Contrasting effects of oncogene expression on two carrier-mediated systems internalizing folate compounds in Fisher rat 3T3 cells.
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.
Program of Molecular Pharmacology and Experimental Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
J M
JM
Kühnel
J H
JH
Chiao
F M
FM
Sirotnak
D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism*; D015870:Gene Expression; D016259:Genes, myc; D011905:Genes, ras; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007939:Leukemia L1210; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism; D051381:Rats; D058978:Reduced Folate Carrier Protein; D014162:Transfection
D002352:Carrier Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; D005492:Folic Acid; D008727:Methotrexate