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 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.
Laethem
R M
RM
International Science Development Group, Glaxo Wellcome Inc, Research Triangle Park, NC 27709, USA.
Hannun
Y A
YA
Jayadev
S
S
Sexton
C J
CJ
Strum
J C
JC
Sundseth
R
R
Smith
G K
GK
D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases; D002518:Ceramides / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D054833:Isoindoles; D015458:Leukemia, T-Cell / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured
D002518:Ceramides; D004075:Diglycerides; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D007211:Indoles; D054833:Isoindoles; D011799:Quinazolines; C079601:1843U89; D013940:Thymidylate Synthase; D013108:Sphingomyelin Phosphodiesterase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D008274:Magnesium