Isolation and characterization of monoclonal antibodies specific for protein conformational epitopes present in prostate-specific membrane antigen (PSMA).

Prostate-specific membrane antigen (PSMA) is a 750-amino acid glycoprotein highly expressed in malignant prostate tissues. PSMA reacts with the murine monoclonal antibody 7E11.C5, whose binding epitope has been mapped to the N-terminal of the protein distributed on the cytoplasmic side of the plasma membrane. We have developed murine monoclonal antibodies specific for extracellular epitopes of PSMA. Three of these antibodies--1G9, 3C6, and 4D4--display distinct binding properties consistent with their recognition of conformational epitopes within native PSMA. Results indicate this panel of antibodies binds to native full-length PSMA, but not to fusion proteins containing portions of the linear sequence of the protein. Antibody binding is greatly reduced upon heat denaturation of native PSMA, and these antibodies do not detect PSMA by Western blot. Immunoprecipitation experiments demonstrate the ability of each to bind to full-length PSMA as well as PSM', a form of the protein missing the first 57 amino acids. These results indicate each antibody is specific for an epitope within the extracellular domain, a region spanning residues 44-750. Flow cytometric experiments indicate strong specific binding to live LNCaP cells. Antibody inhibition studies demonstrate that these antibodies recognize at least two distinct epitopes. Taken together, the results demonstrate that these antibodies are specific for native protein conformational epitopes within the extracellular domain. Their properties, in particular strong binding to live cancer cells, make them ideal candidates that are clearly superior to linear sequence epitope specific antibodies for in vivo applications.

Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain.

Experimental stroke using a focal cerebral ischemia and reperfusion (FCIR) model was induced in male Long-Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30-90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N-glycosylase (Fpg protein)-sensitive sites (FPGSS), as labeled in situ using digoxigenin-dUTP and detected using antibodies against digoxigenin. Because Fpg protein removes 8-hydroxy-2'-deoxyguanine (oh8dG) and other lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS-positive cells in the cortex from the sham-operated control group was 3 +/- 3 (mean +/- SD per mm(2)). In animals that received 90 min occlusion and 15 min of reperfusion (FCIR 90/15), FPGSS-positive cells were significantly increased by 200-fold. Oxidative DNA damage was confirmed by using monoclonal antibodies against 8-hydroxy-guanosine (oh8G) and oh8dG. A pretreatment of RNase A (100 microg/ml) to the tissue reduced, but did not abolish, the oh8dG signal. The number of animals with positive FPGSS or oh8dG was significantly (P<0.01) higher in the FCIR group than in the sham-operated control group. We detected few FPGSS of oh8dG-positive cells in the animals treated with FCIR of 90/60. No terminal UTP nicked-end labeling (TUNEL)-positive cells, as a detection of cell death, were detected at this early reperfusion time. Our data suggest that early oxidative DNA lesions elicited by experimental stroke could be repaired. Therefore, the oxidative DNA lesions observed in the nuclear and mitochondrial DNA of the brain are different from the DNA fragmentation detected using TUNEL.

Prostate-specific membrane antigen (PSMA): current benefits and future value.

We will review the evolution, benefits, and limitations of PSMA testing in the past, as well as its current and future value. Prostate cancer has been the most frequently diagnosed cancer and the second leading cause of cancer death in men in the United States. It has a wide spectrum of biological behavior between latent (indolent) and progressive (aggressive). Further identification of prostate-specific membrane antigen (PSMA) as a prognostic proliferation marker may enhance our understanding of the types of prostate cancer. A review of PSMA testing in the past as well as currently was conducted. Studies were reviewed that deal with detection of PSMA in serum and seminal fluid, reverse transcriptase-polymerase chain reaction (RT-PCR), immunoscintigraphy, and immunohistochemical assays. PSMA is expressed primarily in benign and cancerous prostatic epithelial cells. It is up-regulated in hormone resistant states, and in metastatic situations or other clinical situations where there is tumor recurrence or extension. Based on current results, PSMA detected in the serum by western blotting can assist in the identification, staging, and monitoring of metastatic prostate cancer. In addition, PSMA shows a promising role in directed imaging and therapy of recurrent or metastatic disease.

