EGFR/MEK/ERK/CDK5-dependent integrin-independent FAK phosphorylated on serine 732 contributes to microtubule depolymerization and mitosis in tumor cells.

FAK is a non-receptor tyrosine kinase contributing to migration and proliferation downstream of integrin and/or growth factor receptor signaling of normal and malignant cells. In addition to well-characterized tyrosine phosphorylations, FAK is phosphorylated on several serines, whose role is not yet clarified. We observed that phosphorylated FAK on serine 732 (P-FAKSer732) is present at variable levels in vitro, in several melanoma, ovarian and thyroid tumor cell lines and in vivo, in tumor cells present in fresh ovarian cancer ascites. In vitro P-FAKSer732 was barely detectable during interphase while its levels strongly increased in mitotic cells upon activation of the EGFR/MEK/ERK axis in an integrin-independent manner. P-FAKSer732 presence was crucial for the maintenance of the proliferation rate and its levels were inversely related to the levels of acetylated α-tubulin. P-FAKSer732 localized at the microtubules (MTs) of the spindle, biochemically associated with MTs and contributed to MT depolymerization. The lack of the phosphorylation on Ser732 as well as the inhibition of CDK5 activity by roscovitine impaired mitotic spindle assembly and correct chromosome alignment during mitosis. We also identified, for the first time, that the EGF-dependent EGFR activation led to increased P-FAKSer732 and polymerized MTs. Our data shed light on the multifunctional roles of FAK in neoplastic cells, being involved not only in integrin-dependent migratory signaling but also in integrin-independent MT dynamics and mitosis control. These findings provide a new potential target for inhibiting the growth of tumor cells in which the EGFR/MEK/ERK/CDK5 pathway is active.

Ligand-dependent EGFR activation induces the co-expression of IL-6 and PAI-1 via the NFkB pathway in advanced-stage epithelial ovarian cancer.

The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, is expressed in up to 70% of epithelial ovarian cancers (EOCs), where it correlates with poor prognosis. The majority of EOCs are diagnosed at an advanced stage, and at least 50% present malignant ascites. High levels of IL-6 have been found in the ascites of EOC patients and correlate with shorter survival. Herein, we investigated the signaling cascade led by EGFR activation in EOC and assessed whether EGFR activation could induce an EOC microenvironment characterized by pro-inflammatory molecules. In vitro analysis of EOC cell lines revealed that ligand-stimulated EGFR activated NFkB-dependent transcription and induced secretion of IL-6 and plasminogen activator inhibitor (PAI-1). IL-6/PAI-1 expression and secretion were strongly inhibited by the tyrosine kinase inhibitor AG1478 and EGFR silencing. A significant reduction of EGF-stimulated IL-6/PAI-1 secretion was also obtained with the NFkB inhibitor dehydroxymethylepoxyquinomicin. Of 23 primary EOC tumors from advanced-stage patients with malignant ascites at surgery, 12 co-expressed membrane EGFR, IL-6 and PAI-1 by immunohistochemistry; both IL-6 and PAI-1 were present in 83% of the corresponding ascites. Analysis of a publicly available gene-expression data set from 204 EOCs confirmed a significant correlation between IL-6 and PAI-1 expression, and patients with the highest IL-6 and PAI-1 co-expression showed a significantly shorter progression-free survival time (P=0.028). This suggests that EGFR/NFkB/IL-6-PAI-1 may have a significant impact on the therapy of a particular subset of EOC, and that IL-6/PAI-1 co-expression may be a novel prognostic marker.

E-cadherin directly contributes to PI3K/AKT activation by engaging the PI3K-p85 regulatory subunit to adherens junctions of ovarian carcinoma cells.

E-cadherin (cadh), a member of a family of integral membrane glycoproteins that represent the major component of adherens junctions (AJs), mediates cell-cell adhesion through the calcium-dependent homophilic interaction of its extracellular domain. Metastatic human carcinomas frequently lose E-cadh expression, whereas epithelial ovarian cancer (EOCs) maintain properties characteristic of Müllerian epithelium during tumor progression, including E-cadh expression. Here, we examined the potential role of cell-cell contacts in EOCs through E-cadh homophilic interactions in PI3K/AKT activation whose altered signaling has been implicated in EOC pathogenesis. We show that E-cadh is predominantly expressed at cell-cell contacts and its functionality is necessary and sufficient for the activation of the PI3K/AKT pathway. E-cadh knockdown and phosphoinositide-3-kinase (PI3K) inhibition complement each other in impairing cell-cycle progression and proliferation of ovarian carcinoma cells. E-cadh is stably bound to the PI3K complex, and the de novo formation of E-cadh/beta-catenin complexes following calcium deprivation and subsequent calcium restoration recruits the PI3K p85 subunit to the site of the cell-cell contacts. The finding that E-cadh-mediated AJ formation contributes to PI3K/AKT activation in EOC cells by a mechanism that appears to be restricted to these cells provides the underpinning for therapeutic strategies that exploit PI3K inhibition to halt EOCs.

