concanavalin-a and Melanoma

Suppressor cell activity was determined in 14 patients with stage I melanoma, treated with or without adjuvant Bacillus Calmette-Guerin (BCG) immunotherapy, and in 27 normal healthy volunteers. An in vitro test system was used in which peripheral blood mononuclear cells when stimulated with concanavalin A (ConA) significantly suppress proliferative responses of fresh autologous mononuclear cells. In addition, lymphocyte stimulation capacity to optimal and suboptimal concentrations of phytohemagglutinin (PHA) was determined in 44 BCG treated or not BCG treated melanoma patients and in 40 normal individuals. ConA induced suppressor cell activity was significantly (p less than 0.02) impaired in BCG treated melanoma patients (21.3 +/- 3.1% suppression) when compared to not BCG treated patients (39.8 +/- 5.6%) or to normals (38.3 +/- 9.3%). Lymphocyte stimulation capacity was depressed in all melanoma patients when suboptimal concentrations of PHA were used but was found to be not significantly altered at optimal concentration of PHA. The present study reveals that BCG immunotherapy impairs ConA induced suppressor cell activity in melanoma patients but does not influence lymphocyte stimulation capacity.

Topics: BCG Vaccine; Concanavalin A; Female; Humans; Immunotherapy; Lymphocytes; Male; Melanoma; Middle Aged; Phytohemagglutinins; T-Lymphocytes, Regulatory

Malignant melanoma is one of the most dangerous skin cancer originating from melanocytes. Thus, an early and proper melanoma diagnosis influences significantly the therapy efficiency. The melanoma recognition is still difficult, and generally, relies on subjective assessments. In particular, there is a lack of quantitative methods used in melanoma diagnosis and in the monitoring of tumour progression. One such method can be the atomic force microscopy (AFM) working in the force spectroscopy mode combined with quartz crystal microbalance (QCM), both applied to quantify the molecular interactions. In our study we have compared the recognition of mannose type glycans in melanocytes (HEMa-LP) and melanoma cells originating from the radial growth phase (WM35) and from lung metastasis (A375-P). The glycosylation level on their surfaces was probed using lectin concanavalin A (Con A) from Canavalia ensiformis. The interactions of Con A with surface glycans were quantified with both AFM and QCM techniques that revealed the presence of various glycan structural groups in a cell-dependent manner. The Con A - mannose (or glucose) type glycans present on WM35 cell surface are rather short and less ramified while in A375-P cells, Con A binds to long, branched mannose and glucose types of oligosaccharides.

Topics: Biomarkers, Tumor; Biosensing Techniques; Concanavalin A; Glucose; Glycosylation; Gold; Humans; Lung Neoplasms; Mannose; Melanocytes; Melanoma; Microscopy, Atomic Force; Polysaccharides; Quartz Crystal Microbalance Techniques; Surface Properties

Application of electric pulses (electroporation/electropermeabilization) is an effective method for gene transfer (i.e. gene electrotransfer (GET)) in vitro and in vivo. Currently, the mechanisms by which the DNA enters the cell are not yet fully understood. Experimental evidence is building up that endocytosis is the main mechanism by which the DNA, which is later expressed, enters the cell. Therefore the aim of our study was to elucidate whether inhibitors of endocytosis, methyl-β-cyclodextrin (MβCD), Concanavalin A (ConA) and Dynasore, can impair the transfection efficacy of GET in vitro in B16F1 murine melanoma and in vivo in m. tibialis cranialis in mice. We show that MβCD--general inhibitor of endocytosis--can almost prevent GET of EGFP-N1 plasmid in vitro, that ConA--inhibitor of clathrin mediated endocytosis--also abrogates GET but to a lesser extent, and when using Dynasore--reversible inhibitor of dynamin--there is no effect on GET efficacy, if endocytosis is blocked for only 5 min after GET. Moreover, MβCD also reduced GET efficacy in vivo in m. tibialis cranialis and this effect was long lasting. The results of this study show that endocytosis is probably the main mechanism of entrance of DNA after GET in vitro and also in vivo.

Topics: Animals; beta-Cyclodextrins; Concanavalin A; DNA; Electroporation; Endocytosis; Female; Gene Transfer Techniques; Hydrazones; Melanoma; Mice, Inbred C57BL; Muscles; Plasmids; Transfection; Tumor Cells, Cultured

The objective of this study was to investigate the antiproliferative activity and apoptosis-inducing mechanism of Concanavalin A (ConA) on human melanoma A375 cells. We firstly simulated the three-dimensional structure of ConA. Subsequently, we found that ConA possessed remarkable antiproliferative effect on A375 cells. Further experimental data indicated that there was a link between its hemagglutinating activity, mannose-binding activity and antiproliferative activity. In addition, we showed that ConA induced A375 cell apoptosis in a caspase-dependent manner. Then, we demonstrated that the treatment of ConA caused mitochondrial transmembrane potential (MMP) collapse, cytochrome c release and caspase activation. In conclusion, we report for the first time that there may be a close correlation between carbohydrate-binding activity of ConA and its antiproliferative activity. Also, we demonstrate firstly that ConA induces A375 cell death in a caspase-dependent manner as well as through a mitochondrial apoptotic pathway.

