Loss of Folic Acid Exporter Function with Markedly Augmented Folate Accumulation in Lipophilic Antifolate-resistant Mammalian Cells

Abstract

We previously described a 1,000-fold pyrimethamineresistant Chinese hamster ovary cell line (Pyr R100 ) which retains parental dihydrofolate reductase activity and methotrexate (MTX) sensitivity. This study characterizes the basis for the 14-fold decrease in folic acid and leucovorin concentrations required for clonogenic growth of Pyr R100 cells relative to parental AA8 cells. Under conditions in which folic acid reduction was blocked by trimetrexate, Pyr R100 cells displayed relative to parental AA8 cells a: 1) 17-and 5-fold increase in the net transport of folic acid and MTX, respectively; 2) 23-and 5-fold decrease in the efflux rate constant for folic acid and MTX, respectively; and 3) 2-fold increase in folic acid influx with no significant change in MTX influx. The markedly increased net folic acid transport in Pyr R100 cells could not be explained by cellular folic acid binding, mitochondrial sequestration, polyglutamylation, nor by a decreased membrane potential.The effect of energy deprivation on folic acid and MTX transport in both cell lines was quite different. Glucose and pyruvate deprivation nearly abolished the increase in net folic acid transport in Pyr R100 cells. In contrast, energy deprivation increased net MTX transport in AA8 cells, whereas no change was seen with Pyr R100 cells. Furthermore, while folic acid influx in Pyr R100 and AA8 cells was markedly reduced with energy deprivation, MTX influx was not affected. Provision of glucose and pyruvate to energy-deprived cells resulted in a rapid onset of MTX efflux from parental AA8 cells but not from Pyr R100 cells. Taken together these results indicate that the markedly enhanced net transport of folic acid and MTX in Pyr R100 cells is largely due to the complete loss of exit pump activity. Furthermore, the energy source that sustains the augmented levels of folic acid appears linked to the influx process and is distinct from the energy source that sustains MTX gradients under these conditions. We conclude that the loss of folic acid efflux is an efficient means of augmenting cellular uptake of folate cofactors and subsequent survival on picomolar folate concentrations. This constitutes the first demonstration of the loss of folic acid exporter activity in mammalian cells as a response to lipophilic antifolate selective pressure.

Functional Loss of the Reduced Folate Carrier Enhances the Antitumor Activities of Novel Antifolates with Selective Uptake by the Proton-Coupled Folate Transporter

“…Furthermore, antifolate drug activities are enhanced by decreased intracellular folates resulting from an hRFC genomic deletion (Zhao et al, 2004b,c;Chattopadhyay et al, 2006) or hRFC mutations (Zhao et al, 2000a). Conversely, antifolate effects are reduced by increased intracellular folates resulting from impaired efflux of folic acid (Assaraf and Goldman, 1997) or enhanced folic acid influx by a mutant RFC .…”

Heterogeneous response of different tumor cell lines to methotrexate-coupled nanoparticles in presence of hyperthermia

“…Interestingly, short-time resistances to MTX seem to be related at least to the latter mentioned proteins. 26,[42][43][44][45][46][47][48] Although fundamental knowledge about the mechanisms that are involved in mediating MTX resistance is available, up to now, there is almost no understanding about the feasibility of hyperthermia for overcoming the heterogenic and unfavorable variation in MTX response in tumor cells. Moreover, despite the aforementioned advantages of using MNP for cancer treatment, the potential of MTX-MNP in overcoming the heterogenic cytotoxicity observed for free MTX, especially when combined with hyperthermia, is mostly unknown.…”

Antifolate Resistance Associated with Loss of MRP1 Expression and Function in Chinese Hamster Ovary Cells with Markedly Impaired Export of Folate and Cholate

“…Density was adjusted to 10 7 cells/ml in the same buffer. Transport measurements were performed as described previously (Assaraf and Goldman, 1997). Briefly, after 20-min incubation of cells at 37°C, uptake of [ 3 H]cholic acid (7600-8200 dpm/pmol) at a final concentration of 50 nM was initiated, after which 1-ml samples were drawn at the indicated times and transferred to centrifuge tubes containing 10 ml of ice-cold HEPES-buffered saline.…”

“…Previous studies demonstrated a marked loss of folate export activity in Pyr R100 cells, resulting in a substantial expansion in the intracellular folate pool (Assaraf and Goldman, 1997;Jansen et al, 1999). To explore the basis for the loss of folate efflux function, MRP1 through MRP5 and BCRP expression was examined in the microsomal fraction of wildtype AA8 and Pyr R100 cells (Fig.…”

“…There was marked impairment of folate efflux with enhanced accumulation of physiological folates resulting in decreased activity of pyrimethamine and a variety of other antifolates (Assaraf and Goldman, 1997;Jansen et al, 1999). The current study was undertaken to characterize the basis for the loss of folate exporter function in Pyr R100 cells and to establish how this affected transport of a structurally different organic anion, cholate, an MRP substrate (Henderson et al, 1995;Jedlitschky et al, 1996).…”