Gene Transfection and Expression of the Ovarian Carcinoma Marker Folate Binding Protein on NIH/3T3 Cells Increases Cell Growth

The glycoprotein gp38 is overexpressed in 90% of ovarian carcinomas and recognized by monoclonal antibodies MOvl8 and MOvl9. This molecule is a high affinity folate binding protein (FBP) and a potential marker for ovarian carcinoma. We have developed a model to investigate the biochemical and biological properties of this folate receptor by transfecting NIH/3T3 cells, which do not endogenously express FBP, with a vector containing the complementary DNA for the gp38 cloned from the ovarian carcinoma cell line IGROV1. The FBP expressed shows features identical to those of the protein produced by IGROV1 cell. The FBP is expressed on the cell membrane in a glycosylphosphatidylinositol-linked form, since it is released by treatment with phosphatidylinositol-specific phospholipase C, and is shed into the culture medium of the NIH/3T3 transfectants. Immunoblot analysis with MAbs MOvl8 and MOvl9 showed that both the glycosylphosphatidylinositol-linked and the soluble FBP migrate at the same apparent molecular weight as the respective IGROV1 proteins. The FBP-transfected NIH/3T3 ceils bound folic acid and internalized about 30-fold more folic acid than mock-transfected cells. Growth analysis revealed that FBP-transfected NIH/3T3 cells like IGROV1 maintained their growth rate after 10 days of culture in medium containing physiological or low folate concentration, and tumors arising after transplanting FBPtNIH/3T3 cells in nude mice were 3-fold heavier than those arising after transplantation of non-FBP-expressing NIH/3T3 cells. These results suggest a correlation between human ovarian carcinoma growth and FBP overexpression. I N T R O D U C T I O N Folic acid and its reduced compounds are essential vitamins for cell growth. Several studies have been shown that the uptake of these vitamins into cells involves membrane protein receptors of two different classes: a membrane carrier which binds reduced folate in the /~M range, and a high-affinity FBP 3, which binds preferentially to oxidized folate and other analogues with an affinity below 1 nM (for a review, see Ref. 1). Folate receptors form clusters organized in closed caveolae (2), and their assembly is maintained by cholesterol (3). Once the receptor-vitamin interaction occurs, the whole complex is internalized, and the vitamin, dissociated from the receptor probably by acidification of the caveolae lumen, is delivered to the cell interior, while the FBP recycled to the cell surface (4). Two distinct FBP forms have been isolated: a membrane and a soluble form. The membrane-bound FBP is anchored to the plasma membrane by a fatty acid linkage, which has been identified in some cells as a GPI tail with a molecular weight of about 40,000 (5). The soluble FBP has been found in human serum, in bovine, goat and human milk, and in some biological fluids (6), as well as in the spent medium of the KB epidermoid carcinoma cell line (7) and the IGReceived 6/11/93; accepted 9/28/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. t This work was partially supported by Associazione Italiana per la Ricerca sul Cancro. To whom requests for reprints should be addressed, at Experimental Oncology E, Istituto Nazionate per lo Studio e la Cura dei Tumori, Via Venezian, 1, 20133 Milan. Italy. 3 The abbreviations used are: FBP, folate binding protein; eDNA, complementary DNA; IFBP, transfected FBP; GPI, glycosylphosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; DDIRMA, double-determinant immuno radiometric assay; FBS, fetal bovine serum; FBP-t, FBP transfected. ROV1 ovarian carcinoma cell line (8). Although the biological role of the membrane form is fairly well known, the role of the soluble form is not well defined. Efforts to determine a possible precursor-product relationship between the two forms have suggested the possible involvement of proteases in the generation of the hydrophilic soluble FBP from the membrane form (1), and the GPI-linked FBP has been identified in human placenta and in rat, human, and porcine kidney (9). Two monoclonal antibodies, M O v l 8 and MOv19, derived in mice immunized with a crude membrane preparation from an ovarian carcinoma specimen (10), recognize a glycoprotein, gp38, which is overexpressed in 90% of ovarian carcinomas. Purification of the protein and cloning of the cDNA from IGROV1 have shown that this protein is a FBP (11, 12). Use of these two monoclonal antibodies has identified the FBP in some normal tissues (13), in particular, the fallopian tube and choroid plexus which express the highest levels of FBP compared to other normal tissues tested (13-15). In addition to ovarian carcinomas, brain tumors also express FBP. However, ovarian carcinomas homogeneously overexpress FBP on the membrane, and a soluble form has been detected in ascitic fluids and in the sera of ovarian carcinoma patients (15). In order to study the precursor-product relationship between the membrane and the soluble FBP, and the possible biological role of FBP overexpression in ovarian carcinoma, we analyzed the biochemical and biological characteristics of the murine NIH/3T3 fibroblasts transfected with IGROV1 cDNA with a focus on the possible influence of FBP expression on cell growth. M A T E R I A L S AND M E T H O D S Cell Lines and Reagents. IGROV1 cells (J. Brnard, Institute G. Roussy, Villejuif, France) were cultured in standard RPMI 1640 containing 2.3 /xra folate (Irvine Scientific, Santa Ana, CA) or in folate-depteted RPMI 1640 with 20 nu or less than t nM fo|ate (GIBCO, Paisley, United Kingdom) plus 5% Ft3S (Irvine Scientific). NII-I/3T3 cells were cultured in standard Dulbecco's modified Eagle's medium containing 9.2 /zM folate (Boehringer-Manheim, Germany) supplemented, before transfection, with 10% calf serum (Colorado Serum Company, Denver, CO), or, upon transfection, with 5% FBS and 800 Ixg/ml of geneticin G418 sulfate (GIBCO). Folate-depleted medium consisted of Dulbecco's modified Eagles medium with or without 20 nr~ folic acid (GIBCO) plus 5% FBS (<1 n~ folate). One % of penicillin-streptomycin was added to each medium. Antibodies. Murine monoclonal antibodies MOvl8 and MOvl9 were purified as described (10). Anti-gp38 rabbit antiserum was used as total purified IgG (11). Vector Construction. Human FBP cDNA (11), named FBP-31, was cloned into the EcoRI sites of pBluescript II KS +/(Stratagene, La Jolla, CA), to generate the HindIII/XbaI sites necessary for cloning in the expression vector. pBluescript II KS +/was amplified in Escherichia coli XL-1 Blue. The HindlII/XbaI FBP-31 insert was subsequentIy cloned into the pcDNAI/neo vector and amplified in Escherichia coli MC 1061/P3 (Invitrogen, San Diego, CA). Transcription of the relevant cDNA was under the control of the Citomegalovirus promoter. Transfection. Cells were transfected using the DNA-calcium phosphate coprecipitation technique essentially as described (16). Briefly, NIH/3T3 cells were harvested by trypsinization and replated at a density of 5 • 105 in 90-ram Petri dishes in Dulbecco's modified Eagle's medium plus 10% FBS. After 24 h, the medium was replaced and 3 h later, 10 tzg of either the vector containing