Contrasting effects of oncogene expression on two carrier-mediated systems internalizing folate compounds in Fisher...

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 [(3)H]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-folateH(4), 5lLCH(3)-folateH(4) and folic acid as permeants (influx Ki = 2.7-5.3 microM). The relative inhibition of influx of [(3)H]MTX by the organic anions, probenecid, and PO(4) 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-folateH(4) and PO(4) act as exchange anions for this system but their transpositioning has variable effects on transport. 5lLCHO-folateH(4) inhibits influx (decelerative equilibrium exchange) but stimulates efflux of [(3)H]MTX (accelerative equilibrium exchange) while PO(4) inhibits efflux. In FR3T3 cells transfected with cmyc and Hras, influx V(max) for [(3)H]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 [(3)H]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.