Isolation and characterization of monoclonal antibodies specific for the extracellular domain of prostate specific membrane antigen.

PURPOSE: Monoclonal antibodies specific for protein epitopes of prostate specific membrane antigen (PSMA) expressed on the external surface of prostatic epithelial cells were prepared to provide material for use in the diagnosis or treatment of prostatic cancer. MATERIALS AND METHODS: Mice were immunized with LNCaP cell membranes followed by purified PSMA before fusion. Hybridomas were screened by reactivity with purified PSMA. Resulting antibodies were characterized by enzyme-linked immunosorbent assay, Western blot and fluorescence-activated cell sorter analyses. RESULTS: Monoclonal antibody producing hybridomas designated 3E11, 3C2, 4E10-1.14, 3C9 and 1G3 were obtained which displayed specificities for differing regions of the extracellular domain of the PSMA protein. These antibodies reacted strongly with PSMA from multiple sources and specifically stained unfixed PSMA expressing cells by flow cytometric analysis. CONCLUSIONS: The antibodies obtained displayed strong reactivity and specificity for extracellular epitopes of PSMA. These antibodies will have value in future diagnostic and therapeutic applications focusing on PSMA as a target antigen.

Analysis of glycosylation of prostate-specific membrane antigen derived from LNCaP cells, prostatic carcinoma tumors, and serum from prostate cancer patients.

BACKGROUND: Prostate-specific membrane antigen (PSMA) has been detected in human prostatic cancer tissues, serum, and seminal fluid based on Western blot data with the monoclonal antibody 7E11.C5. The reactive protein is very similar in size to that from human prostatic carcinoma LNCaP cells and corresponds to a protein with a molecular size of about 110,000 daltons. Given that PSMA is known to be a 750 amino acid protein of about 84,000 daltons, a substantial portion, perhaps 20-25% of the native molecular weight, is composed of carbohydrates. METHODS: In this study, we have begun initial analyses of the glycosylation of the PSMA protein from multiple sources using a variety of exo- and endoglycosidase treatments. RESULTS: The results indicate that the carbohydrate is primarily N-linked and in each case the deglycosylated protein has an apparent molecular weight of about 86,000 daltons. The glycan present on in vivo-derived PSMA from tumor tissue or serum was found to be primarily N-linked complex type. A small amount of O-linked glycan also appears to be present. In contrast, only high mannose-type N-linked glycans are present on the PSMA from LNCaP cells. CONCLUSIONS: Oligosaccharides present on PSMA derived from both tissue culture LNCaP cells and in vivo specimens are primarily N-linked and comprise about 20-25% of the native molecular weight. N-linked glycans of PSMA derived from in vivo sources were found to be complex type, lacking polylactosamine structures. In contrast, LNCaP cells express only high mannose-type structures. These results will be useful in our ongoing efforts to develop monoclonal antibodies which are specific for protein epitopes present in the extracellular domain of the protein.

De novo synthesis of type 1 lacto-series glycolipids in human colonic adenocarcinoma cells: efficient synthesis of the Le(a) antigen and absence of brefeldin A-induced inhibition of its synthesis in Colo 205 cells.

