Contrasting effects of oncogene expression on two carrier-mediated systems internalizing folate compounds in Fisher rat 3T3 cells

Abstract

Folate compound transport into Fisher rat 3T3 (FR3T3) cells at physiological pH occurs predominantly by an acid pH‐dependent, mobile carrier system. However, influx of [³H]MTX by this system is 3–4‐fold higher at pH 6 than at pH 7.5, the optimum for RFC‐1–mediated folate compound transport. This acid pH dependency reflects an alteration of influx V_max rather than of influx K_m in these cells at different pH. Acid pH‐dependent folate compound transport interacts effectively with MTX, 5ℓLCHO‐folateH₄, 5ℓLCH₃‐folateH₄ and folic acid as permeants (influx Ki = 2.7–5.3 μM). The relative inhibition of influx of [³H]MTX by the organic anions, probenecid, and PO₄ was different than for RFC‐1 mediated influx. The folate requirements for growth in culture of FR3T3 cells and cytotoxicity of MTX compared to L1210 cells reflects the interactions of these folate compounds with acid pH‐dependent folate transport. 5ℓLCHO‐folateH₄ and PO₄ act as exchange anions for this system but their transpositioning has variable effects on transport. 5ℓLCHO‐folateH₄ inhibits influx (decelerative equilibrium exchange) but stimulates efflux of [³H]MTX (accelerative equilibrium exchange) while PO₄ inhibits efflux. In FR3T3 cells transfected with cmyc and Hras, influx V_max for [³H]MTX is downregulated 4‐fold and 9‐fold, respectively. At the same time, RFC‐1 expression, which is detectable in FR3T3 cells at the level of its mRNA and RFC‐1 mediated folate compound transport, is increased 3–5‐fold in these transfectants. The increase in RFC‐1 expression in FR3T3Hras cells appears to result from a higher rate of transcription of the gene in these cells as determined by a luciferase reporter gene assay of RFC‐1 promoter activity. This downregulation of the acid pH dependent system and concomitant upregulation of the RFC‐1 mediated system markedly altered pH dependency for influx of [³H]MTX in these transfectants compared to that seen in untransfected cells. We conclude that the major route for internalization at a physiological pH of folate compounds in FR3T3 cells is by an acid pH‐dependent carrier‐mediated system independent of RFC‐1 expression and is downregulated by oncogene expression. J. Cell. Physiol. 184:364–372, 2000.