Prior studies from this laboratory documented the prevalence of methotrexate (MTX) transport activity with a low-pH optimum in a variety of human solid tumor cell lines (Clinical Cancer Research, 2004, 10: 718). In HeLa cells, derived from human cervical cancer, this low pH activity has high affinity (Kt=45 nM) for pemetrexed (PMX) and is RFC-independent, since it is not diminished in an RFC-null subline (R5) (Clinical Cancer Research, 2004, 10: 6256). R5 cells also have residual transport activity, with high specificity for PMX, at neutral pH (Cancer Research, 2004, 64: 3313). The current study addresses whether this low pH folate transport activity can be down-regulated under antifolate pressure and the consequence of this down-regulation on the activity of antifolates. An R5 subline, R1, was selected under MTX selective pressure in a medium with a modestly reduced pH (pH 6.9). In R1 cells influx of MTX, PMX and folic acid at pH 5.5 was markedly and equally decreased as compared to R5 cells. PMX influx at pH 7.4 in R1 cells was also markedly reduced while MTX influx at this pH was decreased to a lesser extent. When MTX was removed from the growth medium there was a slow return of transport activity in R1 cells, and when MTX was added back, there was loss of transport at both pHs within eight weeks. PMX influx in R5 cells at both acidic and neutral pH was increased by preloading cells with 5-formyltetrahydrofolate (trans-stimulation), consistent with a process mediated by a facilitative carrier. In R1 cells there was marked decrease in accumulation of PMX, MTX, and folic acid at both pHs as compared to R5 cells. At both pHs R1 cells were more resistant than R5 cells to antifolates, which require a facilitative process to gain entry into cells, although the extent of resistance was different for different antifolates (PMX>MTX>tomudex). These data demonstrate that (i) RFC-independent transport in HeLa cells at low and neutral pH contribute to antifolate activity, in particular, to PMX activity, and can be diminished by antifolate selective pressure and, (ii) the loss of these activities results in marked resistance to PMX, an agent for which there is little or no loss of activity when transport mediated by RFC is abolished. These observations suggest that transport activity in RFC-null HeLa R5 cells at neutral and low pH may reflect the same carrier-mediated process. This study was supported by a grant from the National Institutes of Health, CA-82621, along with a grant from the Eli Lilly Company.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]