Effect of FADD expression during UVC carcinogenesis

To become a cancer cell, a cell must inactivate apoptosis in order to avoid dying. This model predicts that cancer cells are more resistant to DNA damaging agents because they have deactivated the apoptotic pathway. Apoptotic signal caused by UVC radiation triggers intrinsic or mitochondrial apoptotic pathway. Other apoptotic pathway is coupled directly to FADD (Fas-associating protein with death domain), an adaptor protein essential for mediating apoptosis (extrinsic or death receptor pathway). UVC could trigger this extrinsic pathway. Our initial effort was to develop dose-response relationships for survival and apoptosis as a function of UVC radiation using embryonic fibroblasts derived from FADD knockout mice and their genetic counterparts. We examined the effects of UVC exposure on the survival of FADD positive (FADD+/+) and FADD negative (FADD-/-) cells in a time- and dose-dependent manner. FADD+/+ and FADD-/- cells were irradiated with UVC light (254nm) using a germicidal lamp. The culture media was drained before the irradiation and fresh media was added after. The UVC doses in our study were 50 J/m2, 75 J/m2 and 300 J/m2. The cell proteins were isolated 24 and 48 hours after each UVC exposure. The poly (ADP-ribose) polymerase (PARP), 113kDa nuclear enzyme, is the first protein cleaved in fragments of 89 and 24kDa during apoptosis and this event was analysed by Western blotting using antibodies specific for PARP. The results indicated that FADD-/- cells irradiated with a dose of 50 J/m2 do not undergo apoptosis after 24 hours which was not case with FADD+/+ cells. FADD+/+ cells irradiated with 50 J/m2, 75 J/m2 and 300 J/m2 UVC doses undergo apoptosis after 24 hours as well as after 48 hours. All other results for FADD-/- cells (75 J/m2 and 300 J/m2 after 24 hours and 50 J/m2, 75 J/m2 and 300 J/m2 after 48 hours) were identical as results for FADD+/+ cells. We established significant difference in apoptotic response after UVC radiation between FADD+/+ and FADD-/- cells. As we suspected FADD protein is involved in the extrinsic apoptotic pathway caused by UVC radiation, although main apoptotic pathway caused by UVC radiation is intrinsic through mitochondria. (author)