De novo biosynthesis of lacto-series type 1 chain glycolipids was studied by [3H]serine metabolic labeling of ceramide in Colo 205 and SW403 cells. Highly efficient synthesis of III4FucLc4 (Le(a)) from lactosylceramide was found to be characteristic of Colo 205 cells. Although de novo synthesized, 3H-labeled Le(a) was found to accumulate after a 4-h labeling period, a 3H-labeled pool of Le(a) precursors was not detected until after 8 to 12 h. This is due presumably to hydrolysis of the initial Le(a) product. Labeling of neutral glycolipids from SW403 cells instead indicated a stepwise elongation of oligosaccharide chains providing labeled pools of type 1 and 2 chain precursors followed by their fucosylated derivatives. Analysis of neutral glycolipids isolated from Golgi membranes fractionated by sucrose density gradient centrifugation confirmed the efficient synthesis of type 1 antigens in early Golgi fractions of Colo 205 cells along with the results from SW403 cells observed using whole cells. De novo glycolipid synthesis in the presence of brefeldin A indicated a disruption of the efficient Le(a) synthesis in Colo 205 cells with the appearance of biosynthetic intermediates of Le(a) synthesis. However, Le(a) remained the de novo product accumulated in these as well as SW403 cells. The results suggest efficient trafficking of glycolipid precursors between successive glycosyltransferases coupled with the relative distribution of beta 1-->3- vs beta 1-->4galactosyltransferase in intracellular membranes are properties associated with preferential synthesis of type 1 chain antigens as occurs in Colo 205 cells.

Stable expression of a cDNA encoding a human beta 1 --> 3galactosyltransferase responsible for lacto-series type 1 core chain synthesis in non-expressing cells: variation in the nature of cell surface antigens expressed.

Transient expression of a human colonic adenocarcinoma Colo 205 cell derived cDNA in cell lines which ordinarily express only neolacto-series glycolipids has resulted in the expression of a beta 1 --> 3galactosyltransferase gene responsible for synthesis of glycolipids based upon the lacto-series type 1 core chain. Calcium phosphate transfected cells were panned on anti-IgM coated plates after initial treatment with a combination of monoclonal antibodies specific for type 1 chain terminal structures (TE-3) and a very broadly specific antibody reactive with multiple type 1 chain derivatives (TE-2). Adherent cells after panning were capable of efficiently transferring Gal in beta 1 --> 3-linkage to the acceptor glycolipid Lc3. Using these reagents, clones of stably transfected human colonic adenocarcinoma HCT-15 cells were produced and isolated. Parental HCT-15 cells do not express type 1 chain based antigens. The nature of the type 1 chain based antigens produced in each of these clones was analyzed by solid phase antibody binding assays. Three types of behavior were observed. Formation of type 1 terminal structures that were either exclusively sialylated or fucosylated, or a mixture of sialylated and fucosylated determinants occurred. In contrast, no difference in type 2 antigen expression between any clone and the parental cells was observed. These data suggest that coordination of subsequent reactions capable of modifying type 1 chain structures is not the same in all clones. The relationship of these results to aspects of cellular regulation of carbohydrate biosynthesis is discussed.

Isolation and fine-structure characterization of four monoclonal antibodies reactive with glycoconjugates containing terminal GlcNAc residues: application to aspects of lacto-series tumor antigen biosynthesis.

A series of murine monoclonal antibodies, each reactive with terminal GlcNAc residues expressed on glycolipids, have been isolated after immunization with the glycolipid nLc5 (GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1---- 4Glc beta 1----1Cer). The derived antibodies, designated TE-4, TE-5, TE-6, and TE-7, were tested for binding specificity with a variety of terminal GlcNAc-containing oligosaccharides expressed on glycolipids and glycoproteins. Antibody TE-4 was found to be reactive only with linear and branched terminal GlcNAc beta 1----3Gal containing structures present in lacto-series carbohydrates irrespective of core chain length. The binding specificity of TE-7 was similar except that no reactivity was observed with the short chain structure Lc3 and was weakly reactive with branched agalacto-I structures, suggesting a longer recognition epitope than for the TE-4 antibody. Antibodies TE-5 and TE-6 reacted with terminal GlcNAc beta 1----3Gal structures and as well GlcNAc beta 1----2(6)Man structures present on BSA-oligosaccharide conjugates. Weak binding was also observed with GlcNAc beta 1----6Gal structures with these antibodies. TE-5 was found to be particularly sensitive to low amounts of terminal GlcNAc-containing glycolipids in both solid phase assays and in TLC-immunostaining studies of neutral glycolipids extracted from colonic adenocarcinoma cell lines and tumors. No reactivity was observed with internal GlcNAc residues with any antibody tested. The panel of antibodies was applied to studies of binding to Triton X-100-solubilized fractions from normal mucosal and adenocarcinoma cell lines after desialylation and Smith degradation to expose terminal GlcNAc residues on glycoproteins and glycolipids. Binding of antibodies TE-4 and TE-7 was restricted to adenocarcinoma-derived cell fractions. Application of these antibodies in studies of lacto-series core chain synthesis and in immunodiagnostic procedures after initial treatments to concentrate lacto-series antigens into terminal GlcNAc-containing structures is discussed.

