Cell death in response to antimetabolites directed at thymidylate synthase.
OBJECTIVE
Thymidylate synthase (TS) is an indispensable enzyme in the de novo biosynthesis of TMP during DNA replication and cell growth, and has, therefore, been an important target for several classes of antimetabolites used in cancer chemotherapy. While most investigations of the action of TS-directed agents have focused on apoptosis as the primary means of cell death, little is known regarding the role, if any, of non-apoptotic mechanisms. In the present study, we have examined the mode of cell death induced by several TS inhibitors.


METHODS
Apoptosis and necrosis in response to TS inhibitors was assessed. The roles of caspases and the transcriptional regulator nuclear factor kappa B (NFkappaB) in drug-induced cell death were analyzed. Finally, drug-mediated changes in expression of several proteins involved in regulation of apoptosis were analyzed.


RESULTS
Though human colon tumor cells exposed to TS inhibitors undergo classical apoptosis, it is not the predominant mechanism of response; rather, a necrosis-like mechanism prevails. The apoptotic response to TS inhibitors is caspase-dependent, and is promoted by NFkappaB. In contrast, the necrosis-like response is independent of both caspases and NFkappaB. Exposure to TS inhibitors induces PARP cleavage, but does not alter expression of the pro or activated forms of caspases-3 or caspases-8, Fas, or FasL. Treatment with the death-inducing cytokine TNFalpha, like TS inhibitors, results in a limited extent of apoptosis that is both caspase- and NFkappaB-dependent; however, unlike TS inhibitors, the cytokine does not induce necrosis.


CONCLUSIONS
Classical apoptosis occurs to a limited extent in human colon tumor cells exposed to TS inhibitors, with caspase-independent necrosis being the prinicipal mechanism of cell death. We suggest that the role of necrosis and necrosis-like mechanisms should be considered in future studies of the action of TS-directed antimetabolites, as well as other chemotherapeutic agents.
Barbour
Karen W
KW
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA.
Berger
Franklin G
FG
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000502:physiology; D015153:Blotting, Western; D061945:Caspase Inhibitors; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000473:pathology; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007202:Indicators and Reagents; D046529:Microscopy, Electron, Transmission; D016328:NF-kappa B / Q000502:physiology; D009336:Necrosis; D013936:Thymidine / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D013940:Thymidylate Synthase; D013936:Thymidine