SIMULTANEOUS ACTIVITY OF 2 DIFFERENT MECHANISMS OF FOLATE TRANSPORT IN OVARIAN-CARCINOMA CELL-LINES

We investigated whether the folate receptor alpha-isoform (FR alpha), which is overexpressed on ovarian carcinoma cells, is functionally act ive in internalizing the physiological form of folate, 5-methyl tetrah ydrofolate (THF). Six ovarian tumor cell lines, expressing different l evels of FR alpha (COR much greater than OVCAR3 > IGROV1 > OVCAR3 > SK OV3 > OVCAR5), were maintained in folate-depleted medium and internali zation of 10 nM evaluated as acid-resistant radioactivity at 0 degrees and 37 degrees C. The amount of 5-methyl[H-3]THF present in this frac tion was not strictly related to the number of membrane receptors, sin ce even cell lines with low FR alpha expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0 degrees C, at 37 degrees C the internaliz ed fraction showed a slow and constant increase, until 4 h. At this ti me, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl [H-3]THF, which best ensures receptors saturation on cells with highes t FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, th e increase of the uptake was more pronounced, particularly in SKOV3 ce lls. These results, together with the accumulation curves of folic aci d (FA) and 5-methylTHF at 37 degrees C, suggested the presence of a mo lecule on ovarian carcinoma cells with high affinity for reduced folat es, possibly a reduced folate carrier (RFC). Measurement of radioactiv ity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-meth yl[H-3]THF was translocated to the cytosol and, despite differences in membrane levels of FR alpha expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively blo ck FR alpha or RFC activity showed a differential sensitivity of the t wo pathways depending on the cell line examined. Internalization was m ore consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FR alpha activity, e.g., incubation with excess FA and p hosphatidylinositol specific phospholipase C, whereas Probenecid, whic h preferentially inhibits the carrier-mediated pathway, showed a stron g inhibitory effect on both cell lines. These findings suggest that th e internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FR alpha, but another transport route, with features characteristic for RFC, is functional and participates i n relate uptake. J. Cell. Biochem. 65:479-491. (C) 1997 Wiley-Liss. in c.