Increased activity of a novel low pH folate transporter associated with lipophilic antifolate resistance in chinese hamster ovary cells

Abstract

Previous studies described a Chinese hamster ovary cell line, PyrR100, resistant to lipid-soluble antifolates due to the loss of an energy-coupled folate exporter resulting in a marked increase in intracellular folate cofactor accumulation. There was, in addition, an unexplained increase in folic acid influx in PyrR100 cells which is shown in this paper to be mediated by a transporter with a low pH optimum. The pH profile for folic acid influx in parental Chinese hamster ovary AA8 cells indicated peak activity at pH 6; this was increased >3-fold in PyrR100 cells. In contrast, methotrexate (MTX) influx in AA8 cells showed two peaks of comparable activities at pH 6 and 7.5; in PyrR100 cells, the component at pH 6 was increased 2-fold. Folic acid was a potent inhibitor of [3H]MTX or [3H]folic acid influx (1 microM) via the low pH route with IC50 values of approximately 1 microM. Prostaglandin A1 was a potent inhibitor of [3H]MTX influx via the reduced folate carrier 1 at pH 7.5 with only a small inhibitory effect on the low pH route. The addition of 10 microM folic acid to PyrR100 cells resulted in a MTX influx pH profile identical to that of AA8 cells, consistent with suppression of the low pH route. In contrast, addition of 25 microM prostaglandin A1 to PyrR100 cells resulted in a MTX influx pH profile comparable to that of folic acid, consistent with the loss of the reduced folate carrier-mediated component. Inhibition ( approximately 70%) of [3H]folic acid influx by approximately 10 microM unlabeled folic acid at pH 7.5 indicated that the low pH transporter accounts for the majority of folic acid transport at physiological pH. This study demonstrates the functional importance of a low pH folate transporter that is increased when enhanced folic acid entry into cells is required as an adaptive response to antifolate selective pressure. This may represent a mechanism of resistance to new antifolate inhibitors of folate cofactor-dependent enzymes in which cytotoxic activity is limited by expanded cellular folate pools.