CYTOTOXIC MECHANISMS OF 5-FLUOROPYRIMIDINES - RELATIONSHIPS WITH POLY(ADP-RIBOSE) POLYMERASE-ACTIVITY, DNA STRAND BREAKAGE AND INCORPORATION INTO NUCLEIC-ACIDS

A comparative study of the cytotoxic mechanisms of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdUrd) was carried out in Chinese hamster ovary KI (CHO-Kl) cells. The poly(ADP-ribose) polymerase (PADPRP) inhibitor, 3-aminobenzamide (3AB, 3 mM) enhanced the cytotoxicity of FU with a dose enhancement factor at 10% survival of 2. This enhancement was also evident when cells were grown in dThd-free medium, but the IC50 for FU was reduced from 50 to 35 muM. In contrast, 3AB did not enhance the cytotoxicity of FdUrd but exerted a small protective effect. The IC50 for FdUrd was reduced from 35 to 1.25 muM in dThd-free medium. A 55% reduction in NAD levels was seen within 6 hr of 5.0 muM FdUrd treatment in dThd-free medium, and this reduction persisted over 24 hr. This drop was prevented by co-incubation with 3AB, indicating that PADPRP activation was the cause of the NAD depletion. In contrast, FU treatment had little or no effect on NAD levels. Alkaline elution analysis of cells treated with up to 150 muM FU revealed no DNA strand breaks in mature DNA, but an increase in breaks in nascent DNA. Co-incubation with 3AB had little or no effect on strand break levels. FdUrd (up to 40 muM) produced a dose-dependent increase in both mature and nascent DNA strand breaks. Analysis using a ''relative elution'' formula demonstrated that 3AB increased the amount of FdUrd-induced strand breaks (at doses less-than-or-equal-to 5-100 muM) in mature DNA. Whereas FU elution profiles for nascent DNA were biphasic, those for FdUrd were linear. Co-incubation with 3AB increased [H-3]FU incorporation into both RNA (by 50%) and DNA (45%). 3AB also enhanced [H-3]FdUrd incorporation (by 40%) into RNA but had no effect on incorporation into DNA. These data indicate that in addition to acting as an inhibitor of PADPRP, 3AB exerts other metabolic effects.