Method for determining the presence of functional p53 by measuring GADD45 protein expression

• US 5,858,679 A
• Filed: 05/10/1995
• Issued: 01/12/1999
• Est. Priority Date: 11/12/1992
• Status: Expired due to Fees

• Current Assignee: Health and Human Services The Government of The United Sates of America As Represented By, Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Carrier, France, Fornace, Albert J. Jr., Kastan, Michael B.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/432,176
• Time in Patent Office: 1,343 Days
• Field of Search: 435/7.1, 435/6, 530/386, 530/388.1
• US Class Current: 435/7.1
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/47 Assays involving proteins of known structure or function as defined in the subgroups
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/57496 involving intracellular compounds
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

The dependence of ionizing radiation-induced GADD45 mRNA and protein expression on the presence of functional p53 in mammalian cells is disclosed. First and second oligonucleotide sequences are provided which can form a double-stranded oligomer capable of binding to functional p53 protein. The present invention demonstrates that the dependence of ionizing radiation-induced GADD45 mRNA and protein expression on the presence of functional p53 and the binding of functional p53 to a double-stranded oligomer binding sequence can serve as the bases for methods for determining the presence of functional p53 in mammalian cell lines and tumors.

Claims

1. A method for determining the presence of functional p53 in mammalian cells by measuring GADD45 protein expression, comprising the steps of:

(a) stimulating the mammalian cells to increase GADD45 protein expression by irradiating said cells with ionizing radiation or with a radiomimetic compound in an amount sufficient to stimulate GADD45 protein expression; and

(b) comparing the level of GADD45 protein in said stimulated cells to the level of GADD45 protein in unstimulated cells.

2. The method of claim 1, wherein the dose of ionizing radiation sufficient to stimulate GADD45 protein expression is 10 to 20 Gy.

3. The method of claim 1, wherein said comparison step includes the steps of:

(a) providing an anti-GADD45 antibody; and

(b) contacting GADD45 protein with said antibody.

4. The method of claim 3 wherein said contacting step further includes isolating nuclear protein extract from stimulated and unstimulated cells.

5. The method of claim 3, wherein said GADD45 protein is contacted with said antibody in a Western blot assay.

6. The method of claim 3, wherein said GADD45 protein is contacted with said antibody in an immunoprecipitation assay.