INDUCTION OF APOPTOSIS IN HUMAN KERATINOCYTES CONTAINING MUTATED P53 ALLELES AND ITS INHIBITION BY BOTH THE E6 AND E7 ONCOPROTEINS

Induction of apoptosis is a function of external stimuli and cellular gene expression, Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled b y elevation of p53 levels. We have investigated the response of immort alized human keratinocytes (HaCaT) bearing mutated alleles of p53 to g enotoxic stress and the effect of human papillomavirus (HPV) 16 E6 and E7 on this response. UVC irradiation triggered HaCaT's cell death wit h several characteristics of apoptosis, including DNA laddering, chrom atin condensation and fragmentation, and the appearance of cells with a low content of DNA (categorized as sub-G(1) by cell sorter analysis) . This response was accompanied by accumulation of cells in S phase of the cell cycle. HaCaT cells infected with retroviruses carrying HPV16 E6 or E7 showed a significant reduction in their apoptotic response, which was not observed in cells infected with the LXSN vector DNA-carr ying virus. Reduced apoptosis in HaCaT cells expressing E6 or E7 also was observed after treatment with the alkylating agent mitomycin C. We stern blot analysis of p53 and p21/WAF-1/CIP-1, a downstream effector of p53, did not reveal any changes in the levels of these proteins aft er UVC irradiation in either HaCaT cells or HaCaT cells expressing HPV 16 E6 or E7. (C) 1998 Wiley-Liss, Inc.