Ceramide-induced apoptosis is prevented through p53

Abstract

p53 has been known to exert its effects in damaged cell by leading cellular growth arrest and inducing programmed cell death, but in this studies, p53 is thought to play a role in protection against ceramide-induced apoptosis. Using human bladder carcinoma EJ cell and tetracyclin-inducible p53 expression system, EJ-p53 in which exogeneous p53 is fully overexpressed 48hrs after tetracyclin removal from the growth medium, the relation of ceramide and p53 in induction of apoptosis was investigated. 5uM of c2-ceramide has no effect on these cell lines, but at 15-50uM of c2-ceramide, acute(6-12h) induction of apoptosis is observed in EJ cell in contrast to delayed apoptosis in EJ-p53 about 20-24 h. The cytotoxic effects of c2-ceramide is intensified along with increment of concentration of c2-ceramide. When 35 uM of c2-ceramide was treated, Oligonucleosomal DNA ladder is appeared in 6 hr and reached at it``s peak in 36 hr in EJ cells, but there are no appearance of DNA ladder by 48 hr in EJ-p53 cells. For investigating molecular mechanism of this delayed apoptosis, 35uM of c2-ceramide-treated cells were lysed and western blotting was performed for c-jun, which is activated by phosphorylation proceeding apoptosis. In EJ cells, c-jun protein level is elevated and increasingly phosphorylated time-dependently. But p53-overexpressed EJ-p53 cells, c-jun is already phosphorylated to some extent even in control not treated with c2-ceramide. It is suggested that c-jun is not relavant to delay of ceramide-induced apoptosis through p53.