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 K1 (CHO-K1) 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 microM. 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 microM in dThd-free medium. A 55% reduction in NAD levels was seen within 6 hr of 5.0 microM 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 microM 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 microM) 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 < or = 5-100 microM) in mature DNA. Whereas FU elution profiles for nascent DNA were biphasic, those for FdUrd were linear. Co-incubation with 3AB increased [3H]FU incorporation into both RNA (by 50%) and DNA (45%). 3AB also enhanced [3H]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.
Willmore
E
E
Cancer Research Unit, Medical School, University of Newcastle upon Tyne, U.K.
Durkacz
B W
BW
D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D016466:CHO Cells / Q000187:drug effects; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D005467:Floxuridine / Q000633:toxicity*; D005472:Fluorouracil / Q000633:toxicity*; D009243:NAD / Q000032:analysis; D009696:Nucleic Acids / Q000096:biosynthesis*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*
D001549:Benzamides; D009696:Nucleic Acids; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D005467:Floxuridine; D009243:NAD; C025160:3-aminobenzamide; D011065:Poly(ADP-ribose) Polymerases; D005472:Fluorouracil