triphosphate Influences the Commitment of Human Colon Carcinoma Cells to Thymineless Death1

In colon cancers induction of a thymineless state following inhibition of thymidylate synthase (TS) by 5-fluorouracil combined with leucovorin can initiate a cytotoxic response. Using a 5-fluorouracil-leucovorin-treated human colon carcinoma cell line (GC,Icl) and a clonally derived TS mutant, initiation events that dictate the onset of and commitment to thymineless death have been examined. Initial events related to a temporally associated decrease in dTTP and elevation in the dATP pools; no depletion of dGTP or elevation in dCTP was detected. Nucleosomal degradation of DNA commenced at 24 h in TS and 49 h in GCIc1, and was associated with the more rapid development of an imbalance in the dATP and dTTP pools and a higher dATP:dTTP ratio in TS cells. The contribution of elevated dATP or depleted dTTP pools to thymineless death was subsequently determined by treatment of GCIcl or TS cells with deoxyadenosine to elevate the dATP pool either under thymidine-replete or thymineless conditions. Thus, deoxyadenosine supplementation under dTTP-replete conditions elevated the dATP pool for 16 h and was cytotoxic to cells. During dTTP depletion elevated dATP was maintamed, and cytotoxicity was significantly and rapidly enhanced by deoxyadenosine but could be reversed by thymidine. Data suggest that maintenance of elevated dATP and the dATP:dTTP ratio are essential initiation events in the commitment of colon carcinoma cells to thymineless death.