Abstract
To understand the function of the individual oncogenes of HPV16 in modulating the cellular response to apoptogenic signals, we used human keratinocytes immortalized with either E6, E7 or E6/E7 oncoproteins as model system. Applying CD95 antibodies or recombinant CD95 ligand, only the E7-immortalized cells underwent extensive apoptosis. In contrast, E6- and E6/E7-expressing keratinocytes were resistant. Dominance of E6 correlated with significant down-regulation of p53, c-Myc, p21 and Bcl-2. CD95 was found to be reduced in resistant HPV-positive cells, while there were no quantitative differences in expression levels of FADD, FLICE/caspase-8 or caspase-3. Notably, in contrast to primary human keratinocytes, all immortalized cells showed a general reduction of c-FLIP, an inhibitory protein which normally prevents unscheduled CD95-induced apoptosis. E6- and E6/E7-positive keratinocytes, however, can be sensitized to CD95 apoptosis by blocking proteasome-mediated proteolysis. CD95-resistant HPV-positive cells underwent apoptosis within 3–5 h upon co-incubation with MG132 and agonistic antibodies or CD95 ligand, which was preceded by a strong re-expression of p53 and c-Myc, but not of other half-life controlled proteins such as Bax or IκBα. Blockage of proteasomal activity alone did not result in apoptosis, although the same set of pro-apoptotic proteins was up-regulated. Performing similar experiments with cervical carcinoma cells expressing mutated p53 (C33a) or with p53-‘null’ lung carcinoma cells (H1299), no CD95 cell killing occurred eventhough c-Myc was strong induced. These data indicate that the reduced bioavailability of p53 is a key-regulatory event in perturbation of CD95 signaling in HPV16 immortalized keratinocytes.
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Acknowledgements
We thank the Deutsche Akademische Austauschdienst and CONACyT for supporting A Aguilar-Lemarroy. We are also grateful to Dr Petra Boukamp (Deutsches Krebsforschungszentrum, Heidelberg) for providing primary human keratinocytes and to Dr Denise Galloway (Fred Hutchinson Cancer Center, Seattle, U.S.A.) for the amphotropic retroviruses.
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Aguilar-Lemarroy, A., Gariglio, P., Whitaker, N. et al. Restoration of p53 expression sensitizes human papillomavirus type 16 immortalized human keratinocytes to CD95-mediated apoptosis. Oncogene 21, 165–175 (2002). https://doi.org/10.1038/sj.onc.1204979
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DOI: https://doi.org/10.1038/sj.onc.1204979
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