NORMAL HUMAN ORAL KERATINOCYTES ARE MORE SENSITIVE TO N-METHYL-N'-NITRO-N-NITROSOGUANIDINE-INDUCED CYTOTOXICITY AND APOPTOSIS THAN HPV-IMMORTALIZED ORAL KERATINOCYTES - ROLE OF P53

Exposure of HPV-immortalized, but not normal human oral keratinocytes, to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) renders the cells tumorigenic. The underlying mechanism of this differential response of normal and immortalized cells was investigated. Normal pri mary human oral keratinocytes and three HPV-immortalized human oral ke ratinocyte cell lines exposed to MNNG were evaluated for survival rate , single and double-strand DNA breaks, and the expression of p53 and b cl-2 proteins. MNNG exposure for 2 h induced both greater cytotoxicity and a more rapid kinetic of cell death in normal keratinocytes than i n the immortalized cells. Further, normal keratinocytes were more sens itive to lower concentrations of MNNG that were subtoxic for the immor talized cells. Likewise, with lower concentration of MNNG (50 mu M), s ignificant single-strand DNA breaks in normal keratinocytes were induc ed whereas no such effect was seen in the immortalized cells. Double-s trand DNA fragmentation (apoptosis) was observed in normal keratinocyt es exposed to 50 mu M MNNG but not in the immortalized cells. Higher c oncentrations of MNNG (100 mu M) were toxic to both normal and immorta lized cells although the normal cells were still more sensitive and wi th faster kinetics of cell death. MNNG-induced apoptosis was not attri butable to down regulation of the anti-apoptotic product bcl-2 in norm al cells; however, exposure of normal keratinocytes to MNNG did result in induction of the apoptotic gene p53. No change in p53 level was se en in the immortalized cells. These findings suggest that the selectiv e sensitivity of normal keratinocytes to MNNG-induced apoptosis is in part due to the induction of p53. The HPV-immortalized cells are resis tant to MNNG-induced apoptosis and therefore are capable of undergoing mutations affecting cell proliferation and resulting in tumori-genici ty.