HPV 16 E6/7 immortalization sensitizes human keratinocytes to UVB by altering the pathway from caspase-8 to caspase-9-dependent apoptosis

UVB from solar radiation is both an initiating and promoting agent for skin cancer. We have found that primary human keratinocytes undergo an apoptotic response to UVB. To determine if these responses are altered during the course of immortalization, we examined markers of apoptosis in primary human foreskin keratinocytes (HFK) transduced with either a retroviral vector expressing the E6 and E7 genes of HPV 16, or with empty vector alone (LXSN-HFK). Whereas LXSN-HFK, as well as early passage keratinocytes expressing HPV 16 E6 and E7 (p7 E6/7-HFK) were both moderately responsive to UVB irradiation, late passage immortalized keratinocytes (p27 E6/7HFK) were exquisitely sensitive to UVB-induced apoptosis. Following exposure to UVB, enhanced annexin V-positivity and internucleosomal DNA fragmentation were observed in p27 E6/7-HFK compared to either LXSNor p7 E6/7-HFK. Caspase-3 fluorometric activity assays as well as immunoblot analysis with antibodies to caspase3 and PARP revealed elevated caspase-3 activity and processing at lower UVB doses in p27 E6/7-HFK, compared with LXSNor p7 E6/7-HFK. In addition, the caspase inhibitor DEVD-CHO reduced the apoptotic response and increased survival of all three HFK types. Immunoblot analysis revealed that caspase-8 was activated in all three cell types, but caspase-9 was only activated in p27 E6/7-HFK. Cell cycle analysis further showed that only p27 E6/7-HFK exhibit G2/M accumulation that is enhanced by UVB treatment. This accumulation was associated with a rapid down-regulation of Bcl-2 in these cells. The immortalization process subsequent to the expression of HPV E6 and E7 may therefore determine UVB sensitivity by switching the mode of apoptosis 2 by gest on Jauary 1, 2018 hp://w w w .jb.org/ D ow nladed from from a caspase-8 to a Bcl-2-caspase-9-mediated pathway of apoptosis.