Changes in ceramide and sphingomyelin following fludarabine treatment of human chronic B-cell leukemia cells

Fludarabine is used to treat chronic lymphocytic leukemia. Both in vitro an d in vivo studies have indicated that apoptosis is an important mode of flu darabine-induced cell death. However, the apoptotic pathways activated are not known. The effects of apoptotic doses of fludarabine on sphingomyelin, ceramide and the production of reactive oxygen species were investigated in the chronic B-cell leukemia lines WSU and JVM-2. Apoptosis, as assessed by an increase in phosphatidylserine externalization. internucleosomal DNA fr agmentation and caspase-3-like activity, was evident by 18 h after fludarab ine in both cell lines. The general caspase inhibitor t-butoxycarbonyl-Asp( OMe)-fluoromethyl ketone (OMe, methyl ester) significantly inhibited apopto sis supporting a role for caspases in fludarabine-induced cell death. A 2.5 - to threefold elevation in ceramide levels was observed 6 h after fludarab ine treatment. Concomitantly, a decrease in sphingomyelin levels was observ ed. Fumonisin B1 tan inhibitor of ceramide synthase) pretreatment significa ntly prevented fludarabine-induced ceramide generation and apoptosis. Conve rsely, CG-ceramide induced apoptosis in both cell lines. No effect of fluda rabine on indices of oxidative stress (dichlorofluorescin oxidation and glu tathione disulfide formation) were detected, although partial protection fr om apoptosis, and prevention of ceramide generation and caspase-3 activatio n, were achieved with N-acetylcysteine. These findings are consistent With the involvement of caspases and ceramide in fludarabine-induced apoptosis i n WSU and JVM-2 cells. Oxidative stress does not appear to be induced by fl udarabine, although the protective effects of N-acetylcysteine suggest that thiol redox balance may play a role in the apoptotic pathway. (C) 2000 Els evier Science Ireland Ltd. All rights reserved.