Thymidylate synthase inhibition induces S-phase arrest, biphasic mitochondrial alterations and caspase-dependent apoptosis in leukaemia cells

In this study, the downstream effects of thymidylate synthase (TS) inhibiti on in L1210 (p53 mutant) and HL60 (p53 null) leukaemia cells were investiga ted. TS inhibition was induced by the specific TS inhibitor Thymitaq. Withi n 24 h, TS inhibition resulted in S-phase cell cycle arrest in both cell li nes and subsequent apoptotic cell death as characterized by nuclear condens ation, DNA fragmentation and the formation of apoptotic bodies. A biphasic hyper/hypopolarization of the mitochondrial membrane potential (Delta Psim) was also observed. The mitochondrial permeability transition inhibitor, cy closporin A, increased the baseline level of Delta Psim in L1210 cells. How ever, along with bongkrekic acid, it did not influence the changes in Delta Psim induced by TS inhibition in either cell line. In both cell lines the broad spectrum caspase inhibitor, zVAD.fmk as a single agent, induced a sig nificant downward shift in the baseline of Delta Psim. However, only in HL6 0 cells was this accompanied by a slight increase in cytotoxicity. In L1210 cells: zVAD.fink inhibited DNA fragmentation induced by Thymitaq but did n ot influence other cell cycle events (S-phase arrest) or the biphasic mitoc hondrial alterations, indicating caspase involvement downstream but not ups tream of the mitochondria following TS inhibition. In I HL60 cells, zVAD.fm k reduced the fiyperpolarization of Delta Psim observed with Thymitaq alone and failed to inhibit the increase in the sub-G(1) population induced by T hymitaq. Moreover, zVAD.fmk significantly increased the cell death response of these cells following TS inhibition. In conclusion, cell death induced by TS inhibition is mediated via the apoptotic pathway which clearly involv es biphasic alterations ill Delta Psim. In L1210 cells, but not in HL60 cel ls, caspases function as the final executioner of apoptosis.