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By immunizing a BALB/c mouse with a human ovary-carcinoma cell line (IGROV1), grown intraperitoneally in nude mice, a monoclonal antibody (MAb), designated MAR6, was produced and characterized. Immunofluorescence on the immunizing cell line showed a specific labelling by MAR6 at the cell-to-cell contact points. In addition, MAR6 was found to immunoprecipitate the alpha 6 beta 4 integrin complex. Competition tests with MAbs anti-alpha 6, anti-beta 4, anti-beta 1 sub-units demonstrated that the recognized sub-unit is alpha 6. Indirect immunofluorescence on various cell lines gave MAR6 as positive only on alpha 6-positive lines (IGROV1, OVCAR3, SW626, SKOV3, ME4405, Calu3, N592, MDA468, A431 and HT29). Moreover, on IGROV1 and OVCAR3 ovary-carcinoma cells, which normally grow either adhering to the culture flask or forming clumps in suspension in the medium, MAR6 selectively stained the connection points between the cells in clumps, where, in the same position, the presence of the beta 4 sub-unit, laminin and fibronectin was detected. On the contrary, the beta 1 sub-unit was distributed over the whole cell membrane. The same pattern of labelling by these MAbs was observed in 2 cases of ovarian-carcinoma cells present in ascitic fluids obtained from patients. Immunoperoxidase tests performed on cryosections of various normal tissues showed specific reactivity of MAR6 on basal or basolateral membranes of epithelial cells. On cryosections of ovarian tumors, MAR6 reactivity correlated with the degree of tumor differentiation. Indeed, in benign and well-differentiated tumors, a strong basal or basolateral labelling only of cells surrounding the neoplastic nodules was found. On the contrary, on undifferentiated tumors the inner part of the tumor nodules was also progressively labelled, whereas the staining on the border was weak and discontinuous as a result of the alpha 6 sub-unit dispersion on the tumor cell surface.

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.

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...

Eighteen years after the discovery of monoclonal antibodies (MAbs), the debate as to whether these reagents have fulfilled their promise must now be addressed. When MAbs were studied as tools for diagnosis and therapy, problems arose regarding the reagents themselves and the recognized target antigens. Because most MAbs are of murine origin, they usually evoke an immune response in human patients, which reduces their effectiveness. Moreover, their bulky size and dispersal via the blood prevent most of the injected antibodies from substantially penetrating solid, poorly vascularized tumors. However, MAbs that are adequately selected and manipulated to circumvent these problems have been found to successfully image tumors and to be therapeutically active.