Alteration of lacto-series glycolipid glycosyltransferase activities in human colonic adenocarcinoma DLD-1 cells after culture in N,N-dimethylformamide-containing medium.

Human colonic adenocarcinoma DLD-1 cells were grown under conditions which induce characteristics of differentiated cells using medium containing 0.8% N,N-dimethylformamide in order to study alterations in glycosphingolipid glycosyltransferase activities during this process. Analysis of biosynthetic reactions involved in lacto-series antigen synthesis revealed no changes in the specific activities of either beta 1----4galactosyltransferase or alpha 1----3/4fucosyltransferase with N,N-dimethylformamide treatment. However, a dramatic decrease of from 14- to 20-fold in the beta 1----3N-acetylglucosaminyltransferase activity was observed in the treated cells. This enzyme catalyzes the rate-limiting step in lacto-series core chain synthesis. This is consistent with the pattern of regulation of lacto-series antigen expression found to occur during oncogenesis in human colonic mucosa (Holmes EH, Hakomori S, Ostrander GK: J Biol Chem 262:15649, 1987). Total glycolipids from untreated and N,N-dimethylformamide-treated cells were isolated and subjected to TLC immunostain analysis and solid phase radioimmunoassay with a series of monoclonal antibodies specific for lacto-series-based carbohydrate antigens. A decrease of about 2-fold or less in the quantity of lacto-series antigens was observed as a consequence of N,N-dimethylformamide treatment in both neutral glycolipid and ganglioside fractions. The results suggest that only very low levels of beta 1----3N-acetylglucosaminyltransferase activity are required for the steady state expression of significant levels of lacto-series based glycolipids and that modulation of its activity levels by N,N-dimethylformamide treatment in DLD-1 cells represents a convenient in vitro system for studying aspects of regulation of lacto-series antigen expression.

Characterization of two monoclonal antibodies specific for lacto-series type 1 chain Gal beta 1----3GlcNAc-terminal structures.

Murine monoclonal antibodies, TE-1 and TE-3, generated by immunization with a biosynthetic reaction product containing a terminal Gal beta 1----3GlcNAc structure have been produced and found to react specifically with underivatized type 1 chain lacto-series carbohydrate structures. Detailed analysis of these antibodies, both IgM, indicates two differing classes of epitope specificity. Antibody TE-1 was found to bind preferentially to longer chain carbohydrate structures containing a terminal Gal beta 1----3GlcNAc disaccharide, indicating that optimal antibody binding involved more than recognition of this disaccharide. In contrast, antibody TE-3 was found to bind strongly carbohydrate structures containing terminal Gal beta 1----3GlcNAc structures irrespective of chain length. Modification of core chain structures by addition of fucose and/or sialic acid residues completely abolished antibody binding with either antibody. TLC immunostaining of neutral glycolipids isolated from a variety of human colonic adenocarcinoma cell lines indicated intensely stained bands, particularly with antibody TE-3, which correlated with the level of expression of type 1 chain based glycolipid derivatives. These antibodies are applied to the detailed study of the regulation of synthesis of lacto-series type 1 chain based carbohydrate structures.