Polymorphisms in folate pathway genes may influence the susceptibility to acute lymphoblastic leukemia (ALL). DNA was isolated from 245 pediatric ALL patients (cases) and from 500 blood bank donors (controls). Polymorphisms in methylenetetrahydrofolate reductase (MTHFR 677C>T, 1298A>C), methionine synthase (MTR 2756A>G), methionine synthase reductase (MTRR 66A>G), methylenetetrahydrofolate dehydrogenase (MTHFD1 1958G>A), nicotinamide N-methyltransferase (NNMT IVS ؊151C>T), serine hydroxymethyl transferase (SHMT1 1420C>T), thymidylate synthase (TS 2R3R), and the reduced folate carrier (RFC1 80G>A) were detected. In ALL patients, an increased occurrence was observed of the RFC1 80AA variant (odds ratio [OR] ‫؍‬ 2.1; 95% confidence interval [CI] ‫؍‬ 1.3-3.2; P ‫؍‬ .002) and the RFC1 80A allele (OR ‫؍‬ 1.5; 95% CI, 1.1-2.1; P ‫؍‬ .02). Likewise, the NNMT IVS ؊151TT genotype showed a 2.2-fold increased ALL risk (OR ‫؍‬ 2.2; 95% CI, 1.1-4.6; P ‫؍‬ .04). A 1.4-fold reduction in ALL risk was observed for (heterozygous or homozygous) carriers of the TS 2R allele and the MTHFR 677T allele (OR ‫؍‬ 0.7; 95% CI, 0.5-1.0; P < .05). Furthermore, interactions between NNMT and MTHFR 677C>T and RFC1 were observed. NNMT IVS ؊151CC/MTHFR 677CT ؉ TT patients exhibited a 2-fold reduction in ALL risk whereas RFC1 80AA/NNMT IVS ؊151CT ؉ TT subjects had a 4.2-fold increase in ALL risk (P ‫؍‬ .001). For the first time, we associate the RFC1 80G>A and NNMT IVS ؊151C>T variants to an increased ALL susceptibility.(Blood.

Reduced folate cofactors play a key role in one-carbon transfer reactions in the de novo biosynthesis of purines and thymidylate (1). As such, normal and neoplastic dividing cells have an absolute requirement for reduced folates in order to properly initiate and complete DNA replication and mitosis (1). Disruption of one-carbon transfer reactions in the folate metabolic pathway by folic acid antagonists (i.e. antifolates) is the pharmacological basis for the antitumor activity of methotrexate (MTX) 1 (2) as well as the novel antifolates raltitrexed (3) and pemetrexed (4). Because mammalian cells are devoid of de novo biosynthesis of folic acid, they must obtain this folate vitamin from exogenous sources (1). Membrane transport of folates and MTX is mediated by several systems (5, 6). (a) The reduced folate carrier (RFC) is the major uptake route that functions as a bi-directional anion exchanger (7, 8) taking up folates through an antiport exchange mechanism with intracellular organic phosphates (9). (b) Folate receptors mediate the unidirectional uptake of folate cofactors into mammalian cells via an endocytotic process (5, 10). (c) An apparently independent transport system with optimal folate uptake activity at low pH (11-13) is also known. Following uptake, the enzyme folylpoly-␥-glutamate synthetase (FPGS) catalyzes the addition of several equivalents of L-glutamic acid to the ␥-carboxyl group in the side chain of folate cofactors and antifolates like MTX (14). This long chain polyglutamate conjugation (up to 10 glutamate residues) is believed to fulfill three cellular functions as follows. (a) By rendering folates and antifolates polyanionic, it dramatically increases their cellular retention by preventing efflux (15). (b) It increases the binding affinity of folate cofactors and antifolates to some folate-dependent enzymes (4, 16). (c) It appears to facilitate, via mitochondrial FPGS activity, the accumulation in mitochondria of folate polyglutamates that are required for glycine biosynthesis (15).Cellular folate pools are controlled by the above folate influx systems, by FPGS activity, and by ATP-dependent efflux transporters of the ABCC sub-family (17). Recent studies (18 -22) have established an increased energy-dependent folate and MTX transport into inverted membrane vesicles isolated from cell lines with MRP1 (ABCC1) through MRP4 (ABCC4) overexpression. This MTX efflux in various tumor cell lines with MRP overexpression resulted in a significant resistance to MTX predominantly upon a short term (Յ4 h) antifolate exposure (18 -20). The restriction of this antifolate resistance to only a short term drug exposure has been attributed to the ability of these transporters to export only monoglutamate but not polyglutamate conjugates of MTX; indeed, these transport-

Members of the multidrug resistance protein family, notably MRP1-4/ABCC1-4, and the breast cancer resistance protein BCRP/ABCG2 have been recognized as cellular exporters for the folate antagonist methotrexate (MTX). Here we show that MRP5/ABCC5 is also an antifolate and folate exporter based on the following evidence:

The multidrug resistance protein (MRP) is an ATPdependent transport protein for organic anions, as well as neutral or positively charged anticancer agents. In this study we report that dinitrophenyl-S'-glutathione increases ATPase activity in plasma membrane vesicles prepared from the MRP-overexpressing cell line GLC4/ADR. This ATPase stimulation parallels the uptake of DNP-SG in these vesicles. We also show that the (iso)flavonoids genistein, kaempferol and flavopiridol stimulate the ATPase activity of GLC4/ADR membranes, whereas genistin has no effect. The present data are consistent with the hypothesis that certain (iso)flavonoids affect MRPmediated transport of anticancer drugs by a direct interaction with MRP.