UV-induced apoptosis in the p53-deficient SCL-II cell-line is accompanied by the expression of a bax-related protein of 18 kDa

SCL-II is a TP53-deficient squamous cell carcinoma cell line of the skin, showing no G1/S cell-cycle stop and only a weak p53- independent apoptotic response after exposure to ionizing radiation. Additionally, no induction of p21waf-1/cip-1 and bax can be observed after ionizing radiation at any time after exposure (Kriehuber et al. 1999, Kriehuber and Simko 2000, Lange et al. 2002). We examined in UV-irradiated (0.1-2 mJ/cm{sup 2}) SCL II-cells the time-dependent appearance of apoptotic cells (fluorescence microscopy) and the expression status of cell-cycle and apoptosis related proteins (SDS-PAGE, Western- and Immunoblot). The number of apoptotic cells increased 3 h post-irradiation and peaked 6 h after exposure (180/1000 cells). Quantitative analysis of immunoblots (Fluoro-S-Imager, Biorad) revealed a diminished expression of p21waf-1/cip-1 3 h to 6 h post exposure, whereas TP53 expression remains unchanged to control. Four hours after exposure to UV the active form of Caspase-3 was detectable, whereas the expression of the anti-apoptotic bcl-2 was diminished 6 - 9 h post-exposure. A Bax specific antibody detects 4 h to 8 h after UV-irradiation a second yet to characterize 18 kDa protein. Cycloheximide treatment abolished the occurrence of the 18 kDa band. The intensities of the 21 kDa and 18 kDa band equals the intensity of the single 21 kDa band in control lysates, suggesting that the 18 kDa band is a truncated or post-translational modified form of bax. We suggest a p53-independent UV-inducible apoptotic pathway in SCL-II cells, employing p53-downstream proteins bax or bax-related protein. Bcl-2 and p21waf-1/cip-1 seemed to be cleaved or down-regulated during this process. The bax/ bax-related protein have to be further characterized and possible up-stream effectors have to be identified.