Expression of Wild-type p53 Is Required for Efficient Global Genomic Nucleotide Excision Repair in UV-irradiated Human Fibroblasts

James M Ford,Philip C Hanawalt

doi:10.1074/jbc.272.44.28073

Abstract

We have shown previously that Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in the removal of UV-induced cyclobutane pyrimidine dimers from genomic DNA, but still proficient in the transcription-coupled repair pathway (Ford, J. M., and Hanawalt, P. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8876-8880). We have now utilized monoclonal antibodies specific for cyclobutane pyrimidine dimers or 6-4 photoproducts, respectively, to measure their repair in UV-irradiated human fibroblasts. Cells homozygous for p53 mutations were deficient in the repair of both photoproducts, whereas cells heterozygous for mutant p53 exhibited normal repair of 6-4 photoproducts, but decreased initial rates of removal of cyclobutane pyrimidine dimers, compared with normal cells. The specificity of the effect of wild-type p53 on nucleotide excision repair was demonstrated in a p53 homozygous mutant cell line containing a tetracycline-regulated wild-type p53 gene. Wild-type p53 expression and activity were suppressed in the presence of tetracycline, whereas withdrawal of tetracycline resulted in the induction of p53 expression, cell cycle checkpoint activation, and DNA damage-induced apoptosis. The regulated expression of wild-type p53 resulted in the recovery of normal levels of repair of both cyclobutane pyrimidine dimers and 6-4 photoproducts in genomic DNA, but did not alter the transcription-coupled repair of cyclobutane pyrimidine dimers. Therefore, the wild-type p53 gene product is an important determinant of nucleotide excision repair activity in human cells.

Highlights

A wide variety of endogenous and exogenous agents cause damage to DNA, and multiple enzymatic processes exist to repair these different lesions
Our results show that loss of wt p53 activity results in a decrease in the global genomic nucleotide excision repair (NER) of both cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, but not transcription-coupled repair of CPDs, and that the expression of functional wt p53 protein in a p53 mutant human fibroblast cell line results in restoration of normal levels of global genomic repair of both photoproducts, as well as enhanced cell cycle arrest and apoptosis following UV irradiation
These results provide strong evidence that the human wt p53 tumor suppressor gene is involved in the regulation of NER activity in human cells, in vivo

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—All human fibroblasts were maintained as exponentially growing cultures in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and incubated at 37 °C in 5% CO2. TR9-7 were subcloned, and a single clone demonstrating optimal regulation of wt p53 expression was selected for use in all experiments described, and termed 041 TR This cell line was grown continuously in the presence of 600 ␮g/ml G418 and 50 ␮g/ml hygromycin to maintain selection pressure for the two stably integrated plasmid constructs containing the wt p53 cDNA together with the neomycin resistance gene, and the Tetregulated transactivator together with the hygromycin resistance gene, respectively. Cytological Examination by Fluorescence Microscopy—To determine if UV treatment resulted in morphological changes characteristic of apoptosis, cultured cells were irradiated or not with 20 J/m2 UV, incubated for 48 h, fixed in 70% ethanol in PBS, stained with the fluorescent DNA stain Hoeschst 33258 (0.5 ␮g/ml), and examined microscopically. Cells were judged to be apoptotic if they displayed the following morphological characteristics: interphase cells that possessed hypercondensed marginated masses of DNA along the inner surface of their nuclear membranes, cell surface blebbing, and overall cell shrinkage due to reductions in cytoplasmic volume

RESULTS

Findings

DISCUSSION