Evidence for nonspecific adsorption of targeted retrovirus vector particles to cells.

The ability to specifically target a cell-type is important for the development of vectors for in vivo gene therapy. In order to produce retrovirus vectors targeting ovarian cancer cells, which specifically overexpress alpha folate receptor (alphaFR), a single chain antibody was fused as an N-terminal extension of the ecotropic and amphotropic murine leukemia virus (MLV) envelope glycoproteins. Vector particles bearing the modified glycoproteins were produced and analysed. Although conventional FACS studies indicated that viral particles bearing the modified Env could bind to ovarian cancer cells, targeted infection was not achieved. The initial step of virus-cell interaction was further studied using an immunofluorescence technique, which allows visualisation of single retrovirus particles. Vectors bearing chimeric or wild-type glycoproteins bound equally well to cells with or without the targeted receptor, although soluble chimeric glycoproteins bound specifically to FBP. Our results indicate that the incorporation of specific ligands to the virus envelope does not necessarily result in significant enhancement of vector particle binding. A similar interaction was also observed using Env-defective virus particles, suggesting that cellular factors incorporated into the lipid envelope play a dominant role in promoting initial adsorption of virus particles to cells. Significant implications arise from these observations on the interpretation of previous reports on 'targeted' vectors, and for the development of vectors for in vivo gene therapy protocols.

Exon 3 of the alpha folate receptor gene contains a 5' splice site which confers enhanced ovarian carcinoma specific expression.

The human folate receptor (FR) is overexpressed in ovarian carcinoma. FR transcripts are heterogeneous due to the use of two promoters, P1 and P4, and alternative splicing of exon 3. RNase protection assay and RT-PCR revealed higher levels of the transcripts that include exon 3 in lines and specimens from ovarian carcinoma. A P1-chloramphenicol acetyltransferase (CAT) construct containing exon 3 demonstrated efficient reporter expression only in ovarian carcinoma. 5' and 3' deleted variants of the P1-CAT construct were analyzed by RT-PCR of the exogenous transcripts and reporter activity. A 5' splice site and 35 bp downstream intronic region of exon 3 appeared to regulate enhanced FR expression in ovarian carcinoma.

Functional effect of point mutations in the alpha-folate receptor gene of CABA I ovarian carcinoma cells.

The alpha-folate receptor (alpha FR) is overexpressed in 90% of nonmucinous ovarian carcinomas. In addition to the known role of alpha FR binding and mediating the internalization of folates, functional interaction of alpha FR with signaling molecules was recently shown. To identify a model to study the role of alpha FR in ovarian carcinoma, we characterized the alpha FR gene in the ovarian carcinoma cell line CABA I in comparison to a reference line, IGROV1. In CABA I cells, Northern blot analysis revealed an alpha FR transcript of the expected length and FACS analysis indicated receptor expression on the cell membrane; however, RNase protection assay revealed no specific signals. Southern blot and genomic PCR analysis suggested the presence of a rearrangement(s) involving the 5' region of the gene in CABA I cells as compared to IGROV1 cells. Cloning and sequencing of CABA I alpha FR cDNA revealed several point mutations. The partitioning of alpha FR in membrane microdomains from CABA I cells and its association with regulatory molecules was comparable to that of IGROV1 cells. By contrast, the alpha FR expressed on the CABA I cell membrane bound folic acid with lower affinity, and ectopic expression of the corresponding cDNA in CHO cells confirmed impaired folic acid binding. Thus, CABA I cells may provide a tool to delineate functional domains of the alpha FR.

Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to alpha-folate receptor overexpression.

