FUNCTIONAL-ASPECTS OF MEMBRANE FOLATE RECEPTORS IN HUMAN BREAST-CANCER CELLS WITH TRANSPORT RELATED RESISTANCE TO METHOTREXATE

Two methotrexate (MTX)-resistant human breast-cancer cell lines with i mpaired transport via the reduced folate carrier (RFC), one establishe d in vitro (MTX(R)-ZR-75-1) and another inherently resistant (MDA-231) , were adapted to grow in medium containing 2 nM folic acid. This indu ced the expression of pre viously undetectable membrane folate recepto rs (MFR) to levels of 8.2 and 2.3 pmol/10(7) cells, respectively. Poly merase chain reaction (PCR) quantitation revealed that MFR messenger-R NA levels of the isoform first described in human nasopharyngeal carci noma KB cells (MFR-alpha) were increased in low-folate-adapted MTX(R)- ZR-75-1 cells, whereas placental transcripts (MFR-beta) coincided with MFR-alpha expression in low-folate (LF)-adapted MDA-231 cells. These cell lines were used to study the role of MFR in the uptake and growth -inhibitory effects of five different antifolates with varying affinit ies for MFR: N-10-propargyl-5, 8-dideazafolic acid (CB3717) > 5,10-did eazatetrahydrofolic acid (DDATHF) > N-{5- oxoquinazolin-6-methyl)-amin o]-2-theonyl}-glutamic acid (ZD1694) much greater than MTX > edatrexat e (EDX). Expression of MFR only slightly decreased the resistant pheno type for MTX, EDX, and ZD1694, suggesting that these drugs are not tra nsported intracellularly to cytotoxic concentrations at these levels o f MFR expression. On the other hand, both cell lines became from at le ast 180- to 400-fold more sensitive to growth inhibition by CB3717 and DDATHF, which may be correlated with their high affinity for MFR. The se sensitivity/resistance profiles were largely similar following cell culture in medium containing 1 nM L-leucovorin, a folate with an affi nity for MFR 10-fold lower than that of folic acid, the one exception being the increased sensitivity for ZD1694 seen in the LF-adapted cell s with the highest level of MFR expression (MTX(R)-ZR-75-1), These res ults illustrate that the efficacy of MFR in mediating antifolate trans port and cytotoxicity depends on their affinity for the folate antagon ist, their degree of expression, and the levels of competing folates.