Inhibition by methotrexate of the stable incorporation of 5-fluorouracil into murine bone marrow DNA.
As a consequence of the inhibition of de novo purine synthesis by methotrexate (MTX) there is an increase in 5-phosphoribosyl-1-pyrophosphate (PRPP) concentration. In cells where 5-fluorouracil (FUra) is activated via orotate phosphoribosyltransferase (OPRtase), increased PRPP results in greater conversion of FUra to nucleotides. In the murine CD8F1 breast tumor system, MTX markedly enhances the antitumor activity of FUra, increasing both the activation of FUra to FUMP and the incorporation of FUTP into RNA. However, in contrast to reported tumor tissue culture studies, MTX pretreatment in vivo prevents the stable incorporation of FUra into CD8F1 bone marrow DNA. Pretreatment with MTX (300 mg/kg) 2.5 hr prior to [3H]FUra (100 mg/kg), with a 2-hr labeling, reduced the level of FUra in DNA from 921 pmol to 66 pmol/mg of DNA. Without MTX pretreatment, 59% of the total incorporation of FUra into nucleic acids was into DNA when FUra was administered. After MTX the percentage of incorporation into DNA was reduced to 9%. Additionally, the ratio of [3H]FUra to 32P in DNA when both were given simultaneously was reduced by greater than 90%, suggesting that MTX must be specifically blocking the incorporation of FUra rather than nonspecifically preventing its incorporation by inhibiting DNA synthesis. In contrast, MTX failed to reduce the formation of DNA containing fluorouracil residues from FdUrd. To test whether MTX prevents the initial incorporation of FUra into DNA, or acts to enhance removal by the DNA glycosylase repair system, DNA was prelabeled in vivo with [3H]FUra, and MTX or MTX plus dThd was then administered. The level of FUra in bone marrow DNA was not reduced by subsequent treatment with MTX, or MTX plus dThd, indicating that MTX does not enhance the removal of FUra from DNA. The level of total free fluorodeoxynucleotides formed from FUra was reduced by two-thirds following MTX pretreatment, suggesting that the action of MTX in preventing the stable incorporation of FUra into DNA was to reduce the availability of FdUTP.
Sawyer
R C
RC
Cancer Research Laboratories, Catholic Medical Center, Woodhaven, NY 11421.
Stolfi
R L
RL
Martin
D S
DS
Balis
M E
ME
D000818:Animals; D001853:Bone Marrow / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D005472:Fluorouracil / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice
D004247:DNA; D005472:Fluorouracil; D008727:Methotrexate