ROLE OF FOLATE RECEPTOR AND REDUCED FOLATE CARRIER IN THE TRANSPORT OF 5-METHYLTETRAHYDROFOLIC ACID IN HUMAN OVARIAN-CARCINOMA CELLS

Folate receptor-alpha (FR-alpha) is generally over-expressed in non-mu cinous human ovarian carcinomas. The meaning of FR-alpha over-expressi on and its role in the 5-methyltetrahydrofolic acid (N-5-CH3-H(4)PteGl u) transport in such tumors is not clear, especially compared with the reduced folate carrier (RFC), the other known folate transporter. In this study, we analyzed molecular FR-alpha and RFC expression in 16 ov arian carcinoma tissues and in 5 ovarian carcinoma cell lines using co mpetitive PCR. Co-expression of the 2 transporters was found both in v ivo and in vitro. FR-alpha mRNA expression in the cell lines was in go od agreement with the corresponding protein expression evaluated by ce llular folic acid binding and immunofluorescence analysis, using a spe cific monoclonal antibody (MAb) (MOv18). Moreover, RFC mRNA expression levels were consistent with the selective cellular binding of N-hydro xysuccinimide of [H-3]-methotrexate (NHS-MTX). The 5 ovarian carcinoma cell lines (IGROV-1, SW-626, SKOV-3, OVCAR-3 and OAW-42), grown at ph ysiological N-5-CH3-H(4)PteGlu concentrations (20 nM) and expressing F R-alpha and RFC levels superimposable to those observed in vivo, were used as in vitro cellular model to evaluate the different contribution of FR-alpha and RFC to the transport of N-5-CH3-H(4)PteGlu. The cytop lasmic N-5-CH3-[H-3]H(4)PteGlu accumulation observed in each cell line was approximately linear over 4 hr of incubation, but there was no co rrelation between the rate of folate internalization and FR-alpha and RFC expression levels. Furthermore, the selective inhibition of FR-alp ha and RFC functionality allowed us to distinguish their differential role on the overall N-5-CH3-[H-3]H(4)PteGlu intracellular delivery. Tr eatment with the N-hydroxysuccinimide of folic acid, which blocks FR-a lpha activity, showed only a partial inhibition (about 20%) of folate internalization in all the cell lines. In contrast, the inhibition of RFC by NHS-MTX, under conditions that did not affect FR-alpha function ality, generally reduced folate accumulation by more than 70%. Only on e cell line (IGROV-1) showed a comparable contribution of the 2 transp ort systems. Our findings suggest that in ovarian carcinomas, in spite of its over-expression, FR-alpha generally plays a minor role in N-5- CH3-H(4)PteGlu transport compared with RFC. (C) 1998 Wiley-Liss, Inc.