A methotrexate (MTX)-resistant HeLa subline (R5) has been developed in this laboratory with impaired transport due to a genomic deletion of the reduced folate carrier (RFC). In the current study, this cell line was utilized to examine the impact of loss of RFC on the activity of other antifolates. Despite the complete loss of RFC activity, R5 cells were only 2-fold resistant to pemetrexed (PMX), but 200- and 400-fold resistant to ZD1694 and PT523, respectively, as compared to parental HeLa cells when grown with 2 uM folic acid. When folic acid was replaced with the more physiological 25 nM 5-formyltetrahydrofolate, R5 cells were 2-fold collaterally sensitive to PMX while still 40-and 200-fold resistant to ZD1694 and PT523, respectively, as compared to HeLa cells. Under these conditions there was a marked contraction of the cellular folate-cofactor pool in R5 cells. Sensitivity to PT523 and PMX could be completely restored, and to ZD1694 nearly restored, by transfection of RFC cDNA into R5 cells indicating that the defect in drug transport was the only, or major, factor in resistance. The preserved PMX activity in R5 cells could not be related to the very low expression of folate receptors since inclusion of 1 uM folic acid, to abolish any folate receptor-mediated transport, had only a minimal effect on the PMX IC50. Rather, retained PMX activity in R5 cells was associated with residual transport by another process that exhibits good affinity for PMX (Kt=12 uM) with much lower affinities for ZD1694, MTX, and PT523 (Ki’s of ∼ 90, 100, 250 uM, respectively). PMX transported by this route was rapidly converted to higher polyglutamates and, when grown with 25 nM 5-formyltetrahydrofolate, the rate of formation of these derivatives and their net accumulation in R5 cells was comparable to that of wild-type cells. These data suggest that selective preservation of PMX pharmacological activity is due to (i) partial preservation of transport by an unidentified process with a higher affinity for PMX than the other antifolates evaluated, (ii) the contraction of cellular tetrahydrofolate-cofactor pools associated with loss of RFC when the growth substrate is a reduced folate that utilizes this transporter for entry into cells.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]