Human papillomavirus type 16 E6 and HPV-16 E6/E7 sensitize human keratinocytes to apoptosis induced by chemotherapeutic agents: Roles of p53 and caspase activation

We and others have previously reported that human papillomavirus (HPV)-16 E b protein expression sensitizes certain cell types to apoptosis. To confirm that this sensitization occurred in HPV's natural host cells, and to explo re the mechanism(s) of sensitization. we infected human keratinocytes (HKCs ) with retroviruses containing HPV-6 Eb, HPV-16 E6; HPV-16 E7. or HPV-16 E6 /E7. Apoptosis was monitored by DNA fragmentation gel analysis and direct o bservation of nuclei in cells stained with DAPI. Exposure or HKCs to eiopos ide, cisplatin, mitomycin C (MMC), atractyloside, and sodium butyrate, resu lted in a time and dose-dependent induction of apoptosis. Expression of HPV -16 E6 and HPV-16 E6/E7, but not HPV-6 E6 or HPV-16 E7, enhanced the sensit ivity of HKCs to cisplatin-, etoposide- and MMC-, but not atractyloside- or sodium butyrate-induced apoptosis. Expression of both HPV-16 E6 and HPV-16 E6/E7 decreased, but did not abolish, p53 protein levels relative to norma l HKCs, and resulted in cytoplasmic localization or wt p53. p53 induction o ccurred in HPV-16 E6 and HPV-16 E6/E7 expressing cells after exposure to ci splatin or MMC, though never to levels found in normal untreated HKCs. P21 levels were decreased in HPV-16 E6 and HPV-16 E6/E7 expressing HKCs. and no induction of p21 was seen in these cells following exposure to cisplatin o r MMC. Caspase-3 activity was found to be elevated in HPV-16 E6-expressing HKCs following exposure to cisplatin and MMC as documented by fluorometric and Western Blot analysis. Expression or wt CrmA or treatment of HPV-16 Eb expressing HKCs with the caspase-3 inhibitor DEVD.fmk prevented HPV-16 Eb-i nduced sensitization in HKCs. These results suggest that HPV-16 E6 and HPV- 16 E6/E7 expression sensitizes HKCs to apoptosis caused by cisplatin, etopo side and MMC, but not atractyloside or sodium butyrate. The data also sugge st that wt p53 and caspase-3 activity are required for HPV-16 E6 and HPV-16 E6/E7-induced sensitization of HKCs to DNA damaging agents. (C) 2000 Wiley -Liss, Inc.