Ovarian-carcinoma cell lines (OVCAR3, IGROVI, OVCA432, SW626 and SKOV3), grown in standard medium containing supra-physiological (2.3 microM) folate concentration, display different levels of reactivity with the anti-folate-binding-protein (FBP) monoclonal antibody MOv18, which recognizes the alpha-isoform of the protein. Gel-filtration and absorption experiments indicated that on IGROVI cells this molecule accounts for all folic-acid binding at nanomolar concentrations. The aim of the study was to investigate the effect of extracellular folate levels on cells adapted to grow in medium containing physiological folate concentration (20 nM). By the ternary complex assay, all cell lines showed a marked depletion of intracellular reduced folates, compared with those in standard folate medium. The monitoring of FBP by MOv18 showed on IGROVI cells a transient up-regulation of the protein, whereas on the other cell lines, except SKOV3, no changes were detected. These data suggest that in these cells further over-expression of the molecule cannot generally be induced by lowering the extracellular folate concentration. On SKOV3, Scatchard analysis of 125I-MOv18 binding, as well as the evaluation of total folate binding capacity, showed a 2- to 3-fold stable increase of FBP expression after long-term growth in low-folate medium. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated in these cells a 1.5-fold increase in alpha-FBP mRNA. SKOV3 cells, maintained in vitro in medium containing supraphysiological and physiological (i.e., low-folate) concentrations were injected into nude mice. Weight differences, though not statistically significant, were observed in favour of low-folate-derived tumors. Immunohistochemical and immunochemical analysis of the tumor samples showed that in SKOV3 cells the receptor modulation can also be induced by restoring the physiological folate concentration in vivo.

We investigated whether the folate receptor alpha-isoform (FR alpha), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form et folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FR alpha (COR > > OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0 degree and 37 degrees C. The amount of 5-methyl[1H]THF present in this fraction was not strictly related to the number of membrane receptors, since 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 degree C, at 37 degrees C the internalized fraction showed a slow and constant increase, until 4 h. At this time 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[3H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37 degrees C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[3H]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 block FR alpha or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FR alpha activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the 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 in folate uptake.

We investigated whether the folate receptor alpha-isoform (FR alpha), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form et folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FR alpha (COR > > OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0 degree and 37 degrees C. The amount of 5-methyl[1H]THF present in this fraction was not strictly related to the number of membrane receptors, since 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 degree C, at 37 degrees C the internalized fraction showed a slow and constant increase, until 4 h. At this time 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[3H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37 degrees C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[3H]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 block FR alpha or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FR alpha activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the 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 in folate uptake.

Summary It has been suggested that sensitivity of ovarian carcinomas to cisplatin is in part related to an endogenous folate deficiency. In this work, we investigated whether overexpression of the folate-binding protein (FBP), a receptor involved in folate transport, might be associated with cisplatin sensitivity. The results obtained on a panel of ten ovarian carcinoma cell lines that overexpress different levels of the FBP showed a statistically significant relationship between FBP overexpression and cisplatin responsiveness, with the most sensitive cell lines expressing higher FBP levels on their membrane than the less sensitive ones. The relationship was observed both in cells growing in standard medium-containing high-folate concentrations (2.3 gM) and in cells adapted to growth in low-folate (20 nM) medium. Analysis of two cisplatin-resistant cell lines derived from the cisplatin-sensitive IGROV1 ovarian carcinoma cell line indicated that resistance was associated with a significant decrease in FBP expression. However, the receptor does not appear to be directly responsible for drug sensitivity per se as different cell lines transfected with FBP cDNA did not become more sensitive to the drug. Together, the data suggest the possible predictive value of FBP in ovarian carcinoma, as higher levels of expression can be indirectly but significantly associated with increased drug sensitivity.Keywords: ovarian carcinoma; folate receptor; cisplatin Platinum-derived compounds have occupied a dominant place in cancer therapy, particularly for the treatment of ovarian malignancies (Rosenberg, 1985;Ozols, 1992). Unfortunately, not all tumours are responsive, and initially sensitive tumours often become resistant in a short time Ozols et al, 1991).Various mechanisms have been proposed to contribute to cisplatin resistance, including altered drug accumulation (Loh et al, 1992;Nakagawa et al, 1993;Jekunen et al, 1994;Misawa et al, 1995), enhanced drug detoxification by elevated metallothionein or glutathione levels (Andrews et al, 1987;Morrow et al, 1990;Tedeschi et al, 1990;Mistry et al, 1991), enhanced DNA repair capability (Masuda et al, 1988;Eastman et al, 1988) or upregulation of specific biochemical pathways . Concerning, in particular, the last possibility, an association between cisplatin resistance and changes in folate metabolism has been observed. Indeed, cisplatin can stimulate endogenous methionine and folate metabolism (Gross et al, 1986;Shionoya et al, 1986;Scanlon et al, 1989a). Enhanced 5,10-methylenetetrahydrofolate (5,10-methylene THF) and THF pools and enhanced gene expression of enzymes of the dTMP synthase cycle, which is the sole source of de novo dTMP, have been observed in drug-resistant cells (Scanlon et al, , 1989bNewman et al, 1988;Lu et al, 1988 Correspondence to: S Miotti increased dTMP synthase activity might be required for the repair of cisplatin-induced DNA damage.Previous reports have shown that the 38-KDa folate-binding protein (FBP) is overexpressed in a majority of non-mucinous ovaria...

The chromosomal localisation of the human gene coding for peroxisomal 3-oxoacyl-CoA thiolase (ACAA) was determined by human-hamster somatic cell hybrids and fluorescence in situ hybridisation, using cDNA and genomic probes, respectively. The results allowed an exclusion of the previously suggested presence of a second site for ACAA on chromosome 11 and an assignment of the gene to a single chromosome band (3p22).