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
Ronald M. Laethem,
Yusuf A. Hannun,
Supriya Jayadev,
Connie J. Sexton,
Jay C. Strum,
Rebecca Sundseth, and
Gary K. Smith
From the International Science Development Group, the Departments of
Molecular Sciences, Functional Genetics, and Molecular Biochemistry,
Glaxo Wellcome Inc, Research Triangle Park; the Department of Medicine,
Duke University Medical Center, Durham; the National Institute of
Environmental Health Sciences, Research Triangle Park; and AndCare Inc,
Durham, NC.
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 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 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 blebbing 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.
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4350-4360
© 1998 by The American Society of Hematology.
