Evidence for direct inhibition of de novo purine synthesis in human MCF-7 breast cells as a principal mode of metabolic inhibition by methotrexate.

C J Allegra,K Hoang,G C Yeh,J C Drake,J Baram

doi:10.1016/s0021-9258(19)76458-8

Abstract

We have investigated the role of dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo purine synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Previous studies have shown that cytotoxic concentrations of MTX that inhibit dihydrofolate reductase produce only minimal depletion of the reduced folate cofactor, 10-formyltetrahydrofolate, required for purine synthesis. At the same time, de novo purine synthesis is totally inhibited. In these studies, we show that 10 microM MTX causes inhibition of purine synthesis at the step of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase, as reflected in a 2-3-fold expansion of the intracellular AICAR pool. The inhibition of purine synthesis coincides with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of AICAR transformylase. When the generation of H2PteGlu is blocked by pretreatment with 50 microM 5-fluorodeoxyuridine (FdUrd), an inhibitor of thymidylate synthase, MTX no longer causes inhibition of purine synthesis. Intermediate levels of H2PteGlu produced in the presence of lower (0.1-10 microM) concentrations of FdUrd led to proportional inhibition of purine biosynthesis, and the exogenous addition of H2PteGlu to breast cells in culture re-established the block in purine synthesis in the presence of FdUrd and MTX. The early phases of inhibition of purine biosynthesis could be ascribed only to H2PteGlu accumulation. MTX polyglutamates, also known to inhibit AICAR transformylase, were present in breast cells only after 6 h of incubation with the parent compounds and were not formed in cells preincubated with FdUrd. The lipid-soluble antifolate trimetrexate, which does not form polyglutamates, produced modest 10-formyltetrahydrofolate depletion, but caused marked H2PteGlu accumulation and a parallel inhibition of purine biosynthesis. This evidence leads to the conclusion that MTX and the lipid-soluble analog trimetrexate cause inhibition of purine biosynthesis through the accumulation of H2PteGlu behind the blocked dihydrofolate reductase reaction.

Highlights

(H,PteGlu) accumulation in the inhibition of de nouo nouo synthesis of purines, thymidylate, and certain amino purinesynthesis by methotrexate (MTX) inhuman acids [1]
Inthe face of dihydrofolate portionalinhibition of purine biosynthesis, and the reductase inhibition, the intracellulapr ools of reduced folate exogenous addition of H,PteGlu to breastcells in cul- are converted to H,PteGlu by the thymidylate synthase reture re-established the block in purine synthesis in theaction and cannotbe reduced to theH,PteGlu state required presence of FdUrd and MTX
In but caused marked HzPteGlu accumulationand a par- particular, we found that 5-methyl-H4PteGlu and H,PteGlu allel inhibition of purine biosynthesis

Summary

THEJOURNALOF BIOLOGICACLHEMISTRY

Val. 262,No 28,Issue of October5,pp. 13520-13526, 1987 Printed in U.S.A. Evidence for Direct Inhibition of de N o v o Purine Synthesis in Human MCF-7 Breast Cells as a Principal Mode of Metabolic Inhibition by Methotrexate*. AICAR transformylase was purified 300-400-fold from the MCF-7 human breast cancer cell line (specific activity, 0.3 pmol/min/mg) [17]. All cells weregrown in Intracellular nucleotide pools were measured by plating MCF-7 cells dialyzed fetal bovine serum for at least two passages prior to their as outlined above with the addition of["C]glycine One h prior to termination of each experiment, Associates HPLC model 6000-A solvent delivery system, Model 440 the cells were incubated with 10 pCi of ["Clglycine (final specific absorbance detector, and Model 720 system controller anddata activity, 5.17 mCi/mmol) in order to label the purine nucleotides module and a Whatman Partisil-10 SAX anion-exchange column. Protein Measurement-Cytosolic protein was measured according to the method of Bradford [22] using bovine serum albumin as the standard

RESULTS

DISCUSSION

MTX polyglutamation by FdUrd pretreatment as a potential