INCREASES IN NEUTRAL, MG2-DEPENDENT AND ACIDIC, MG2+-INDEPENDENT SPHINGOMYELINASE ACTIVITIES PRECEDE COMMITMENT TO APOPTOSIS AND ARE NOT A CONSEQUENCE OF CASPASE 3-LIKE ACTIVITY IN MOLT-4 CELLS IN RESPONSE TO THYMIDYLATE SYNTHASE INHIBITION BY GW1843()

Thymidylate synthase (TS) inhibition causes cell death, and this enzym e is the target for the important chemotherapy regime 5-fluorouracil/l eucovorin. GW1843 (1843U89) is a potent and specific folate analog TS inhibitor in clinical development. Because of the importance of TS as a chemotherapy target, we are studying the mechanism of TS inhibition- induced cell death by GW1843. Ceramide:is a regulatory lipid generated by the action of sphingomyelinase and is believed to signal apoptosis . The role of the ceramide in apoptotic signaling was studied in Molt- 4 human T-cell leukemia cells undergoing cell death after treatment wi th GW1843. In response to GW1843, Molt-4 cells undergo apoptosis with both acidic pH, Mg2+-independent sphingomyelinase (ASMase) and neutral pH, Mg2+-dependent sphingomyelinase (NSMase) activities elevated as e arly steps in the initiation of apoptosis before Molt-4 commitment to death. These activities lead to ceramide production with kinetics cons istent with a role as an effector molecule signaling the initiation of apoptosis in Molt-C cells. These changes were found to be independent of caspase 3-like (CPP32/apopain) activity and DNA degradation, but w ere not separable from membrane blabbing or cell lysis in this cell li ne. In this report, kinetic evidence is provided for a role of ceramid e in initiating GW1843-induced cell death of Molt-4 T cell leukemia ce lls. (C) 1998 by The American Society of Hematology.