Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-05-10
1999-01-12
Campbell, Eggerton A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
530386, G01N 3353, C07K 1528
Patent
active
058586792
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention is in the field of tumor cell biology. More specifically, this invention relates to the first identification of a gene, GADD45, whose expression has been shown to be altered by the presence of functional p53 protein and to the development of methods for determining the presence of functional p53 protein in mammalian cells.
BACKGROUND OF THE INVENTION
The p53 protein was first detected in a complex with the SV40 large T antigen in rodent cells transformed by simian virus SV40 (Lane, D. P. et al. (1979) Nature, 278:261-263). Subsequently, p53 was shown to be complexed with adenovirus and oncogenic papillomavirus oncoproteins (Sarnow, P. et al. (1982) Cell, 28:387-394; Werness, B. A. et al. (1990) Science, 248:76-79). Initially, p53 protein was considered to be a cellular proto-oncogene but recent observations have indicated that the gene encoding p53 in its native form is a tumor suppressor gene. Experimental support for the role of p53 as a tumor suppressor has been provided by the demonstration that the p53 gene can suppress the growth of transformed murine or human cells and that mutation or deletion of the p53 gene results in loss of this suppressor function (Eliyahn, D. et al. (1989) Proc. Natl. Acad. Sci. U.S.A., 86:8763-8767; Baher, S. J. et al. (1990) Science, 249:912-915; Mercer, W. E. et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87:6166-6170). To date, such mutations of the p53 gene have been demonstrated in tumors of the colon, breast, lung, ovary, bladder, and several other organs, making the p53 gene the most commonly mutated gene yet identified in human cancers (Vogelstein, B., (1990) Nature, 348:681-682). Based on the association of tumor progression with alterations in the p53 gene, major research efforts have been devoted to elucidating the potential biological function of p53.
Recent evidence strongly suggests that one function of p53 protein may be in the regulation of gene transcription. Several groups have demonstrated sequence-specific binding of p53 to DNA (Bargonetti et al. (1991) Cell, 65:1083-1091); Kern et al. (1991) Science, 252:1708-1711) and a genomic consensus sequence has been elucidated that consists of two copies of a symmetric 10 base pair (bp) motif separated by 0-13 bp (El-Deiry et al. (1992) Nature Genet., 1:45-49). Placement of this consensus sequence adjacent to a basal promoter linked to chloramphenicol acetyltransferase (CAT) or luciferase reporter genes resulted in induction of the reporter gene when these constructs were cotransfected with a p53 expression vector into mammalian cells (Kern et al. (1992) Science, 256:827-830; Funk W. D. et al. (1992) Mol. Cell. Biol., 12:2866-2871). In addition, the amino-terminus of p53 has been shown to behave as an acidic transcriptional activation domain when fused to GAL4 (Fields, S. et al. (1990) Science, 249:1046-1049).
More recently, wild-type (wt) p53 protein has been shown to directly activate transcription in vitro (Farmer, G. et al. (1992) Nature, 358:83-86). However, despite the experimental evidence supporting a role for p53 protein in transcriptional activation and the high interest in the potential involvement of p53 in tumorigenesis, there are currently only a few methods available for determining the presence of wt or mutant p53 protein in mammalian cells. One widely used method involves laborious DNA sequencing of the p53 gene itself. A major drawback of this approach is that the presence of a normal p53 DNA sequence is not necessarily an accurate predictor of the presence of functional p53 protein in the cells assayed since interference of p53 function by viral proteins or by abnormal binding of p53 protein to endogenous cellular proteins can occur (Momand, J. et al. (1992) Cell, 69:1237-1245; Oliner, J. D. et al. (1992) Nature, 358:80-83). In addition, this approach is both costly and time-consuming.
Another method used for determining the presence of wt or mutant p53 involves the use of antibodies capable of distinguishing between these two forms of p53. However, this a
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Carrier France
Fornace, Jr. Albert J.
Kastan Michael B.
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