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Rate-limiting steps in the interactions of fluoropyrimidines and methotrexate
Abstract:
Rate-limiting steps are defined between methotrexate (MTX) and 5-fluorouracil (FU) or 5-fluorodeoxyuridine (FUdR) and [14C]-formate incorporation into RNA, DNA and protein as a function of the basal rate of dTMP synthesis. When Ehrlich cells are incubated with 0.1 microM FU dR, 1 microM FU and 50 microM MTX for 1-35 min. [3H]-deoxyuridine (UdR) incorporation into DNA is maximally inhibited within 1, 10 and 15 min respectively. The delay in suppression of [3H]-UdR incorporation into MTX-exposed cells compared to cells exposed to FU or FUdR is related to the slow transport of MTX and the increasing free intracellular MTX levels. Influx of MTX is 4 and 10 times slower than FU and FUdR respectively. At 2.5, 5, 10 and 15 min the free intracellular MTX levels (nmol/g dry wt) are 5.8, 7.4, 8.7 and 8.8 respectively. Free intracellular FdUMP is identified 1 min after exposure of cells to FU and FUdR. Antagonism to MTX-suppression of [14C]-formate incorporation into RNA, DNA and protein occurs when cells are simultaneously exposed to MTX and FU or FUdR. However, [14C]-formate incorporation into RNA, DNA and protein is maximally inhibited when Ehrlich tumor cells are incubated with 50 microM MTX for 10 min and then exposed to 1 microM FU for 1 min (a time in which free intracellular MTX is maximal and [3H]-UdR incorporation is maximally suppressed). Hence the sequence and time of administration of FU or FUdR and MTX inhibition of formate incorporation into RNA, DNA and protein is related to the rate of (a) FU, FUdR and MTX transport, (b) FU and FUdR metabolism to FdUMP and (c) generation of maximal free intracellular MTX.
Insights
Methotrexate (MTX) and 5-fluorouracil (FU) or 5-fluorodeoxyuridine (FUdR) inhibit DNA synthesis. The timing of drug administration significantly impacts the inhibition of [14C]-formate incorporation into RNA, DNA, and protein.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Methotrexate (MTX) and 5-fluorouracil (FU)/5-fluorodeoxyuridine (FUdR) are chemotherapeutic agents targeting DNA synthesis.
- Understanding the kinetics of their action and interaction is crucial for optimizing cancer treatment strategies.
Purpose of the Study:
- To define the rate-limiting steps in the inhibition of dTMP synthesis by MTX, FU, and FUdR.
- To investigate the impact of drug administration sequence and timing on the inhibition of macromolecular synthesis.
Main Methods:
- Ehrlich cells were incubated with varying concentrations and durations of MTX, FU, and FUdR.
- Incorporation of [3H]-deoxyuridine (UdR) into DNA and [14C]-formate into RNA, DNA, and protein was measured.
- Intracellular drug levels were quantified.
Main Results:
- [3H]-UdR incorporation into DNA was rapidly inhibited by FU and FUdR, with a delayed but sustained inhibition by MTX.
- MTX transport into cells was significantly slower than FU and FUdR.
- Simultaneous administration showed antagonism, while sequential administration (MTX followed by FU) led to maximal inhibition of [14C]-formate incorporation.
Conclusions:
- The sequence and timing of MTX and FU/FUdR administration are critical for maximal inhibition of DNA synthesis and formate incorporation.
- Drug transport rates, metabolism to active forms (FdUMP), and intracellular MTX accumulation dictate the overall inhibitory effect.