Topics: Apoptosis; Caspase 3; Caspase 8; Caspase 9; Cell Division; Cell Line, Tumor; Computer Simulation; Concanavalin A; Hemagglutination; Humans; Mannose; Melanoma; Membrane Potentials; Mitochondrial Membranes; Models, Molecular; Molecular Conformation

Dendritic cell (DC) and DC-derived exosomes (EXO) have been used extensively for tumor vaccination. However, its therapeutic efficiency is limited to only production of prophylactic immunity against tumors. T cells can uptake DC-released EXO. However, the functional effect of transferred exosomal molecules on T cells is unclear. In this study, we demonstrated that OVA protein-pulsed DC-derived EXO (EXO(OVA)) can be taken up by Con A-stimulated, nonspecific CD4(+) T cells derived from wild-type C57BL/6 mice. The active EXO-uptaken CD4(+) T cells (aT(EXO)), expressing acquired exosomal MHC I/OVA I peptide (pMHC I) complexes and costimulatory CD40 and CD80 molecules, can act as APCs capable of stimulating OVA-specific CD8(+) T cell proliferation in vitro and in vivo and inducing efficient CD4(+) Th cell-independent CD8(+) CTL responses in vivo. The EXO(OVA)-uptaken CD4(+) aT(EXO) cell vaccine induces much more efficient CD8(+) T cell responses and immunity against challenge of OVA-transfected BL6-10 melanoma cells expressing OVA in wild-type C57BL/6 mice than EXO(OVA). The in vivo stimulatory effect of the CD4(+) aT(EXO) cell to CD8(+) T cell responses is mediated and targeted by its CD40 ligand signaling/acquired exosomal CD80 and pMHC I complexes, respectively. In addition, CD4(+) aT(EXO) vaccine stimulates a long-term, OVA-specific CD8(+) T cell memory. Therefore, the EXO(OVA)-uptaken CD4(+) T cells may represent a new, effective, EXO-based vaccine strategy in induction of immune responses against tumors and other infectious diseases.

Topics: Animals; Antigens, Neoplasm; B7-1 Antigen; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD40 Antigens; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Concanavalin A; Dendritic Cells; Histocompatibility Antigens Class I; Humans; Immunologic Memory; Melanoma; Mice; Mice, Knockout; Mitogens; Ovalbumin; Peptides; Signal Transduction; Time Factors

Ecto-5'-nucleotidase is a GPI-anchored enzyme localized in cell membrane lipid rafts. Although it is highly expressed in many tumour cells, its specific function during tumorigenesis is unclear. We have found that, among different melanoma cells, upregulated expression of ecto-5'-nucleotidase is associated with a highly invasive phenotype. Analysis of other cell membrane proteins involved in melanoma adhesion and metastasis demonstrated that expression of alpha5, beta1, beta3-integrin subunits and CD44 was elevated gradually in accordance with increasing metastatic potential. Expression of alphav-integrin and caveolin-1 was seen mostly in cells derived from metastatic melanomas. Furthermore, in contrast to N-cadherin, which was unaltered in all lines, we could not detect E-cadherin in any cell type. Functional assays demonstrated that highly expressed ecto-5'-nucleotidase is a catalytically competent protein that is very sensitive to inhibition by concanavalin A. The interaction with concanavalin A also caused increased association of ecto-5'-nucleotidase-rich lipid rafts with much heavier cytoskeletal complexes as determined by density gradient centrifugation. A similar shift towards heavier cytoskeletal fractions also took place with other proteins coexpressed with ecto-5'-nucleotidase, such as alphav, alpha5, beta1 and beta3-integrins, caveolin-1 and CD44. As ConA-induced clustering may reflect the interactions of membrane proteins with extracellular matrix, we also analysed the effect of several extracellular matrix proteins on the in-situ activity of ecto-5'-nucleotidase in WM9 cells and found that tenascin C strongly inhibited ecto-5'-nucleotidase activity and adenosine generation from AMP. We also developed WM9 cells with reduced ecto-5'-nucleotidase expression and tested differences in cell adhesion on various extracellular matrix proteins. WM9 cells attached significantly weaker to tenascin C layer. These observations indicate that expression of ecto-5'-nucleotidase correlates with a number of metastasis-related markers and thus may have a function in this process. Furthermore, our data suggest that, in addition to generating adenosine, ecto-5'-nucleotidase may have independent roles in adhesion and interaction with extracellular matrix components in melanoma.

Topics: 5'-Nucleotidase; Cell Adhesion; Cell Line, Tumor; Centrifugation, Density Gradient; Concanavalin A; Extracellular Matrix Proteins; Humans; Hyaluronan Receptors; Integrin alpha3; Iohexol; Melanocytes; Melanoma; Membrane Microdomains; Membrane Proteins; Neoplasm Staging; Tenascin

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: alpha5-, beta1-, beta3-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. alphav-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.

Topics: 5'-Nucleotidase; Antigens, Neoplasm; Cell Adhesion; Cell Line, Tumor; Cluster Analysis; Concanavalin A; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Integrin alphaV; Melanoma; Membrane Microdomains; Neoplasm Metastasis

Tissue factor (TF), the membrane-bound glycoprotein that normally initiates the coagulation pathway, is expressed on the surface of various cells including endothelial cells, fibroblasts, monocytes and tumor cells. We recently reported that hemoglobin (Hb) enhances TF expression and procoagulant activity on TF-bearing human A375 malignant melanoma cells. To elucidate the mechanism of Hb-induced TF expression, we studied the interaction between purified TF from human A375 malignant melanoma cells and Hb. Selective binding of highly purified melanoma cell TF-apoprotein to Hb was demonstrated under native conditions using a dot-immunobinding assay and under denaturing conditions by Western blotting. The complex formation between purified melanoma cell TF-apoprotein and Hb was also demonstrated by the binding of fluid-phase Hb to immobilized TF-apoprotein (0-2.0 microg/ml) in an enzyme-linked immunosorbent assay. The binding was specific, concentration-dependent, saturable and inhibited significantly (60%) by Concanavalin-A. Hb enhanced the factor X-activating procoagulant activity of melanoma cell TF in a concentration-dependent manner, but had no effect on recombinant human TF. Concanavalin-A and wheat germ agglutinin significantly (60%) inhibited the Hb-induced procoagulant activity of malignant cell TF. We conclude that TF-apoprotein selectively binds Hb, most probably via the carbohydrate moieties (alpha-d-glucosyl; alpha-d-mannosyl and N-acetyl-beta-d-glucosaminyl residues) of TF, and enhances its procoagulant activity. The physiological significance of this interaction remains to be established.

