Molecular Analysis of Murine Leukemia Cell Lines Resistant to 5,10-Dideazatetrahydrofolate Identifies Several Amino Acids Critical to the Function of Folylpolyglutamate Synthetase

Rongbao Zhao,Steven Titus,Feng Gao,Richard G Moran,I David Goldman

doi:10.1074/jbc.m002580200

Abstract

Four L1210 murine leukemia cell lines resistant to 5, 10-dideazatetrahydrofolate (DDATHF) and other folate analogs, but sensitive to continuous exposure to methotrexate, were developed by chemical mutagenesis followed by DDATHF selective pressure. Endogenous folate pools were modestly reduced but polyglutamate derivatives of DDATHF and ALIMTA (LY231514, MTA) were markedly decreased in these mutant cell lines. Membrane transport was not a factor in drug resistance; rather, folypolyglutamate synthetase (FPGS) activity was decreased by >98%. In each cell line, FPGS mRNA expression was unchanged but both alleles of the FPGS gene bore a point mutation in highly conserved domains of the coding region. Four mutations were in the predicted ATP-, folate-, and/or glutamate-binding sites of FPGS, and two others were clustered in a peptide predicted to be beta sheet 5, based on the crystal structure of the Lactobacillus casei enzyme. Transfection of cDNAs for three mutant enzymes into FPGS-null Chinese hamster ovary cells restored a reduced level of clonal growth, whereas a T339I mutant supported growth at a level comparable to that of the wild-type enzyme. The two mutations predicted to be in beta sheet 5, and one in the loop between NH(2)- and COOH-terminal domains did not support cell growth. When sets of mutated cDNAs were co-transfected into FPGS-null cells to mimic the genotype of drug-selected resistant cells, clonal growth was restored. These results demonstrate for the first time that single amino acid substitutions in several critical regions of FPGS can cause marked resistance to tetrahydrofolate antimetabolites, while still allowing cell survival.

Highlights

Folate cofactors play an essential role in the biosynthesis of purines, thymidylate, glycine, and methionine by providing one-carbon moieties at a variety of oxidation levels
When sets of mutated cDNAs were co-transfected into Folylpoly-␥-glutamate synthetase (FPGS)-null cells to mimic the genotype of drug-selected resistant cells, clonal growth was restored. These results demonstrate for the first time that single amino acid substitutions in several critical regions of FPGS can cause marked resistance to tetrahydrofolate antimetabolites, while still allowing cell survival
We investigate the mechanisms which allow resistance to develop to the prototypical GART inhibitor DDATHF after treatment with the mutagen N-methyl-N-nitrosourea

Summary

Introduction

Folate cofactors play an essential role in the biosynthesis of purines, thymidylate, glycine, and methionine by providing one-carbon moieties at a variety of oxidation levels. Resistance of tumor cells to MTX can involve any of several mechanisms, including mutation or down-regulation of the reduced folate carrier (14 –21), amplification or mutation of the dihydrofolate reductase gene [22, 23], and/or decreased formation of polyglutamate derivatives by FPGS [24]. Relevant compounds that fall into these three groupings are lometrexol (DDATHF) and L309887 [25,26,27], raltitrexed (Tomudex, ZD1694) [28], and ALIMTA (LY231514, MTA) [29, 30], respectively Most of these agents are strongly activated by polyglutamation, because they are better inhibitors of their target enzymes as polyglutamates, and because they are retained in target cells and accumulate to high levels as these metabolites (26 –32). The in vivo function of polyglutamation of the naturally occurring folates was sufficient to sustain cell growth and replication

Methods

Results

Conclusion