Caveolin (cav-1) and the GPI-anchored alpha-folate receptor (alphaFR) are membrane proteins both found associated to caveolar structures. Several studies in tumor cells independently reported cav-1 downregulation and alphaFR overexpression. Here we analysed the expression of the two molecules in normal and tumor ovarian samples derived from fresh specimens and from cultured cell lines. Whereas normal ovary surface epithelial cells displayed only cav-1 expression, ovarian tumor surgical samples and cell lines (COR, IGROV1, OVCAR3 and OVCA432) displayed high alphaFR and low-level or no cav-1 expression, except those cell lines (SKOV3 and SW626) with the lower alphaFR expression. SKOV3, but not two alphaFR-negative non-ovarian cell lines, exhibited down-regulation of cav-1 expression following stable alphaFR cDNA transfection. Conversely, cav-1 transfection in IGROV1 cells led to downregulated alphaFR expression, together with formation of caveolar structures and reduction of growth capability. Moreover, cav-1 expression was induced in IGROV1 cells by transfection with intracellular anti-alphaFR antibodies to downmodulate alphaFR expression. In cav-1 transfected cells, transcriptional activity of the alphaFR-specific promoter P1 was reduced by 70% and an additional specific DNA-protein complex was identified by gel-shift assay, indicating that cav-1 expression influences alphaFR gene transcription. Together these results support the notion that alphaFR and cav-1 protein expression is reciprocally regulated in ovary cancer cells.

Interaction of folate receptor with signaling molecules lyn and G(alpha)(i-3) in detergent-resistant complexes from the ovary carcinoma cell line IGROV1.

Using as a model the ovary carcinoma cell line IGROV1, we analyzed the partitioning of the glycosyl-phosphatidylinositol-anchored folate receptor into lipid rafts based on its relative detergent insolubility, with a focus on physically and functionally associated signaling molecules. A variable amount (40-60%) of folate receptor was found in low-density Triton X-100 insoluble complexes together with subunits of heterotrimeric G-proteins and the src-family non-receptor tyrosine kinases p53-56 lyn. In the same fraction the structural component of caveolae, caveolin, was not detected at the protein level, although the corresponding mRNA was detected in trace amounts. Comodulation of folate receptor and signalling molecules was observed in the detergent-insoluble complexes during cell proliferation or induced by phosphatidylinositol-specific phospholipase C treatment or by interaction with anti-folate receptor monoclonal antibodies. Moreover, complexes of folate receptor, lyn and the G(&agr;)(i-3) subunit were immunoprecipitated using either anti-folate receptor or anti-lyn antibodies. In vitro kinase assay of the immunoprecipitates revealed stimulation of phosphorylation of common and specific proteins. In particular, the p53 form of lyn appeared to be enriched and phosphorylated in the anti-folate receptor MOv19 monoclonal antibody immunoprecipitate, whereas a 40 kDa band common to anti-folate receptor and anti-lyn immunoprecipitates was the phosphorylated form of the G(&agr;)(i-3) subunit. These findings point to the functional interaction between folate receptor and associated signaling molecules.

DNA vaccination against the ovarian carcinoma-associated antigen folate receptor alpha (FRalpha) induces cytotoxic T lymphocyte and antibody responses in mice.

Human folate receptor alpha (FRalpha) is a folate-binding protein that is selectively overexpressed in ovarian carcinoma and has been regarded as a suitable target antigen for immunotherapy purposes. To study the possible use of this antigen in DNA vaccination, FRalpha cDNA was ligated into the VR1012 (Vical) expression vector under the transcriptional control of the cytomegalovirus promoter. A total of 100 microg of purified plasmid DNA was injected intramuscularly in BALB/c mice three times at 14-day intervals. At 10 days after the second injection, the sera of the animals (100%) displayed significant antibody titers (by indirect immunofluorescence and fluorescence-activated cell sorter analysis) against syngeneic C26 cells transduced with FRalpha, but not against unmodified C26 cells. Immunoglobulin G2a was the predominant isotype. In addition, specific cytotoxic T lymphocyte activity against FRalpha-transduced C26 cells could be detected in splenocytes from all immunized animals. Coinjection of a plasmid containing interleukin-2 cDNA increased both antibody titers and cytotoxic T lymphocyte activity. Challenge by subcutaneous injection with FRalpha-transduced C26 cells (performed 10 days after the third injection) showed a statistically significant delay in tumor growth. Vaccination with the FRalpha and interleukin-2 cDNA mixture, which was performed after an intravenous injection of FRalpha-transduced cells, enhanced the mean survival time and reduced the number of lung metastases, thus suggesting that such vaccination is effective even against preexisting tumor cells.

Molecular requirements for attachment of the glycosylphosphatidylinositol anchor to the human alpha folate receptor.

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.

Simultaneous activity of two 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 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.

