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-

ABCG2 is an ATP-binding cassette transporter that confers resistance to various chemotherapeutic agents. Recent studies have established that an Arg (wild-type) to Gly mutation at amino acid 482 in ABCG2 alters substrate specificity. Here, we explored the role of this G482 mutation in antifolate resistance using a clinically relevant 4-hour drug exposure. Stable transfectants overexpressing the mutant G482 transporter displayed 120-, 1,000-, and >6,250-fold resistance to the antifolates methotrexate, GW1843, and Tomudex, respectively, relative to parental human embryonic kidney cells. Moreover, although overexpressing equal transporter levels at the plasma membrane, G482-ABCG2 cells were 6-, 23-, and >521-fold more resistant to methotrexate, GW1843, and Tomudex, respectively, than R482-ABCG2 cells. In contrast, upon a continuous (72-hour) drug exposure, both the G482-and R482-ABCG2 cells lost almost all their antifolate resistance; this result was consistent with the inability of ABCG2 to extrude long-chain antifolate polyglutamates. Ko143, a specific and potent ABCG2 inhibitor reversed methotrexate resistance in both G482-and R482-ABCG2 cells. Consistently, whereas the pool of free methotrexate in parental human embryonic kidney cells was prominent after 4 hours of transport with 1 Mmol/L [ 3 H]methotrexate, in R482-and G482-ABCG2 cells, it was minimal. Furthermore, G482-ABCG2 cells contained marked decreases in the di-and triglutamate species of [ 3 H]methotrexate at 4 hours of incubation with methotrexate and in the tetra-and pentaglutamates at 24 hours. These changes were not associated with any significant decrease in folylypoly-;-glutamate synthetase activity. These results provide the first evidence that the G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates. (Cancer Res 2005; 65(18): 8414-22)

Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120-6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414-8422, 2005). Here we explored the ability of the wild type (WT) R482-as well as the mutant G482-and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed.