Expression of glutathione transferase pi in benign and malignant lesions of the melanocyte lineage.

Intrinsic and acquired resistance to chemotherapeutic agents represents the major clinical obstacle in the control of most tumours. In vitro studies have established that multiple mechanisms, including changes in drug uptake and efflux and in detoxifying enzymes, are responsible for drug resistance. Among the latter, glutathione S transferases (GST) have been recognized to play a relevant role. In the present study we have evaluated GST pi immunohistochemically as well as enzymatically in benign and malignant primary and metastatic lesions of the melanocyte lineage. A parallel analysis of the multiple drug resistance (MDRI) gene product was performed in a representative number of specimens. Results of this study demonstrate that while GST pi is constitutively expressed by the melanocyte lineage, independently from the transformed stage, MDRI p-glycoprotein is detected with a significantly lower frequency. These findings clearly indicate that GST pi represents the major detoxifying metabolic pathway of the melanocyte lineage and may be responsible for the high degree of inherent resistance of malignant melanoma to available cytostatic treatments.

Interferon-gamma gene expression in human B-cell lines: induction by interleukin-2, protein kinase C activators, and possible effect of hypomethylation on gene regulation.

Human interferon-gamma (IFN-gamma) is an important immunomodulatory protein produced predominantly by T cells and large granular lymphocytes (LGLs). Whereas large amounts of data have been accumulated regarding IFN gamma gene expression in these two cell types, little information about IFN gamma expression in other cell types exists. In this study, we have analyzed the production of IFN gamma by the Epstein-Barr virus (EBV)-positive B-cell line, JLP(c), derived from a patient with Burkitt's lymphoma, and another human B-cell line, PA682BM-1, which was derived from an acquired immunodeficiency syndrome patient. Southern blot analysis indicates the presence of an Ig heavy chain gene rearrangement, but no rearrangement of the T-cell receptor beta chain gene or IFN gamma gene in these B-cell lines. Both cell lines were found to express surface IgD and other B-cell surface markers, thus confirming their B-cell lineage. Analysis for surface Ig, cytoplasmic Ig, and secreted Ig indicates that the two cell lines are in relatively early stages of the B-cell differentiation pathway. We now report that PA682BM-1 can be triggered by the protein kinase C (PKC) activators, phorbol 12-myristate 13-acetate (PMA) and (-)Indolactam-v, to secrete IFN gamma, whereas JLP(c) cells spontaneously produce low levels of IFN gamma that can be enhanced by PKC activators and interleukin-2 (IL-2). After activation of the cell lines with IL-2, (-)Indolactam-v, and PMA, increases in cytoplasmic messenger RNAs (mRNAs) of IFN gamma and the IL-2 receptor chains were also observed. The induction of IFN gamma mRNA and protein by IL-2 was completely blocked by a monoclonal antibody to IL-2 receptor p75 (beta chain), but not by the monoclonal antibody to p55 (alpha chain). Analysis of IFN gamma genomic DNA indicates that the gene is not amplified, but that hypomethylation in the 5' noncoding region of the IFN gamma gene has occurred in the B-cell line from the Burkitt's lymphoma patient that spontaneously produces IFN gamma. This finding suggests that the methylation state of the promoter region may play an important role in the control of IFN gamma gene expression in B cells.

Two antigens detected on human ocular melanomas with the mouse monoclonal antibodies 2/10SN and 10/12SN.

Seven human ocular melanoma cell lines were established in vitro and 3 of these, GU-4, LLN-40 and its subline C17-11, were characterized. Mice were immunized with these ocular melanoma cell lines, and 2 hybridomas producing monoclonal IgG1 antibodies (MAb) were produced. MAb 2/10SN recognizes a 44-kDa monomeric protein, whereas MAb 10/12SN reacts with an 83/65-kDa heterodimeric protein. These melanoma-associated antigens (MAA) are detected at high concentrations in the cytoplasm of ocular melanoma cells. However, cell-surface labelling techniques suggest that these MAA are also associated with the cell-surface membrane. These 2 ocular MAA are also expressed by several skin melanoma cell lines. Immunohistochemical studies have localized these antigens to ocular and skin melanomas, to sweat ducts and basal squamous cells in normal skin, with limited expression in several other normal tissues and some carcinomas. Biodistribution studies in nude mice with human ocular melanomas have demonstrated good localization of 125I-labeled MAb 2/10SN at the tumor sites. Therefore, these 2 MAbs, 2/10SN and 10/12SN, recognize MAA which appear to be unique and may prove useful for imaging purposes.

Mechanism of target cell recognition by natural killer cells: characterization of a novel triggering molecule restricted to CD3- large granular lymphocytes.

In an attempt to identify a molecule in target recognition by CD3- large granular lymphocytes (LGL), we have generated a rabbit antiidiotypic (anti-ID) serum against a monoclonal antibody (mAb 36) that reacted with the cell membrane of K562. Flow cytometry analysis demonstrated that the anti-ID serum bound selectively to CD3- LGL and that F(ab')2 fragments of the anti-ID serum blocked both target cell binding and lysis by NK cells. Stimulation of CD3- LGL with F(ab')2 fragments resulted in the release of serine esterases and the secretion of interferon gamma. Furthermore, anti-ID F(ab')2 antibodies crosslinked to anti-DNP F(ab')2 mediated directed cytotoxicity of a non-natural killer (NK)-susceptible mouse target (YAC-1) via this surface ligand. These functional reactivities were only removed by adsorption with the specific idiotype. Protein analysis showed that the anti-ID serum immunoprecipitated 80-, 110-, and 150-kD proteins. Using this anti-ID, a partial cDNA was cloned and an antipeptide antiserum was made against the portion of the predicted amino acid sequence that corresponded to a portion of the ID binding region. This antipeptide serum exhibited similar functional and biochemical reactivities to those observed with the anti-ID serum. These data suggest that the cell surface moiety recognized by the anti-ID and anti-p104 is novel and is selectively involved in both recognition and triggering of NK-mediated lytic function.

Monoclonal antibodies to glutathione S-transferase pi-immunohistochemical analysis of human tissues and cancers.

Mouse monoclonal antibodies (MAb) have been generated against the anionic isozyme of human glutathione S-transferase (GST pi). MAb AGST I can inhibit 50-70% of GST pi enzymatic activity and reacts with a 3-dimensional epitope which includes a putative glutathione binding site on GST pi. A sandwich enzyme-immunoassay established using MAb AGST I and a polyclonal antibody displayed a sensitivity of 0.5 ng/ml. Immunohistochemical analysis of human tissues demonstrated marked increases in GST pi levels in cancers of the brain, cervix, endometrium, colon, rectum and testis and in fibro- and chondrosarcomas.

Immunohistochemical localization of glutathione S-transferase in preneoplastic and neoplastic lesions of the human uterine cervix.

A mouse monoclonal antibody and a rabbit polyclonal antibody prepared against the placental form of the enzyme glutathione S-transferase (GST-pi) were used to immunohistochemically stain normal and neoplastic human uterine cervical tissues from 88 cases. Of 65 cases of preneoplastic squamous lesions and invasive carcinomas of the cervix, 94% stained with the monoclonal antibody and 100% with the polyclonal antibody. In the 23 benign tissues, staining of ectocervical squamous epithelium was generally not observed; however, areas of reserve-cell hyperplasia, immature squamous metaplasia and adjacent endocervical cells did show staining (68% with the monoclonal antibody and 95% with the polyclonal antibody). Many of the positive tissue types showed a variety of staining patterns and intensities. These findings do not support the concept that GST-pi staining can be used to distinguish preneoplastic lesions of the cervix from benign reactive or proliferative processes. These results are of interest in the investigation of cervical carcinogenesis since GST-pi may be involved in an early stage of neoplastic transformation of the cervical epithelium. The correlation of these findings with the results of human papillomavirus testing and DNA content analysis should be of interest in determining the relationship of this enzyme to cervical neoplasia.

Biochemical analysis of two cell surface glycoprotein complexes, very common antigen 1 and very common antigen 2. Relationship to very late activation T cell antigens.

Two mouse monoclonal antibodies (mAb), AJ2 and J143, define two related human cell surface protein complexes, very common antigen 1 (VCA-1) and very common antigen 2 (VCA-2). In the present report, these complexes have been defined with respect to: (i) subunit arrangement; (ii) monoclonal antibody binding sites; (iii) carbohydrate content; (iv) homology to other cell surface protein complexes; and (v) possible functional roles. Analysis of the antigens from a human melanoma cell line, MeWo, reveals that VCA-1 is a noncovalently linked heterodimer of 170- and 140 (designated 1401)-kDa polypeptides. mAb AJ2 reacts with an epitope on the 1401-kDa polypeptide. VCA-2 is composed of the same 1401-kDa polypeptide as VCA-1 and another 170-kDa species; this 170-kDa species consists of a second distinct 140-kDa (designated 140(2)) and a 30-kDa polypeptide which are disulfide-bonded. Indirect evidence indicates that mAb J143 reacts with an epitope on this 170-kDa complex. Peptide mapping has shown that the complexes are members of a family of cell surface proteins that include antigens present on activated T cells (designated very late activation antigens). Since VCA-2 is found predominantly on the basal membrane of adherent cells and its expression increases 12-fold when HUT-102 lymphoblastoid cells are grown as an adherent cell culture, we suggest that VCA-2 plays a role in cellular adherence.

DNA-mediated gene transfer of a human cell surface 170-kilodalton glycoprotein. Evidence for association with an endogenous murine protein.

We have previously reported the identification and characterization of two related human cell surface protein complexes, very common antigens 1 and 2 (VCA-1, VCA-2) (Kantor, R. R. S., Mattes, M. J., Lloyd, K. O., Old, L. J., and Albino, A. P. (1987) J. Biol. Chem. 262, 15158-15165). We now report the transfection of DNA sequences encoding the 170-kilodalton heterodimer of VCA-2 from human SK-RC-41 renal cancer cells to B78H1 mouse melanoma cells. B78H1 cells were cotransfected with high molecular weight renal cancer DNA and a plasmid vector containing the neomycin resistance gene. Antibiotic-resistant transfectants were screened for the expression of the 170-kDa heterodimer with mouse monoclonal antibody (mAb) J143. Analysis of mAb J143-positive (J143+) transfectants showed that they expressed a 170-kDa heterodimer with an identical molecular weight, isoelectric point, two-dimensional peptide map, and spatial orientation of surface-exposed epitopes to the homologous 170-kDa species seen in human donor cells. The 170-kDa heterodimer in SK-RC-41 cells is associated with a 140-kDa (designated 140(1] polypeptide to form the VCA-2 complex. The 170-kDa complex and the 140(1)-kDa polypeptides are encoded by genes located on different human chromosomes. J143+ transfectants display a molecule of 140 kDa associated with the 170-kDa complex which is biochemically similar, but non-identical, to the human 140(1)-kDa polypeptide on VCA-2. This evidence supports our interpretation that the transfected human 170-kDa heterodimer associates with a murine counterpart of the human 140(1)-kDa polypeptide in J143+ transfectants.

Class II histocompatibility antigen expression in human melanocytes transformed by Harvey murine sarcoma virus (Ha-MSV) and Kirsten MSV retroviruses.

Human melanocytes infected with Ki-MSV or Ha-MSV, but not amphotropic MuLV, undergo a series of transformation-related changes that are characteristic of malignant melanoma. These are (a) expression of Ia antigens, in particular DP, DQ, and DR class II histocompatibility gene products, (b) a transformed morphology and ability to grow in soft agar, and (c) a 5-10-fold increase in the cell surface expression of GD3 ganglioside. However, other characteristics of melanoma, such as independence from specific growth factors and loss of adenosine deaminase binding protein were not observed. We conclude that viral ras oncogenes initiate early transformation events in melanocytes, and that Ia antigen expression is a transformation marker in this system.

Biosynthesis and intracellular processing of four human melanoma associated antigens.

By analyzing human melanoma cells with monoclonal antibodies (MoAb) we have identified four melanoma associated antigens with distinct tissue distribution and structural properties. They include the high molecular weight melanoma associated antigen (MAA), the Mr 120,000 MAA, the Mr 100,000 MAA, and the cytoplasmic MAA defined by MoAb 465.12. Previous studies have shown that these antigens may be useful markers to characterize the biology of melanoma cells and to develop immunodiagnostic and immunotherapeutic approaches to melanoma. In the present investigation pulse-chase intrinsic labeling studies combined with the biosynthetic inhibitor tunicamycin and with enzymatic degradations with endoglycosidase H have shown that the determinants recognized by the MoAb utilized are expressed on the core protein of the molecules. Furthermore the four MAAs are highly glycosylated with N-linked carbohydrate chains and are synthesized as precursors which bear endoglycosidase H-sensitive chains. The high molecular weight (Mr 500,000/280,000) MAA displays a major precursor with a molecular weight of 240,000 which expresses the epitopes recognized by the anti-high molecular weight MAA MoAbs 149.53, 225.28S, and 763.74T. This precursor has an apparent molecular weight of 220,000 when cells are grown in the presence of tunicamycin. The Mr 89,000 and Mr 36,000 subunits of the Mr 125,000 MAA have biosynthetic precursors with molecular weights of 76,000 and 25,000. Endoglycosidase H digestion of the Mr 76,000 precursor produces a Mr 46,000 polypeptide. The Mr 100,000 MAA has a Mr 87,000 biosynthetic precursor. The cytoplasmic MAA (Mr 100,000, 75,000, 72,000, and 25,000) has a single precursor with a molecular weight of 75,000 which appears as a Mr 60,000 polypeptide after endoglycosidase H digestion. Characterization of the biosynthesis of the four MAAs will contribute to the development of approaches to modulate their expression and shedding by melanoma cells.

DNA-mediated transfer of human melanoma cell surface glycoprotein gp130: identification of transfectants by erythrocyte rosetting.

DNA sequences encoding a human melanoma membrane-bound sialoglycoprotein of 130,000 molecular weight (gp130) were introduced into a clonal derivative of mouse B-16 melanoma cells with the selectable neomycin resistance gene (aminoglycoside phosphotransferase). Mouse transfectants were identified by a rapid and precise screening method with mouse monoclonal antibodies and erythrocyte rosetting. The frequency of gp130 transfectants was approximately 1 in 2,000 to 5,000 colonies with neo+ cells. Analysis of secondary mouse transfectants has revealed that the transfected gp130 has a molecular weight, isoelectric point, intracellular processing, peptide map, and spatial orientation of surface-exposed epitopes indistinguishable from those seen with gp130 from human melanoma cells. In contrast to primary transfectants, secondary transfectants expressing gp130 lack demonstrable human repetitive sequences.

Double determinant immunoassay to measure a human high-molecular-weight melanoma-associated antigen.

Using monoclonal antibodies to distinct determinants of a human high-molecular weight melanoma-associated antigen (HMW-MAA), a double determinant immunoassay has been developed. The assay is specific and reproducible. Its sensitivity is influenced by the incubation time of antibodies with antigen sources and the combination of antibodies, as well as by the pH of the buffer and the incubation time used to coat plates with antibodies. Testing with the double determinant immunoassay of Nonidet P-40 extracts of human cell lines and of surgically removed normal and malignant tissues has confirmed the restricted tissue distribution of the HMW-MAA. In addition, significant differences have been found in the level of HMW-MAA in melanoma cell lines, as well as in melanoma lesions removed from different patients and from different sites of a given patient. The amount of HMW-MAA shed by various melanoma cell lines does not correlate with their cell surface expression and with their level in Nonidet P-40 extracts. Interferon and hyperthermia increase the shedding of the HMW-MAA by melanoma cells.

Inhibition of lymphocyte activation by antisera to embryonic antigens shared with human placental trophoblast.

In order to further examine the inhibition of lymphocyte activation reported with conventional antisera to human trophoblast membranes, we studied their effects on cells stimulated by antigen in mixed lymphocyte culture (MLC) and by the mitogen phytohemagglutinin. The results were compared with the effects of antisera known to recognize intrinsic membrane determinants on activated T cells (DR antigens and placental alkaline phosphatase) and with those of antisera to normal human serum components such as transferrin which may bind to the activated lymphocyte membrane. The results indicated that antibodies to placental alkaline phosphatase, which constitutes the predominant specificity of conventional trophoblast membrane antisera, caused inhibition both of MLC response and, to a lesser extent, of activation induced by mitogen. Similar inhibition was obtained with antisera to human DR antigens, while antisera to normal human serum and transferrin were not suppressive. These findings, together with time course studies and immunocytochemical studies of the homologous antigens, suggest that these antisera mediate their inhibitory effects in part through binding to antigens which appear on cells as they undergo activation.

Analysis of the NIH workshop monoclonal antibodies to human melanoma antigens.

Reagents exchanged at the 2nd workshop on monoclonal antibodies (MoAb) to human melanoma antigens were analyzed using both serological and immunochemical assays. The analysis by laboratories participating in the workshop of our MoAb 225.28S, 345.134S, 376.96S, 465.12S, and 763.24TS reaffirms our own analysis of these reagents in that (1) they all react with the majority of melanoma cell lines tested and (2) the reactivity of MoAb 225.28S and 763.24TS is much more restricted than that of MoAb 345.134S 376.96S, and 465.12S. Our serological analysis revealed that the majority of workshop reagents reacted with cultured melanoma cells. Immunochemical analysis of these monoclonal antibodies allowed for their division into three groups according to the molecular weights of the antigens recognized in immunoprecipitation experiments, greater than 100 kd, 80-100 kd, and DR antigens. Further analysis of the first two groups of monoclonal antibodies by immunodepletion and antibody binding inhibition assays revealed that MoAb 9.2.27, 225.28S, and 763.24TS recognize distinct determinants with a heterogeneous distribution on subpopulations of a high molecular weight melanoma associated antigen. MoAb 376.96S and 705.F6 recognize either the same or spatially close determinant(s).

Studies of antigenic components of the human syncytiotrophoblast membrane.

In order to initiate characterization of potentially immunogenic moieties on the human trophoblast membrane, antisera to native and detergent-solubilized normal trophoblast membranes were prepared in rabbits. By indirect immunofluorescence, these antisera reacted strongly with all villous structures in normal placentae and also with a panel of normal adult human tissues. Specificities to normal human serum components were then removed by solid-phase immunoabsorption. However, the resultant antisera still reacted weakly with differentiated, normal adult tissues, and additional absorption with normal adult liver homogenate was necessary to remove cross-reactivity completely. Such absorbed antisera gave undiminished and specific fluorescence of the syncytiotrophoblast plasma membrane, and specifically precipitated two protein species with approximate relative molecular masses of 148,000 and 62,000. However, they also reacted with PHA-activated peripheral blood lymphocytes, in addition to Chang liver and HeLa cell lines. These results indicate that, even after extensive absorption, trophoblast membrane antisera may retain reactivity for determinants shared with normal and transformed adult cells.

Studies of the interaction between human transferrin and specific receptors on the trophoblast membrane.

The concept of specific receptors for maternal transferrin on the human syncytiotrophoblast membrane has been generally accepted for many years, but definitive evidence of their existence has been established only recently. In this study, experiments were performed to characterize transferrin receptors further, both on intact membranes and after solubilization. Intact, isolated membrane fragments were examined for transferrin binding, both qualitatively by immunofluorescence and quantitatively by radiobinding. The amounts of ligand bound varied inversely with the quantities of residual maternal transferrin remaining at the time of testing. Scatchard plots revealed that the calculated affinity (K alpha) and the number of receptors increased substantially when transferrin was removed by initial washing with chaotropic agents. After dissociation from the membrane by detergents, occupied receptors largely retained bound transferrin, and transferrin binding by unoccupied receptors was also preserved. The stability of such ligand:receptor complexes was considerably enhanced at pH 5.0, and was apparently unaffected by prior treatment with chaotropic agents. Specific immunoprecipitation of solubilized radioiodinated membrane with rabbit antiserum to human transferrin, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate under reducing conditions, indicated a relative molecular mass for the unoccupied receptor of 90,000. These results confirm the existence of high-affinity receptors for transferrin on the trophoblast, and also delineate certain potential pitfalls in studies of their interactions with ligand.