Topics: Apoproteins; Concanavalin A; Enzyme Activation; Factor X; Glycosylation; Hemoglobins; Humans; Lectins; Melanoma; Neoplasm Proteins; Phytohemagglutinins; Protein Binding; Protein Interaction Mapping; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Thromboplastin; Tumor Cells, Cultured; Wheat Germ Agglutinins

Nitric oxide (NO) is known to facilitate tumour metastasis through the promotion of angiogenesis, vascular dilation, platelet aggregation, etc. In the present study we explored its novel role in producing dysfunction of the host immune system in the metastasis of murine metastatic melanoma B16-BL6 cells. A significant reduction in the mixed lymphocyte reaction (MLR) was observed in the spleen cells from B16-BL6-bearing mice, but not in those from mice bearing the parent cell B16. When B16-BL6 cells were added in vitro to the MLR, a significant decrease was also found, even when they were co-cultured with the lymphocytes in two compartments of a Transwell chamber separated by an 8.0 microm filter. The supernatant from cultured B16-BL6 but not B16 cells, which had a greatly increased NO activity, significantly inhibited concanavalin A- and lipopolysaccharide-induced lymphocyte proliferation. A remarkably higher expression of inducible NO synthase (iNOS) was detected in B16-BL6 cells than in B16 cells. Nomega-Nitro-l-arginine (l-NNA), a NO synthase inhibitor and superoxide dismutase, significantly antagonized the above inhibition by B16-BL6 cells, while l-arginine, a NO precursor, and S-nitroso-N-acetyl-d,l-penicillamine, a NO donor, strengthened the inhibition. Furthermore, l-NNA significantly inhibited lung metastasis of B16-BL6 cells, while l-arginine tended to enhance the metastasis. The cytotoxicity of B16-BL6-specific T-cells was significantly decreased by pre-culture with B16-BL6 cells in a Transwell chamber or the culture supernatants of B16-BL6 cells, whereas l-iminoethyl-lysine, a selective inhibitor of iNOS, showed a significant recovery from the disease. These results suggest that NO released by metastatic tumour cells may impair the immune system, which facilitates the escape from immunosurveillance and metastasis of tumour cells.

Topics: Animals; Arginine; Blotting, Western; Coculture Techniques; Concanavalin A; Cytotoxicity Tests, Immunologic; Female; Lipopolysaccharides; Lung Neoplasms; Lymphocyte Activation; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; T-Lymphocytes; Tumor Cells, Cultured; Tumor Escape

Lectin binding is known to be able to elicit signalling events relevant for various aspects of cell physiology. The influence of lectin binding on melanoma cells remains relatively unexplored. The aim of our study was to investigate the in vitro effects of five plant lectins, namely peanut (PNA), wheat germ (WGA), concanavalin A (Con-A), Griffonia simplicifolia (GSA-IA4) and Phaseolus vulgaris (PHA-L) agglutinins, on the cell proliferation of melanoma cell lines (SK-MEL-28, HT-144 and C32) cultured in media supplemented with either 10% or 1% fetal calf serum (FCS). Cell proliferation was assessed by means of the tetrazolium derivative reduction (MTT) assay. Four lectin concentrations were tested, namely 0.05, 0.5, 5 and 50 micrograms/ml, in four experimental settings, namely 1, 3, 5 and 7 days after the addition of each lectin to the culture media. Determination of the cell gain compartment (percentage of cells in the S and G2 phases of the cell cycle) was done by means of digital cell image analysis assessed on Feulgen-stained nuclei. Our results demonstrated that of the five lectins under study, four had a globally significant dose-dependent toxic effect on melanoma cell proliferation. The fifth lectin, PNA, had a significant stimulatory effect on the C32 cell line. Low doses of lectins may produce a transient increase in cell proliferation. Increasing the FCS from 1% to 10% in the culture media significantly antagonized lectin-induced toxicity in the three cell lines. The cell kinetics measurements showed that the inhibition of cell growth was merely due to cell death. The present data strongly suggest that some lectins might influence the proliferation of melanoma cells. In addition, because lectins are present in our diet and are able to pass into the systemic circulation, we speculate that lectins may exert an influence on melanoma growth under clinical conditions.

Topics: Cell Division; Cell Line; Cell Nucleus; Concanavalin A; Culture Media; DNA, Neoplasm; Humans; Kinetics; Lectins; Melanoma; Peanut Agglutinin; Phytohemagglutinins; Time Factors; Tumor Cells, Cultured; Wheat Germ Agglutinins

Autotaxin is a 125kD autocrine motility factor that stimulates both random and directed motility in producing the human A2058 melanoma cell line. The recently cloned autotaxin has been demonstrated to bind strongly and specifically to concanavalin A (con A). In this study, we show that the oligosaccharide side chains on autotaxin are exclusively asparagine linked, since N-glycosidase F, but not neuraminidase or O-glycosidase, decreases the protein molecular mass to 100-105kD, which is the calculated molecular mass of the deduced autotaxin polypeptide. Furthermore, removal of oligosaccharide side chains by N-glycosidase F can be performed under mild conditions that retain motility-stimulating activity, suggesting that the oligosaccharide side chains are not necessary for autotaxin to activate its receptor. Finally, when melanoma cells are treated with inhibitors of carbohydrate processing, such as N-methyl-1-deoxynojirimycin, 1-deoxymannojirimycin and swainsonine, they still secrete a motility-stimulating autotaxin. Therefore, the carbohydrate side chains on autotaxin are not necessary to stimulate motility; however, they may still play a role in folding, secretion or maintenance of the active conformation of the protein.

Topics: Amidohydrolases; Blotting, Western; Cell Movement; Concanavalin A; Glucose-6-Phosphate Isomerase; Glycoproteins; Glycosylation; Humans; Melanoma; Multienzyme Complexes; Neoplasm Invasiveness; Oligosaccharides; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Phosphodiesterase I; Phosphoric Diester Hydrolases; Protein Folding; Pyrophosphatases; Stimulation, Chemical; Structure-Activity Relationship; Tumor Cells, Cultured

In the present study we tested the phenotypic profile as well as several immunological responses of peripheral blood mononuclear cells (PBMC) isolated from melanoma patients. These patients underwent chemotherapy with dacarbazine and carboplatin from day 1 to day 22, followed by immunotherapy of low-dose recombinant interleukin-2 and recombinant interferon alpha administered subcutaneously from day 36 to day 75. The PBMC from 14 patients were isolated on day 0 before chemotherapy, on day 36 after chemotherapy and on day 76 after immunotherapy. After chemotherapy, a decrease in CD16+ cells and increase in CD3+ and CD4+ cells correlated with a significant decrease in the generation of lymphokine-activated killer (LAK) activity. After immunotherapy, an increase in CD16+ cells correlated with an increase in the induction of LAK activity. A comparison between responding and non-responding patients revealed statistically significant differences in LAK activity of PBMC and response to concanavalin A following chemotherapy, and in the percentage of CD8+ cells following immunotherapy. Our results point toward the value of continuing such a study on a larger population of cancer patients in order to select the appropriate bioassays for monitoring and predicting the clinical responsiveness to combined therapies.

Topics: Adult; Aged; Antigens, CD; Antineoplastic Combined Chemotherapy Protocols; Carboplatin; Cell Division; Chemotherapy, Adjuvant; Concanavalin A; Dacarbazine; Dose-Response Relationship, Drug; Female; Humans; Immunophenotyping; Immunotherapy; Interferon Type I; Interleukin-2; Killer Cells, Lymphokine-Activated; Leukocytes, Mononuclear; Male; Melanoma; Middle Aged; Phytohemagglutinins; Pokeweed Mitogens; Recombinant Proteins

We studied in a homologous system the procoagulant activity of human tumor cells cultured "in vitro" (1402 primary melanoma, Me 7110/2 metastatic melanoma, Hep G2 hepatoma and GLC1 small cell lung carcinoma) or of cells freshly isolated from different human tumor tissues. Tumor cells cultured "in vitro" possessed and released a factor VII dependent procoagulant activity, which was inhibited by concanavalin A and unaffected by iodoacetamide or HgCl2. The activity released by the cells of metastatic melanoma was higher than that released by the cells of the primary tumor. On the contrary, cancer cells isolated from tumor tissues possessed and released a factor VII independent activity which was inhibited by iodoacetamide of HgCl2 and was not modified by concanavalin A. Therefore, different methods for the preparation of tumor cell suspensions have to be used for the study of tumor procoagulants, since their expression depends very largely on the source of tumor cells. Furthermore, cultured human tumor cells are not an appropriate model for the "in vivo" procoagulant effect of tumor cells.

Topics: Blood Coagulation Factors; Concanavalin A; Cysteine Endopeptidases; Factor VII; Factor X; Humans; Iodoacetamide; Melanoma; Mercuric Chloride; Neoplasm Proteins; Neoplasms; Neoplastic Stem Cells; Tumor Cells, Cultured

Only a few autoantigenic human tumor antigens have been purified and characterized. We employed the monoclonal antibody (MAb) TA99 to isolate, purify and partially characterize an autoantigenic intracellular glycoprotein, gp75, from human melanoma cells. The gp75 antigen is the most abundant glycoprotein expressed in human melanocytes and pigmented melanomas and is the human homologue of the mouse brown locus gene product. Differential solubilization of melanoma membrane fraction and subcellular fractionation of pigmented melanoma cells showed that gp75 is an integral membrane protein localized to melanosomes. The gp75 glycoprotein eluted as a broad peak during ion exchange chromatography and appeared as a protein with broad pI (pI 5.5-5.9), consistent with charge microheterogeneity. gp75 also exhibited heterogeneity of binding to concanavalin A. Tyrosine hydroxylase (tyrosinase) activity co-purified with gp75 during membrane solubilization and anion exchange and Con A chromatography. However, most tyrosine hydroxylase activity could be dissociated from gp75 antigen during MAb TA99 affinity chromatography. TA99 did not immunoprecipitate or deplete tyrosine hydroxylase activity from lysates of human melanoma cells. Attempts to obtain N-terminal amino acid sequence of purified gp75 were not successful due to blocked N-terminus. Amino acid composition of gp75 was similar to that of tyrosinase. Physicochemical similarities and limited identity in the primary structure between gp75 and tyrosinase support the conclusion that the gp75 antigen does not exhibit tyrosine hydroxylase activity, but is a member of a tyrosinase-related family of proteins.

Topics: Amino Acids; Antibodies, Monoclonal; Antigens, Neoplasm; Autoantigens; Cell Membrane; Chromatography; Concanavalin A; Electrochemistry; Glycoproteins; Humans; Immunosorbent Techniques; Isoelectric Point; Melanocytes; Melanoma; Molecular Weight; Tyrosine 3-Monooxygenase

The concept of cell adhesiveness was analyzed by looking for correlations between the adhesive behavior and measurable biological properties of different cell populations. Ten established lines of melanoma cells were assayed for passive deformability (by micropipet aspiration), active spreading (by measuring the height/diameter ratio after incubation on different surfaces), density and mobility of concanavalin A binding sites (by quantitative analysis of fluorescence microscopic images), spontaneous and concanavalin A-mediated agglutination (by measuring the number of cell conjugates resisting calibrated shearing forces), and binding to glass capillary tubes (with a quantitative assay of binding strength). Forty-four different parameters were thus measured, and each set of determinations was repeated 2 or 3 t at different days on each cell line. Analysis of variance was performed to assess the capacity of each parameter to discriminate between different lines. Correlations between different parameters were studied in order to understand a possible influence of cell intrinsic properties on the behavior of individual cells. The following conclusions were suggested by experimental data 1. Cell spreading ability, resistance to slow deformation within a micropipette and ability to form shear-resistant bonds, are independent properties. It is therefore suggested that different mechanisms rule the cell deformations on time scales of several minutes, tens of seconds, and fractions of a second. 2. Cell spreading ability may effectively influence binding strength only when adhesive stimuli are low, since in this case, cell stiffness is likely to impair the formation of extensive contact areas. 3. Individual cells may display marked heterogeneity within a given population, that emphasizes the danger of using averaged parameters to predict rare events (such as metastasis formation). 4. The most useful parameters to discriminate between different cell lines were, spreading ability and shear-resistant lectin agglutination, and substrate adhesion. It is concluded that cell adhesion is influenced by several measurable cellular properties that may display independent variations. The importance of a given parameter depends on the conditions of bond formation and rupture.

Topics: Cell Adhesion; Cell Aggregation; Cell Membrane; Concanavalin A; Humans; Melanoma; Microscopy, Fluorescence; Tumor Cells, Cultured

Anti-tumour effector cells were generated through 4 days culture of normal C57BL/6 splenocytes in a medium containing concanavalin A supernatant and then fractionated with Dolichos biflorus lectin (DBA) into DBA+ (agglutinable with DBA) and DBA- (non-agglutinable with DBA) cells. The DBA- cells, infused intravenously into mice together with B16 melanoma cells, or adoptively transferred into mice 3 days after the injection of B16 cells, caused a marked decrease in the number of lung nodules, while the DBA+ cells exerted no effect. On the other hand, the DBA+ cells exhibited higher cytolytic activity in vitro than the DBA- cells in short-term 51Cr-release assays. Then, we analysed the mechanism of the strong anti-tumour activity of DBA- cells in vivo. We found that DBA- cells showed higher response to recombinant interleukin-2 (rIL-2) than DBA+ cells and proliferated very well with a small amount of IL-2. In addition, DBA- cells adhered more strongly to lung endothelial cells than DBA+ cells in response to rIL-1 or rTNF. Furthermore, DBA- cells produced larger amounts of macrophage activating factor (MAF) including IFN-gamma when cultured with B16 melanoma. Taken together, our results show that DBA- cells are effective in reducing experimental pulmonary metastases not only by the direct lytic activity but also by the indirect killing activity through the activated macrophage.

Topics: Animals; Cell Adhesion; Cell Separation; Concanavalin A; Endothelium, Vascular; Female; Immunization, Passive; Interleukin-2; Killer Cells, Lymphokine-Activated; Lectins; Lung Neoplasms; Lymphokines; Macrophage-Activating Factors; Melanoma; Mice; Mice, Inbred Strains; Plant Lectins; Spleen

Human melanoma-specific, HLA restricted, cytotoxic T-cell lines can be generated by in vitro stimulation and culturing of peripheral lymphocytes, or lymph node cells, with autologous or HLA-A region matched melanomas in the presence of a low concentration (5 U/ml) of IL-2. Stimulation is followed by a period of clonal expansion and differentiation into cytotoxic T-cells specific for melanoma. We investigated the effect of the PKC modulating drug phorbol dibutyrate combined with the calcium ionophore Ionomycin on growth and differentiation of the cell lines. The growth of the T-cell lines was substantially augmented in the presence of the drugs with increases of 10-fold or more in clonal expansion by 3 weeks of culture. The cell lines were IL-2 dependent for growth in the presence or absence of the drugs and the phenotypic distribution remained predominantly CD3+ T-cells of mixed CD4 and CD8 phenotypes. In spite of the increased rate of growth in the presence of the drugs, autologous melanoma-specific cytotoxicity was almost completely abrogated in those cultures. The cells were, however, nonspecifically lytic in the presence of concanavalin A. The melanoma-specific cytotoxic response was completely restored following culture with IL-2 alone. The results suggest that the human tumor-specific cytotoxic T-cell response can be induced and amplified in the presence of immune modulating drugs.

Topics: Antigens, CD; Cell Line; Concanavalin A; Cytotoxicity, Immunologic; Humans; Interleukin-2; Ionomycin; Melanoma; Phorbol 12,13-Dibutyrate; T-Lymphocytes, Cytotoxic

A simple and sensitive chromogenic microtitre plate assay for glycoprotein enzymes is described, using melanoma tissue plasminogen activator (t-PA) as a model enzyme. The assay is based on the binding of t-PA to immobilised lectin and quantitating the bound enzyme with plasminogen, fibrinogen fragments and chromogenic substrate S-2251 on an ELISA plate reader. Seven different lectins were examined for the binding of t-PA, and of these, concanavalin A was chosen for subsequent studies. The specificity of this binding can be inhibited dose-dependently in the presence of D-mannose and methyl alpha-D-mannoside, but not by D-glucose and D-lactose. The lower limit of the sensitivity of this assay is about 0.5 IU/ml. Comparison of the dose-response curves indicates that the sensitivity of this assay method is very similar to that of bioimmunoassay using anti-t-PA IgG to capture the antigen. The applicability of this method to other glycoprotein enzymes was also evaluated using alkaline phosphatase from bovine mucosa. The specificity of this method was related to the choice of substrate and this was shown by analysis of a mixture of t-PA and alkaline phosphatase. It is suggested that this assay can be adapted for the analysis of complex glycoprotein mixtures with the appropriate choice of lectin and substrate.

Topics: Alkaline Phosphatase; Biological Assay; Concanavalin A; Fibrinogen; Glycoproteins; Lectins; Melanoma; Plasminogen; Tissue Plasminogen Activator; Tumor Cells, Cultured

The method of skin window was used in the investigation of cell reaction of healthy volunteers (25 persons) and patients with malignant melanoma (12 cases), breast cancer (18 cases), and with various malignant lymphomas (6 cases) to a local application of the lectin Concanavalin A (Con A, concentration of 10 mg/ml of saline, 0.02 ml per scarification). The obtained results were compared with cell reaction after the application of saline alone. Correlation between cell reaction and clinical state of patients was studied as well. No significant difference in the amount of lymphocytes after the application of Con A was observed between the group of healthy individuals and the group of patients with malignant tumors. On the contrary, the reaction of neutrophil granulocytes to Con A was significantly lower in cancer patients than in healthy volunteers. After the application of Con A, all preparations showed a higher absolute amount of all cells. Con A induced a significant increase in the amount of lymphocytes in patients after therapy and in poor clinical state. The persons with good clinical state reacted by a slight increase or decrease in the number of lymphocytes to the application of lectin.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Concanavalin A; Humans; Leukocyte Count; Lymphocyte Activation; Lymphocytes; Lymphoma; Melanoma; Mitogens; Skin; Skin Tests

We studied the effects on platelet function of cells isolated from freshly dissociated human tumor tissues (11 breast carcinomas, 9 colon carcinomas and 1 lymph node metastasis from melanoma) obtained at surgery as compared with cultured human tumor cells: namely, human melanoma 1402 cell line derived from a primary tumor and two lines derived from lymph node metastases (ME 7110/2 and Me 665/1) as well as a human hepatoma cell line (Hep G2). The three melanoma cell lines activated platelets by producing ADP, as evidenced by the inhibitory effect of apyrase and by the direct measurement of the agonist in the supernatants of tumor cell suspensions; this production was much greater by the cells derived from metastases than by the cells derived from the primary tumor. On the other hand, aggregation induced by Hep G2 hepatoma cells was unaffected by apyrase and was inhibited by hirudin or concanavalin A, suggesting that the cells aggregate platelets by producing thrombin, probably through tissue factor activity of the cells themselves. Cells isolated from 16 of the 21 human tumor tissues possessed a potent platelet-aggregating effect, which was not inhibited by apyrase, hirudin or concanavalin A, but was virtually abolished by the cysteine protease inhibitors iodoacetic acid or p-hydroxymercuri-phenylsulfonate. Collectively, our data demonstrate that cells isolated from freshly dissociated tumor tissues activate platelets through tumor-associated cysteine proteinases rather than by the ADP- or thrombin-dependent mechanisms characteristic of cultured human tumor cell lines.

Topics: Adenosine Diphosphate; Adult; Apyrase; Blood Platelets; Breast Neoplasms; Cell Communication; Cell Separation; Colonic Neoplasms; Concanavalin A; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Hirudins; Humans; Melanoma; Platelet Aggregation; Tumor Cells, Cultured

We previously demonstrated that the cells of lymph nodes near to a melanoma respond well to stimulation by mitogens, alloantigens, and interleukin 2 than do nodes further away. In this study we examined suppressor T-cell activity in nodes at different distances from primary melanoma, using a concanavalin A (Con A) suppressor cell assay. Tumor-free regional nodes were classified as proximal, intermediate, and distal relative to primary melanoma. Lymph node lymphocytes (LNL) were stimulated with 50 micrograms/ml Con A for 48-72 h, inactivated, and then mixed with autologous peripheral blood lymphocytes. The peripheral blood lymphocyte-LNL mixtures were stimulated with phytohemagglutinin for 3 days. Proliferation was measured by [3H]thymidine uptake during the final 18 h of culture. In 13 patients, Con A-treated LNL from nodes near to tumor were more suppressive of the peripheral blood lymphocyte response to phytohemagglutinin than those from nodes located further from tumor. T-lymphocyte subset assessment before and after Con A treatment of LNL showed no significant changes in T4:T8 ratios. Con A-induced suppressor cells could be maintained in culture in the presence of recombinant interleukin 2 and retained their suppressive activity. LNL not exposed to Con A and maintained in culture with interleukin 2 did not show suppressor cell activity. Suppressor cell activity thus contributes to the weak immune reactivity of lymph nodes near to melanoma.

