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Fas-dependent and -independent Mechanisms of Cell Death following DNA Damage in Human Colon Carcinoma Cells¹

Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105

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 µM doxorubicin, 0.3–3 µM topotecan, and 10–90 µM 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.

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