Fas-dependent and -independent mechanisms of cell death following DNA damage in human colon carcinoma cells.
In thymidylate synthase-deficient (TS-) colon carcinoma cells, thymineless death is mediated via Fas/Fas ligand (FasL) interactions after thymidine deprivation and inhibited by the Fas-inhibitory monoclonal antibody NOK-1. The objective of the study was to elucidate whether other modes of DNA damage induced by doxorubicin, topotecan, and etoposide (VP-16) could elicit a similar cytotoxic response in TS- cells by signaling via the Fas death receptor. After a 72-h drug exposure, a loss in clonogenic survival that was not prevented by NOK-1 was induced by each agent in the absence of acute apoptosis, yielding IC50 values of 5 (doxorubicin), 10 (topotecan), and 150 nM (VP-16). Furthermore, TS- cell clones selected for resistance to Fas-mediated apoptosis (CH-11) were cross-resistant to the induction of thymineless death after thymidine deprivation but were not cross-resistant to doxorubicin, topotecan, or VP-16. A close correlation was found between acute induction of apoptosis (24 h) and up-regulated expression of FasL at high concentrations of each of the three agents (0.3-3 microM doxorubicin, 0.3-3 microM topotecan, and 10-90 microM VP-16), which was caspase dependent but Fas independent. At all drug concentrations, cell cycle distribution analyses demonstrated marked accumulation of cells in the G2-M phase. At nanomolar drug concentrations, prolonged arrest of TS- cells in G2-M phase resulted in the up-regulation of FasL expression and the delayed appearance of apoptotic cells (6 days), which could also be inhibited by the general caspase inhibitor Z-VAD-FMK, but not by NOK-1 or Fas-Fc. In clonogenic assays, Z-VAD-FMK did not rescue cells treated with VP-16 in contrast to treatment with CH-11 or thymineless stress, suggesting an irreversible commitment to cell death in G2-M phase. Expression of FasL at all drug concentrations appeared to be unrelated to the mechanism of drug-induced apoptosis. This was in contrast to the Fas-dependent regulation of thymineless death, which could be inhibited by blocking Fas/FasL interactions.
Petak
I
I
Department of Molecular Pharmacology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Tillman
D M
DM
Harwood
F G
FG
Mihalik
R
R
Houghton
J A
JA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000954:Antigens, Surface; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D002999:Clone Cells; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology*; D004249:DNA Damage; D053222:Fas Ligand Protein; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D015398:Signal Transduction; D013941:Thymine / Q000378:metabolism; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000502:physiology*
D000590:Amino Acid Chloromethyl Ketones; D000954:Antigens, Surface; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D020169:Caspases; D013941:Thymine