Role of p53 in Apoptosis, DNA Repair and Cellular Sensitivity to DNA Damage.

James M. Ford
Department of Biological Sciences
Stanford University
Stanford CA, 94305
E-mail: jmf@leland.stanford.edu

The p53 tumor suppressor gene product is critical in mediating certain cellular responses to DNA damage in mammalian cells' including cell cycle regulation, DNA repair and apoptotic cell death The effect of the p53 tumor suppressor gene on apoptosis' cell cycle regulation and the cellular sensitivity to DNA damage induced by W-irradiation and various cancer chemotherapeutic agents, and on nucleotide excision repair (NER) following UV-induced DNA damage, was investigated in tissue culture using primary human skin fibroblasts wild-type (wt)' heterozygous or homozygous for mutant p53 (p53 mut), p53 mut cells into which wt p53 expression vectors were introduced, and tumor cells carrying various mutations within the p53 gene.

The p53 homozygous mutant fibroblasts were more resistant to UV-cytotoxicity and exhibited less W-induced apoptosis than p53 wt fibroblasts. Analysis of global NER demonstrated a decrease in both the rate and overall extent of removal of W photoproducts from overall genomic DNA in p53 mut cells compared to p53 wt cells. The p53 mut cells retained the ability to preferentially repair damage in the transcribed strand of expressed genes (transcription-coupled repair) similar to normal cells' but were deficient in the repair of the nontranscribed strands of expressed genes.

A specific role for the expressed wtp53 gene product in the induction of global NER and apoptosis was demonstrated in p53 homozygous mutant fibroblasts into which an exogenous wt p53 gene under control of a tetracycline (Tet)-repressible promoter was introduced. In the presence of Tet, wt p53 expression and activity was suppressed, whereas withdrawal of Tet resulted in the induction of wt p53 gene expression, transcriptional activation of p21 and cell cycle checkpoint activation. Induction of wt p53 resulted in the recovery of normal levels of global repair of W-induced DNA damage from genomic DNA, and increased levels of UV-induced apoptosis, compared to the NER-deficient p53 mutant cells. Similarly, expression of human papilloma virus E6 protein (which promotes p53 degradation) in normal human fibroblasts resulted in deficient global NER Therefore, the wt p53 gene product is an important determinant of NER activity, in addition to cell cycle progression and apoptosis, following DNA damage to human cells. These results suggest that loss of wt p53 function may lead to greater genomic instability by reducing the efficiency of DNA repair, but that cellular resistance to the cytotoxic effects of DNA damaging agents may be enhanced through elimination of apoptosis and maintenance of efficient transcription-coupled repair.

To further explore the effect of p53 mutations on cellular sensitivity to chemotherapeutic agents, analysis of cellular viability and apoptosis was performed following treatment of these cells with the DNA damaging agents nitrogen mustard, doxorubicin, and cisplatin, and the microtubule-stabilizing agent Taxol. The p53 mutant cell lines were 2- to 10-fold more resistant to the cytotoxic effects of DNA damaging agents than were cells expressing wt p53. However, p53 mutant cells were 100-fold more sensitive to the cytotoxic effects of Taxol than cells expressing wt p53. These results suggest that human cells mutant for p53 may be resistant to the cytotoxic effects of DNA damaging agents due to loss of p53-dependent apoptosis' but that certain agents may be preferentially toxic to these cells through p53-independent mechanisms of cell death.

Therefore' the consequences of mutations in the p53 gene' common in human tumors' on the cellular sensitivity to cytotoxic agents' are dependent on the particular set of complex cellular responses to the cytotoxic insult. Understanding of the role and mechanisms of the p53 gene in cell death and DNA repair may allow for strategies exploiting the DNA repair deficiency found in p53 mutant cells by restoring the apoptotic response to DNA damage. Alternatively' identifying and employing cytotoxic agents which selectively target cells deficient in p53 dependent biological processes may result In a therapeutic advantage in tumors deficient in this tumor suppressor gene.

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