Toxicology 154 (2000) 45 – 53 Changes in ceramide and sphingomyelin following fludarabine treatment of human chronic B-cell leukemia cells Shyam S. Biswal, Kaushik Datta, George K. Acquaah-Mensah, James P. Kehrer * Diision of Pharmacology and Toxicology, College of Pharmacy, The Uniersity of Texas at Austin, Austin, TX 78712 -1074, USA Received 7 July 2000; accepted 25 July 2000 Abstract Fludarabine is used to treat chronic lymphocytic leukemia. Both in vitro and in vivo studies have indicated that apoptosis is an important mode of fludarabine-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 fragmentation and caspase-3-like activity, was evident by 18 h after fludarabine in both cell lines. The general caspase inhibitor t -butoxycarbonyl-Asp(OMe)- fluoromethyl ketone (OMe, methyl ester) significantly inhibited apoptosis supporting a role for caspases in fludara- bine-induced cell death. A 2.5- to threefold elevation in ceramide levels was observed 6 h after fludarabine treatment. Concomitantly, a decrease in sphingomyelin levels was observed. Fumonisin B1 (an inhibitor of ceramide synthase) pretreatment significantly prevented fludarabine-induced ceramide generation and apoptosis. Conversely, C6-ceramide induced apoptosis in both cell lines. No effect of fludarabine on indices of oxidative stress (dichlorofluorescin oxidation and glutathione disulfide formation) were detected, although partial protection from apoptosis, and prevention of ceramide generation and caspase-3 activation, were achieved with N-acetylcysteine. These findings are consistent with the involvement of caspases and ceramide in fludarabine-induced apoptosis in WSU and JVM-2 cells. Oxidative stress does not appear to be induced by fludarabine, although the protective effects of N-acetylcysteine suggest that thiol redox balance may play a role in the apoptotic pathway. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Apoptosis; Fludarabine; Ceramide; Sphingomyelin; Oxidative stress www.elsevier.com/locate/toxicol Abbreiations: ara-C, cytosine arabinoside; Boc-D-FMK, t -butoxycarbonyl-Asp(OMe)-fluoromethyl ketone (OMe, methyl ester); CLL, chronic lymphocytic leukemia; EBV, Epstein – Barr virus; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; SM, sphingomyelin. * Corresponding author. Tel.: +1-512-4711107; fax: +1-512-4715002. E-mail address: kehrerjim@mail.utexas.edu (J.P. Kehrer). 0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII:S0300-483X(00)00296-1