Sensitivity to Fas-mediated apoptosis in high-risk HPV-positive human cervical cancer cells: relationship with Fas, caspase-8, and Bid.

ABSTRACT

OBJECTIVE: Binding of Fas ligand or agonistic anti-Fas antibody to the death receptor Fas can activate a caspase-cascade resulting in apoptosis. In the present study, the functionality of the Fas pathway was studied in human cervical cancer cells with different HPV and p53 status. METHODS: HeLa (HPV-18 positive), CaSki, and SiHa (both HPV-16 positive) contain wild-type p53, while C33A (HPV negative) expresses mutant p53. Fas cell surface expression was determined by flow cytometry. Expression of proteins involved in the apoptotic pathway was analyzed by Western blotting and apoptosis was measured by acridine orange staining of nuclear chromatin. RESULTS: Despite high Fas membrane expression in the HPV-positive cells, CaSki was highly sensitive, HeLa slightly sensitive, and SiHa and C33A were resistant for agonistic anti-Fas antibody. Almost undetectable Fas membrane levels can explain the non-responsiveness of C33A for anti-Fas. Although interferon-gamma (IFNgamma) strongly and cisplatin to a lesser extend enhanced Fas membrane expression in all HPV-positive cells, sensitization to anti-Fas by IFNgamma or cisplatin was only observed in HeLa. Analysis of the Fas apoptotic pathway showed that anti-Fas treatment induced caspase-8 activation and concomitantly Bid cleavage, caspase-9 and caspase-3 activation, PARP cleavage and apoptosis in HeLa and CaSki. IFNgamma plus anti-Fas treatment, in contrast to anti-Fas alone, facilitated caspase-8 activation in HeLa and SiHa, while an increase in Bid cleavage, caspase-9 activation and apoptosis was only observed in HeLa. Apoptotic failure in SiHa (even in the presence of IFNgamma) was probably due to low caspase-8, almost undetectable Bid protein levels and therefore lack of caspase-9 activation. CONCLUSION: Sensitivity to anti-Fas depends on Fas, caspase-8, and Bid protein levels in cervical cancer cells. Additionally, IFNgamma and cisplatin can increase sensitivity to anti-Fas in a subset of HPV-positive cervical cancer cell lines by upregulation of Fas and caspase-8 expression without major changes in p53 levels.