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

Autor: Jean‐Marc Kühnel, Judy H. Chiao, Francis M. Sirotnak
Rok vydání: 2000
Předmět:
Zdroj: Journal of Cellular Physiology. 184:364-372
ISSN: 1097-4652
0021-9541
DOI: 10.1002/1097-4652(200009)184:3<364::aid-jcp11>3.0.co;2-n
Popis: 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 Vmax rather than of influx Km 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 Vmax 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. © 2000 Wiley-Liss, Inc.
Databáze: OpenAIRE
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