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 enzyme is the target for the important chemotherapy regime 5- fluorouracil/leucovorin. GW1843 (1843U89) is a potent and specific foists 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 with 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 early steps in the initiation of apoptosis before Molt-4 commitment to death. These activities lead to ceramide production with kinetics consistent with a role as an effector molecule signaling the initiation of apoptosis in Molt-4 cells. These changes were found to be independent of caspase 3-like (CPP32/apopain) activity and DNA degradation, but were not separable from membrane blabbing or cell lysis in this cell line. In this report, kinetic evidence is provided for a role of ceramide in initiating GW1843-induced cell death of Molt-4 T- cell leukemia cells.