HUMAN PAPILLOMAVIRUS (HPV)-16-E6 SENSITIZES CELLS TO ATRACTYLOSIDE-INDUCED APOPTOSIS - ROLE OF P53, ICE-LIKE PROTEASES AND THE MITOCHONDRIAL PERMEABILITY TRANSITION

Infection of cervical epithelial cells with certain high risk HPV geno types is thought to play an etiologic role in the development of cervi cal cancer. In particular, HPV type 16 and 18 early protein 6 (E6) is thought to contribute to epithelial transformation by binding to the t umor suppressor protein p53, targeting it for rapid proteolysis, resul ting in loss of its cell cycle arrest and apoptosis-inducing activitie s. Recent data indicate that factors responsible for triggering apopto sis reside in the cytoplasm of cells, and not in the nucleus. In parti cular, the findings that mitochondria are required in certain cell-fre e models for induction of apoptosis and that bcl-2 is localized to mit ochondria have focused attention on the role of the mitochondrial memb rane permeability transition (MPT) in apoptosis. Here we present data to indicate that HPV 16 E6 expression sensitizes cells to MPT-induced apoptosis. We also report that HPV 16 E6 sensitization of cells to MPT -induced apoptosis occurs only in the presence of wildtype (wt) p53 ex pression. The extent of apoptosis induced by atractyloside (an inducer of the MPT) in normal, temperature-sensitive (ts) p53, and HPV-16 E6 transfected J2-3T3 cells, and the HPV expressing cervical carcinoma ce ll lines SiHa, Hela and CaSki was determined. C33A cells, which expres s mutant p53 but not HPV, were also exposed to atractyloside in the pr esence or absence of HPV 16 E6 expression. Dose-dependent apoptosis in duced by atractyloside in normal J2-3T3 cells and cervical carcinoma c ells was measured by loss of cell viability, nuclear fragmentation and DNA laddering. The sensitivity of cells to atractyloside-induced apop tosis was found to be: HPV 16 E6-J2-3T3 > CaSki > normal-J2-3T3 cells approximate to ts p53-J2-3T3 approximate to vector-J2-3T3 cells > Hela > SiHa > C33A approximate to C33A 16 E6. Cyclosporin A (CsA), an inhi bitor of the MPT, and ICE-I, a protease inhibitor, provided protection against atractyloside-induced apoptosis. These findings indicate that : 1) high risk HPV 16 E6 protein is capable of sensitizing cells to ap optosis; 2) HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis occurs in a p53-dependent fashion; 3) the target of HPV 16 E 6 sensitization of cells to atractyloside-induced apoptosis is the mit ochondria; and 4) HPV 16 E6 sensitization of cells to atroctycoside-in duced apoptosis involves an ICE-like protease-sensitive mechanism, reg ulating the onset of the MPT. These findings constitute the first evid ence that mitochondria play a role in HPV 16 E6 modulation of apoptosi s. (C) 1997 Wiley-Liss, Inc.