Role of membrane folate-binding protein in the cytotoxicity of 5,10-dideazatetrahydrofolic acid in human ovarian carcinoma cell lines in vitro.

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 alpha-folate-binding protein (FBP) complementary DNA to examine the role of the receptor. In FBP-transfected NIH/3T3 (FBP-tNIH/3T3) cells, which internalised about three times more 5-methyltetrahydrofolic acid than the mock-transfected cells, the cytotoxtic potential of DDATHF showed a clear increase. Subsequently, we analysed four ovarian carcinoma cell lines (OVCAR3, IGROV1, SKOV3, and SW626) expressing different amounts of FBP. Cells were conditioned to grow in medium depleted of folic acid then tested by MOv18 and folic acid binding. Only SKOV3 and SW626 cells grown in folic acid-depleted medium showed increased FBP expression, about 3- and 8-fold respectively. The cytotoxic potential of DDATHF was evaluated by a standard clonogenic assay. In a medium containing 2.27 microM folic acid the DDATHF IC50 values were 50 nm on OVCAR3, 500 nM on SW626 and 1000 nM on IGROV1. In folic acid-free medium IC50 values were 2 nM on OVCAR3 and Sw626 and 40 nM on IGROV1. Only on SKOV3 cells was DDATHF cytotoxicity the same regardless of the amount of folic acid in the medium (IC50 8 nM). Thus, DDATHF did not inhibit the growth of IGROV1 cells depleted of folic acid after stripping FBP with phosphatidylinositol-phospholipase C, even at a dose toxic for cells constitutively expressing FBP. Although FBP expression is certainly one of the parameters affecting drug toxicity, taken alone it is not a sufficiently reliable predictor of cancer cell sensitivity to DDATHF.

Growth of ovarian-carcinoma cell lines at physiological folate concentration: effect on folate-binding protein expression in vitro and in vivo.

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.

Unidirectional potentiation of binding between two anti-FBP MAbs: evaluation of the involved mechanisms.

The monoclonal antibody MOv19 directed to a folate binding protein shows temperature-dependent potentiation of binding of the noncompeting monoclonal antibody MOv18 to the relevant antigen, but the mechanism involved in this phenomenon had remained unclear. Use of chimeric versions of both monoclonal antibodies and the F(ab')2 and Fab fragments of MOv19 revealed an increment in MOv18 binding in all combinations irrespective of the origin of the Fc portion of the monoclonal antibody. The potentiating effect of bivalent MOv19 fragments on 125I-MOv18 binding was similar to that of the entire monoclonal antibody and occurred at saturating concentrations of both reagents at which monovalent binding prevails. Similarly, the monovalent fragment also induced a significant increase in MOv18 binding. However, the potentiation occurred only at very high concentrations of antibody fragment. Homologous inhibition was drastically reduced using MOv19 Fab fragment, suggesting a low binding stability of the monovalent reagent. Immunoblotting analysis and binding in the presence of exogenous purified folate binding protein indicated a cross-linking between soluble and cell surface molecules mediated by the bivalent monoclonal antibodies. The extent of the increase in MOv18 binding at 0 degrees C with high amounts of exogenous folate binding protein was lower than that obtained at 37 degrees C in the absence of added molecule. Release of 125I-MOv18 from the cell surface was significantly higher in the absence of MOv19 than in its presence.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene transfection and expression of the ovarian carcinoma marker folate binding protein on NIH/3T3 cells increases cell growth in vitro and in vivo.

The glycoprotein gp38 is overexpressed in 90% of ovarian carcinomas and recognized by monoclonal antibodies MOv18 and MOv19. 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 MOv18 and MOv19 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 cells 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 FBP-tNIH/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.

Isolation and biochemical characterization of the soluble and membrane forms of folate binding protein expressed in the ovarian carcinoma cell line IGROV1.

The human ovarian carcinoma cell line, IGROV1, produces two forms of folate binding protein (FBP), the membrane form that is anchored to the cell surface by a glycosylphosphatidylinositol tail and the soluble form that is shed into the tissue culture medium. Both forms are recognized by the monoclonal antibodies MOv18 and MOv19. Here we describe their purification and biochemical characterization. The purified soluble protein appeared as a single band with an apparent Mr of 36 kDa after SDS-PAGE, whereas the membrane form appeared as a single band with an apparent Mr of 38 kDa. The size difference between the two forms of FBP was confirmed by gel filtration of both the native and the N-glycanase-treated proteins. Both purified proteins had equal capacity to bind folic acid. The immunological cross-reactivity and the folic acid binding capability of the FBPs extracted from IGROV1 gave more evidence of the possible existence of a precursor-product relationship between them.