Topics: Concanavalin A; Humans; Interleukin-2; Lymph Nodes; Melanoma; Mitomycin; Mitomycins; T-Lymphocytes; T-Lymphocytes, Regulatory

Twenty patients with malignant melanoma were treated with cyclophosphamide (100, 300 or 500 mg/m2 i.v.) in pilot studies to determine whether such treatment affected suppressor-cell activity. Delayed hypersensitivity to dinitrochlorobenzene and other recall antigens, the serological response to primary immunization with pneumococcal or influenza virus antigens, and the serological response to melanoma antigens were found to be normal and were not changed by cyclophosphamide (Cy) treatment. In vitro assays for production of antibodies against sheep red blood cell (SRBC) antigens, reactivity in the mixed lymphocyte culture reaction, and induction of suppressor cells by Concanavalin-A (Con-A) yielded abnormal results as a consequence of increased suppressor-cell activity in eleven, three, and nine patients, but no concordance was seen between results with the three assays prior to treatment. After treatment with Cy, the results of these tests became normal in seven, three, and seven of the patients with previously abnormal results, independent of the dose given. Examining all patients as a group, a statistically significant effect was seen after treatment with Cy on days 14 and 21 in the assay for the production of antibodies against SRBC, and days 7, 14, 21, 28, and 35 in the assay for Con-A-induced suppressor cells. The decrease in suppressor-cell activity was largely restricted to patients who showed increased suppressor-cell activity prior to treatment with Cy. Our results suggest that increased suppressor-cell activity in patients with malignant melanoma does not affect immune reactions generally but is selective, and that the anti-suppressor-cell effect of Cy is restricted to reactions with increased suppressor-cell activity to start. Based on these results, attempts at increasing the immune response to melanoma antigen vaccines administered between 7 and 35 days after treatment with Cy seem justified.

Topics: Antibody Formation; Concanavalin A; Cyclophosphamide; Dermatitis, Contact; Humans; Hypersensitivity, Delayed; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Melanoma; Phytohemagglutinins; T-Lymphocytes, Regulatory

Topics: Adult; Breast Neoplasms; Concanavalin A; Female; Humans; Immunity, Cellular; Male; Melanoma; Middle Aged; Skin; Skin Neoplasms; Skin Window Technique

Clinical observations suggest that tumors grow more slowly in aged subjects. To investigate the influence of age on tumor growth, we injected the same number of cultured B16 melanoma cells into C57BL/6 mice of various ages. B16 melanoma cells, inoculated s.c., grew more slowly in old (18-20-month-old) as compared to young (6-8-week-old) mice. In young tumor-bearing mice there was a significant increase in the number and the proliferative response to phytohemagglutinin and concanavalin A of splenic T-cells as compared to old tumor-bearing animals. There was no difference in the response of B-lymphocytes from old and young tumor-bearing mice to lipopolysaccharide. The positive association between T-cells and the rate of tumor growth was also suggested by the slower growth of melanoma cells in thymectomized or thymectomized and anti-theta antiserum-treated young mice. Finally, the age-associated difference in tumor growth could be transferred by spleen cells from old or young mice to thymectomized and lethally irradiated syngeneic young animals. Young mice with rapidly growing B16 melanoma tumors have increased numbers and proliferative responses of thymic-derived lymphocytes. It is likely that T-cells or their products facilitate the growth of B16 tumor cells.

Topics: Aging; Animals; Cell Division; Cell Line; Concanavalin A; Melanoma; Mice; Mice, Inbred C57BL; Phytohemagglutinins; Spleen; T-Lymphocytes; Thymus Gland

Monoclonal antibodies (MoAb) to human melanoma have demonstrated a limited ability to cause tumor regression in humans when used alone or when coupled to gamma-emitting radioisotopes. We have evaluated heteroaggregates containing antilymphocyte antibodies crosslinked to antimelanoma monoclonal antibodies recognizing p97, a transferrin-like molecule (MoAb 96.5). When coupled to antibodies recognizing T3 (CD3, part of the T-cell receptor complex for antigen) or to 3G8, an antibody recognizing the Fc receptor present on large granular lymphocytes and granulocytes (CD16), significant induction of effector target crosslinks and target cell lysis could be obtained. Effector cells incubated for 24 hr with recombinant IL-2 were coated with the crosslinked reagents and tested for conjugate formation and for cytotoxicity in a 4-hr assay with chromium-labeled targets. Marked increases in conjugation to autologous tumor (47.0% compared to 11.8%) was demonstrated with E+ cells using the T3-coupled MoAb and with E- cells using the Fc receptor-coupled MoAb (22.6% compared to 11.2%). When tested in sequential cytotoxicity assays using unseparated effector cells incubated for 1, 2, and 3 days in IL-2, lytic activity was less than 2, less than 2, and 3.3 LU/10(6) cells for cells incubated in monomeric 96.5; 2.6, 5.3, and 50 LU/10(6) cells incubated in 96.5 crosslinked T3; and less than 2, 3.6, and 8.0 LU/10(6) cells for cells incubated with 96.5 crosslinked to 3G8. Similar findings were noted in two other experiments. Heteroaggregates such as these may be useful in conjunction with the transfer of IL-2-activated cells or with IL-2 alone in immunotherapy trials.

