.
A method of detecting the presence of a wild-type p53 protein in a cell, comprising the steps of:
- providing a histological section from a human;
- incubating the section with a detectably-labeled p53-specific binding DNA fragment to bind said DNA fragment to wild-type p53 present in the histological sample;
- removing unbound DNA fragment from the histological section;
- and
- determining the amount of DNA fragment which is bound to the histological sample.
A method of providing the physiological effect of wild-type p53 protein to a cell, comprising the steps of:
- providing to a cell a compound which is is able to complex specifically with a p53-specific binding site.
A double-stranded DNA fragment which comprises a p53-specific DNA binding site, wherein the fragment comprises more than one monomer of the sequence RRRCWWGYYY and wherein the fragment is covalently attached to an insoluble polymeric support.
A linear or circular single-stranded oligonucleotide or oligonucleotide containing nucleotide analogs which is able to complex specifically with a p53-specific binding site, said binding site comprising more than one monomer of the sequence RRRCWWGYYY.
A method of identifying compounds which specifically bind to p53-specific DNA binding sequences, comprising the steps of:
- contacting a p53-specific-binding DNA fragment with a test compound to bind the test compound to the DNA fragment;
- determining the amount of test compound which is bound to the DNA fragment.
A method of identifying compounds which specifically bind to p53-specific DNA binding sequences, comprising the steps of:
- contacting a p53-specific-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 indicating binding of the test compound to the p53-specific DNA binding sequences.
A method of supplying wild-type p53 gene function to a cell which has lost said gene function by virtue of mutation in a p53 gene, comprising:
- introducing a wild-type p53 gene into a cell which has lost said gene function such that said gene is expressed in the cell.
A method of supplying a wild-type p53 gene function to a cell which has lost said gene function by virtue of a mutation in a p53 gene, comprising:
- introducing a portion of a wild-type p53 gene into a cell which has lost said gene function such that said portion is expressed in the cell, said portion encoding a part of the p53 protein which is required for non-neoplastic growth of said cell.
A method of pre-screening agents for use in cancer therapy, comprising:
- measuring the amount of binding of a p53 protein encoded by a mutant gene found in cancer cells of a patient to a DNA molecule which conforms to the consensus binding site having more than one monomer of RRRCWWGYYY;
- 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.
A method of pre-screening 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, 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.
A method of pre-screening agents for use in cancer therapy, 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 at least two monomers of RRRCWWGYYY, 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;
- and
- 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.
A DNA construct for use in screening potential chemotherapeutic agents, comprising:
- a reporter gene which encodes an assayable product;
- a sequence which conforms to the p53 consensus binding site having more than one monomer of RRRCWWGYYY 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 isolated molecule of DNA.
A method of diagnosing tumor-inducing or hyperplastic-inducing strains of human papilloma virus (HPV) comprising:
- contacting cells or cell extracts of patients suspected of being infected by HPV with a p53-specific binding DNA fragment;
- detecting the amount of wild-type p53 in said cells or cell extract which binds to said DNA fragment, absence of bound p53 indicating infection by strains of HPV which sequester p53.
A method of pre-screening agents for use in cancer therapy, comprising:
- contacting a transfected cell with a test substance, said transfected cell containing a wild-type p53 protein 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, 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.
A method of pre-screening oligonucleotides for use in cancer therapy, comprising:
- adding a p53 protein which is encoded by a mutant gene found in a cancer patient and a preparation of random oligonucleotides to a p53-specific-binding DNA fragment immobilized on a solid support;
- recovering the olignucleotides which bound to the solid support.
Dependencies are inferred from the claim text.