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J. Biol. Chem., Vol. 275, Issue 34, 26599-26606, August 25, 2000
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From the 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
Molecular Analysis of Murine Leukemia Cell Lines Resistant to
5,10-Dideazatetrahydrofolate Identifies Several Amino Acids
Critical to the Function of Folylpolyglutamate Synthetase*
,,¶
Albert Einstein College of Medicine,
Comprehensive Cancer Center, Bronx, New York 10461 and the
§ Massey Cancer Center and the Department of Pharmacology
and Toxicology, Medical College of Virginia, Virginia Commonwealth
University, Richmond, Virginia 23298
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 sheet 5, and one in the loop
between NH2- 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.
*
This work was supported in part by National Institues of
Health Grants CA-39687 (to R. G. M.) and CA-39807 and
CA-82621 (to I. D. G.) from the Department of Health and
Human Services.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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