title: Restoration of p53 expression sensitizes human papillomavirus type 16 immortalized human keratinocytes to CD95-mediated apoptosis.
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 IkappaBalpha. 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 even though c-Myc was strongly induced. These data indicate that the reduced bioavailability of p53 is a key-regulatory event in perturbation of CD95 signaling in HPV16 immortalized keratinocytes.
authors.affiliation: Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany.
authors.forename: Adriana
authors.initials: A
authors.lastname: Aguilar-Lemarroy
authors.forename: Patricio
authors.initials: P
authors.lastname: Gariglio
authors.forename: Noel J
authors.initials: NJ
authors.lastname: Whitaker
authors.forename: Sören T
authors.initials: ST
authors.lastname: Eichhorst
authors.forename: Harald
authors.initials: H
authors.lastname: zur Hausen
authors.forename: Peter H
authors.initials: PH
authors.lastname: Krammer
authors.forename: Frank
authors.initials: F
authors.lastname: Rösl
mesh_terms: D000328:Adult; D017209:Apoptosis / Q000502:physiology*; D002478:Cells, Cultured; D002675:Child, Preschool; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D016158:Genes, p53 / Q000187:drug effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D008297:Male; D008562:Membrane Glycoproteins / Q000494:pharmacology; D027383:Papillomaviridae / Q000276:immunology*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000166:cytology / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000276:immunology*
chemical_list: C494826:BAX protein, human; D017931:DNA Primers; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D003513:Cycloheximide