CYTOTOXICITY AND DNA FRAGMENTATION ASSOCIATED WITH SEQUENTIAL GEMCITABINE AND 5-FLUORO-2'-DEOXYURIDINE IN HT-29 COLON-CANCER CELLS

The combined cytotoxic effects of the antimetabolites gemcitabine (dFd Cyd) and 5-fluoro-2'-deoxyuridine (FdUrd) were studied. Cytotoxicity, biochemical perturbations, and DNA damage seen with dFdCyd and FdUrd a lone and in combination were evaluated in HT-29 human colon cancer cel ls. A 4-h exposure to dFdCyd followed by FdUrd for 24 h produced more than additive cytotoxicity and marked S-phase accumulation. Cells prog ressed through the cell cycle, however, after a 22-h drug-free interva l. [H-3]dFdCyd was rapidly metabolized to the 5'-triphosphate and inco rporated into DNA, [H-3]FdUrd was anabolized exclusively to FdUrd mono phosphate, and preexposure to dFdCyd did not affect FdUrd monophosphat e formation. Thymidylate synthase catalytic activity was inhibited by 48% after a 4-h exposure to 10 nM FdUrd and by 80% after exposure to t he combination, Sequential 4-h exposures to 15 nM dFdCyd and 10 nM FdU rd led to greater depletion of dTTP pools (29% of control) than with e ither drug alone. Greater effects on nascent DNA integrity were seen w ith sequential dFdCyd followed by FdUrd, Although parental DNA damage was not evident immediately after exposure to 15 nM dFdCyd for 4 h fol lowed by 10 nM FdUrd for 24 h, high molecular mass DNA fragmentation w as evident 72-96 h after drug removal. Sequential dFdCyd/FdUrd was ass ociated with prominent disturbance of the cell cycle, dTTP pool deplet ion, dATP/dTTP imbalance, and nascent DNA damage. Induction of double- strand parental DNA damage and cell death was delayed, consistent with postmitotic apoptosis.