P53 INACTIVATION BY HPV16 E6 RESULTS IN INCREASED MUTAGENESIS IN HUMAN-CELLS

To study the pathways associated with genomic instability in cancer, w e examined UV-induced and spontaneous mutagenesis in clonal cell lines expressing human papillomavirus (HPV) proteins, either high-risk (HPV 16) E6 or E7 or low-risk (HPV11) E6, in comparison to the parental RKO cells, a colon carcinoma cell line expressing only normal p53. High-r isk E6 and E7 bind and functionally inactivate tumor suppressor protei ns p53 and Rb, respectively, and both disrupt the G(1) arrest in respo nse to DNA damage. Low-risk HPV E6 proteins bind p53 with much lower a ffinity than high-risk E6 and fail to mediate p53 degradation or to di srupt the G(1) checkpoint. We found that cells expressing HPV16 E6 had reduced survival and increased mutagenesis at the hprt locus when tre ated with low doses of UV. However, this analysis was complicated by t he unexpected observation of a very high background of spontaneous mut agenesis in the unirradiated cells expressing the HPV16 E6 gene. Fluct uation analysis revealed a 5-fold elevated mutation rate in the cells expressing HPV16 E6. HPV11 E6 conferred a 2-fold elevation in the muta tion rate, but HPV16 E7 had no effect. The increased spontaneous mutag enesis, therefore, appeared to be mediated by p53 inactivation and to be independent of Rb (which acts downstream of p53 in the G(1) arrest pathway following DNA damage). Taken together, these findings suggest that the effect of p53 inactivation on spontaneous mutagenesis is mani fested at the level of DNA repair, recombination, or coupling of trans cription with one of these processes instead of by an alteration in G( 1) arrest.