We have examined the ability of gamma-irradiation and bleomycin to induce apoptosis in a model system consisting of cell lines derived from naturally occurring human head-and-neck squamous-cell carcinomas with contrasting p53 status and expression levels of pro- and anti-apoptotic molecules. Following exposure to gamma-irradiation (20 Gy) or bleomycin (3.5 microM) for 0 to 96 hr, cells expressing either transcriptionally inactive mutant p53 (HN6) or a truncated p53 molecule (HN19) underwent apoptosis, as assessed by fluorescence-activated cell sorting and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, in contrast to cells that express wild-type p53 (HN30), suggesting that apoptosis induced by these agents occurs by p53-independent mechanisms. Apoptosis in HN6 and HN19 cells was preceded by a G(2)/M cell-cycle block, as analyzed by DNA content and BrdU staining. In contrast, HN30 cells remained blocked in both G(1) and G(2)/M and failed to re-enter the cell cycle. Levels of Bcl-2 were elevated in 3 of 10 cell lines, and only marginal differences were observed for Bcl-x(L). Pro-apoptotic proteins bax and Bcl-x(S) were detectable in normal keratinocytes and 4 tumor cell lines. Bax-delta (16 kDa) was highly represented in normal keratinocytes, and levels of bak were variable between cell lines. Elevated expression of Bcl-2 failed to protect HN19 cells from either gamma-irradiation or bleomycin-induced apoptosis. Our data support the existence of p53- and Bcl-2-independent pathways regulating apoptosis in keratinocytes and suggest that efficacy of either radiotherapy or bleomycin treatment for oral squamous-cell neoplasms may not, therefore, be influenced solely by endogenous p53 status.
Copyright 2000 Wiley-Liss, Inc.