Characterization of a mouse-human chimeric antibody to a cancer-associated antigen.

In an attempt to obtain a therapeutic antibody, the murine monoclonal antibody (MAb) MBrI (IgM,k), directed against human carcinomas, was converted in a mouse/human chimeric MAb of gamma I isotype. The chimeric MAb, gamma I CHI-MBrI, retains the ability to specifically bind tumor cells and tissues with no modification in its binding to the normal material tested. gamma I CHI-MBrI recognizes mucins and high-molecular-weight glycoproteins carrying the antigenic determinant and stains a neutral glycolipid extracted from MCF-7 cells. The chimeric and the murine MBrI efficiently cross-inhibit each other on the reference cell line MCF-7 and the calculated affinity constants amount to 3.8 x 10(7) and 1.7 x 10(8) M-1, respectively. The human constant region allows gamma I CHI-MBrI to bind with the FcR on the human monocytic cell line U937 and to efficiently mediate antibody-dependent cellular cytotoxicity in the presence of human lymphocytes activated by IL2. In addition, gamma I CHI-MBrI, like the murine MBrI, mediates complement-dependent tumor-cell lysis. Thus, by modelling a molecule with reduced size and increased functional characteristics, we have obtained a reagent which is more suitable for in vivo therapeutic approaches.

Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein.

Monoclonal antibodies MOv18 and MOv19, raised against a membrane preparation of an ovarian carcinoma surgical specimen, react with a surface antigen present on the majority of nonmucinous ovarian malignant tumors tested but not with normal adult tissue (S. Miotti, S. Canevari, S. Ménard, D. Mezzanzanica, G. Porro, S. M. Pupa, M. Regazzoni, E. Tagliabue, and M. I. Colnaghi, Int. J. Cancer, 39: 297-303, 1987). This surface antigen was purified as a soluble glycoprotein (molecular mass, 36-38 kDa) released from the cell surface of an ovarian carcinoma cell line (IGROV1) by digestion with Bacillus thuringiensis phospholipase C. Immunoblotting demonstrated that the purified protein reacted with MOv18 and MOv19 and that treatment of the purified preparation with N-glycanase resulted in a protein with a molecular mass of 27 kDa. The NH3-terminal amino acid sequence of the purified antigen was determined. This sequence is highly homologous to an internal stretch of 27 amino acids located near the NH3 terminus of human folate-binding protein. An oligonucleotide probe was synthesized and used to screen an IGROV1 ovarian carcinoma, lambda gt11 complementary DNA library to obtain three complementary DNA clones. The complete nucleotide sequence of one of these complementary DNA clones was determined. This sequence is nearly identical to that of a folate-binding protein clone obtained from the Caco-2 human carcinoma cell line. In addition, the nucleotide sequence of the 5'-untranslated region of the other two clones was determined. This region of all three clones was different. The product of the Caco-2 folate-binding protein clone expressed in Chinese hamster ovary cells was recognized by the MOv18 and MOv19 antibodies, confirming that the antigen and folate-binding protein are one and the same. Furthermore, a cell line that binds the MOv18 and MOv19 antibodies expressed increased levels of folate-binding protein mRNA compared with a cell line that does not bind these antibodies. These results indicate that the MOv18 and MOv19 monoclonal antibodies bind to at least one form of folate-binding protein and that this protein, which is evidently overexpressed in certain malignant tumors, may provide a suitable target for immunotherapy with these antibodies.

Polypeptide composition and an immunological analysis of DNA methyltransferases from different species.

The cross-reactivity of the monoclonal anti-human placental DNA methyltransferase antibody M2B10 with DNA methyltransferases isolated from other species was investigated. This antibody immunoprecipitates DNA methyltransferases from mammalian cells, i.e., human placenta, mouse P815 cells, and rat liver cells. No cross-reactivity is observed with DNA methyltransferases from wheat germ and with bacterial DNA methyltransferases HpaII and EcoRI. The mammalian enzymes are characterized by polypeptides of molecular mass 150-190 kDa. Polypeptides smaller than 190 kDa are presumably generated by proteolysis of the native 190-kDa DNA methyltransferase. Trypsin digestion of the 190-kDa polypeptide isolated from mouse cells results in progressive appearance of DNA methyltransferase polypeptides of 150-190, 110, 100, and 52-60 kDa.