Human papillomavirus (HPV) 16 E6 sensitizes cells to atractyloside-induced apoptosis: Role of p53, ICE-like proteases and the mitochondrial permeability transition

Abstract

Infection of cervical epithelial cells with certain high risk HPV genotypes is thought to play an etiologic role in the development of cervical cancer. In particular, HPV type 16 and 18 early protein 6 (E6) is thought to contribute to epithelial transformation by binding to the tumor suppressor protein p53, targeting it for rapid proteolysis, resulting in loss of its cell cycle arrest and apoptosis-inducing activities. Recent data indicate that factors responsible for triggering apoptosis reside in the cytoplasm of cells, and not in the nucleus. In particular, the findings that mitochondria are required in certain cell-free models for induction of apoptosis and that bcl-2 is localized to mitochondria have focused attention on the role of the mitochondrial membrane 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 expression. 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 cell lines SiHa, Hela and CaSki was determined. C33A cells, which express mutant p53 but not HPV, were also exposed to atractyloside in the presence or absence of HPV 16 E6 expression. Dose-dependent apoptosis induced by atractyloside in normal J2-3T3 cells and cervical carcinoma cells was measured by loss of cell viability, nuclear fragmentation and DNA laddering. The sensitivity of cells to atractyloside-induced apoptosis was found to be: HPV 16 E6-J2-3T3 > CaSki > normal-J2-3T3 cells approximately ts p53-J2-3T3 approximately vector-J2-3T3 cells > Hela > SiHa > C33A approximately C33A 16 E6. Cyclosporin A (CsA), an inhibitor 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 apoptosis; 2) HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis occurs in a p53-dependent fashion; 3) the target of HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis is the mitochondria; and 4) HPV 16 E6 sensitization of cells to atroctycoside-induced apoptosis involves an ICE-like protease-sensitive mechanism, regulating the onset of the MPT. These findings constitute the first evidence that mitochondria play a role in HPV 16 E6 modulation of apoptosis.

Human papillomavirus type 16 E7 protein sensitizes cervical keratinocytes to apoptosis and release of interleukin-1α

“…Apoptosis induced by other agents may be mediated by dierent pathways which may dier in their requirements for p53. Previous studies have shown that high level expression of HPV-16 E6 sensitizes cultured human ®broblasts, immortalized breast epithelial cells, or cervical carcinoma cell lines to apoptosis (Wahl et al, 1996;Xu et al, 1995;Brown et al, 1997). Our in vitro results show that E6 promotes a low level of apoptosis in normal human cervical keratinocytes, the natural target of HPV infection.…”

Interaction of viral proteins with host cell death machinery

“…Such regulation of host DNA synthesis is exemplified by human adenoviruses (Li et al, 1997), Simian virus 40 (Conzen et al, 1997), human papillomaviruses (HPV) (Brown et al, 1997) and hepatitis B viruses (HBV) (Chirillo et al, 1997). Certain viral genomes encode proteins with highly conserved domains characteristic of Bcl-2-related proteins.…”

Pleiotropic effects of human papillomavirus type 16 E6 oncogene expression in human epithelial cell lines.

“…Other reports have also indicated that E6 can mediate its antiapoptotic activity in a p53-independent manner (Brown et al, 1997 ;Steller et al, 1996). HaCaT (Lehman et al, 1993) and C33A (Crook et al, 1991) cells have mutations in the p53 loci and the resistance to serum starvation-induced apoptosis is likely to be mediated by a p53-independent pathway.…”