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 Lometrexol (5,10-dideazatetrahydrofolic acid; DDATHF), is a specific inhibitor of glycinamideribonucleosyl (GAR) transformylase with anti-tumour activity in murine and human carcinomas. The cytotoxicity activity of DDATHF was evaluated in vitro in NIH/3T3 cells transfected with human a-folatebinding protein (FBP)
The alpha isoform of the folate receptor (FR) is a 38-KDa glycosylphosphatidylinositol (GPI) protein which mediates the internalization of folates. The FR amino acid sequence has features typical of GPI-linked proteins, including the presence of a hydrophobic carboxyl-terminus, a hinge region, and a stretch of small and uncharged amino acids. Substitution of predicted cleavage/attachment Ser234 with arginine or threonine, or replacement of Gly235 with proline by site-directed mutagenesis had no effect on GPI processing. In fact, CHO cells transfected with each of the three cDNA variants or with FR wild-type showed comparable amounts of phosphatidylinositol-specific phospholipase C-resistant FR in double-determinant radioimmunoassay. Western blot analysis of total cell lysates from all transfectants consistently revealed the 38-KDa FR band. Deletion of residues 233-237 in the amino-terminal portion of the FR cDNA constructs derived by a polymerase chain reaction strategy abrogated GPI processing, with only a small proportion of the FR remaining in the cytoplasm in four of the five clones tested. This finding suggests that FR residues 233-237 are essential in properly juxtaposing the FR hydrophobic domain. Together, these data support the hypothesis that the postulated Ser234 is not the only potential cleavage/attachment site of the alpha isoform of FR.
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