Topics: Antibodies, Monoclonal; Antibodies, Neoplasm; Concanavalin A; Cross-Linking Reagents; Cytotoxicity, Immunologic; Flow Cytometry; Humans; Immunotherapy; Interleukin-2; Lymphocytes; Melanoma; Receptors, Antigen, T-Cell; Receptors, Fc

Interleukin-2 (IL-2) production following concanavalin A stimulation and the response of peripheral blood mononuclear cells (PBMC) to both IL-2 alone and IL-2 plus indomethacin, a prostaglandin synthetase inhibitor, were examined in 16 melanoma patients and 12 healthy controls. Mean IL-2 production by PBMC in 11 melanoma patients with metastatic disease (Stage III) was significantly decreased compared with controls and was moderately decreased compared with five patients with resected nodal disease (Stage II). Indomethacin restored IL-2 production in Stage III PBMC to levels equivalent to that produced by control PBMC. The PBMC of stage III patients also produced 40 times more prostaglandin E2 than PBMC from controls or Stage II patients. Indomethacin plus IL-2, but not IL-2 alone, was capable of restoring the low blastogenic response of PBMC of Stage III patients to normal levels. Hence, these data emphasize the importance for using IL-2 along with indomethacin for in vivo immunorestoration in disseminated melanoma.

Topics: Concanavalin A; Dinoprostone; Female; Humans; In Vitro Techniques; Indomethacin; Interleukin-2; Lymphocyte Activation; Lymphocytes; Male; Melanoma; Prostaglandins E

Thirty radically operated patients with a locally advanced malignant melanoma were given adjuvant levamisole orally 50 mg three times a day on two days a week for one to 40 months in order to prevent recurrence of melanoma. During levamisole treatment the number of E- and EAC-rosette forming cells, proliferative responses of lymphocytes to phytohemagglutinin, concanavalin A and to purified protein derivative of tuberculin were followed at two to four month intervals. All patients were clinically followed at least for five years or to the recurrence of melanoma. Only slight variations occurred in the number of E- and EAC-rosette forming cells and in the responses to mitogens during levamisole treatment. Five out of 30 patients were alive without a recurrence at five years after starting adjuvant levamisole treatment. We conclude that adjuvant levamisole treatment is of no benefit in radically operated melanoma with satellites or metastases in the regional lymph nodes.

Topics: B-Lymphocytes; Concanavalin A; Female; Humans; Levamisole; Lymphatic Metastasis; Male; Melanoma; Middle Aged; Phytohemagglutinins; Recurrence; Rosette Formation; T-Lymphocytes; Time Factors; Tuberculin

It has repeatedly been proposed that fibrin plays a role in tumor growth and metastasis. Among tumor cell products or activities which may promote clot formation, cancer procoagulant (CP), a direct activator of coagulation factor X, has been suggested to be selectively associated with the malignant phenotype. We report here the enzymatic and immunological identification of this cysteine proteinase procoagulant in extracts and cells from human melanoma. CP activity was independent of both the intrinsic and extrinsic pathways of blood coagulation, using factor IX and factor VII deficient plasmas, and was inhibited by the cysteine proteinase inhibitors iodoacetamide and HgCl2. CP activity was detectable in extracts and cell suspensions from all 32 patients studied and was higher in extracts from metastases (14.8 +/- 3.9 units/mg protein) than from the primary tumors (3.7 +/- 1.0 units/mg protein). CP activity was not affected by an anti-apoprotein III antibody or by concanavalin A, a known inhibitor of thromboplastin. In contrast, no CP activity or antigen was detected in extracts from six benign melanocytic lesions. The procoagulant activity was dependent on factor VII and was inhibited by anti-apoprotein III antibody and by concanavalin A, properties that suggest that the procoagulant was tissue thromboplastin. These data indicate that CP can be expressed by human tumor cells and that, among melanotic lesions, its presence is associated with the malignant phenotype and its activity is particularly high in metastatic cells.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Blood Coagulation Factors; Concanavalin A; Cysteine Endopeptidases; Endopeptidases; Female; Humans; Male; Melanoma; Mercuric Chloride; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins

Lectin binding glycoproteins of 5 human malignant melanoma cell lines (HMMCL), differing in their ability to grow subcutaneously in athymic nude mice, were compared by electrophoresis of total cellular proteins and subsequent incubation of SDS-poly-acrylamide gel with 125I-labelled lectins. Despite the similarity between the protein profiles of the different HMMCL, Concanavalia ensiformis agglutinin (ConA), wheat-germ agglutinin (WGA) and peanut agglutinin (PNA) revealed differences in their glycoprotein expression, in contrast with Ulex europaeus agglutinin I (UEA I). A great diversity was observed in the electrophoretic mobilities and/or staining intensities of ConA and WGA binding glycoproteins of HMMCL. However, neither ConA-reactive glycoproteins nor WGA-reactive glycoproteins could be detected that were characteristic of HMMCL with high tumorigenicity (HT) or low tumorigenicity (LT). In contrast, the expression of two cell-surface PNA binding glycoproteins appeared to be related to the tumorigenic phenotype of HMMCL. One of them, with an apparent molecular weight of 190 kDa, was only detected in the LT cell lines. The other, with an apparent molecular weight of 60 kDa, was detected in all HMMCL but became strongly labelled after neuraminidase treatment only in the HT cell lines. Thus, the expression of glycoproteins rich in terminal galactose residues may characterize human melanoma cells with different tumorigenic behavior.

Topics: Concanavalin A; Glycoproteins; Humans; Lectins; Melanoma; Membrane Proteins; Molecular Weight; Neoplasm Proteins; Peanut Agglutinin; Plant Lectins; Wheat Germ Agglutinins

We have reported previously that pulmonary alveolar macrophages (PAMs) from individuals with lung cancer and active chronic pulmonary diseases were cytotoxic to tumor cells in vitro, whereas PAMs from normal individuals or patients with acute pulmonary disorders were noncytotoxic. In the present study, we evaluated 20 PAM preparations for both suppressor and cytotoxic functions to determine if PAMs could function as suppressor cells and, if so, whether a correlation between the two functions exists. Cytotoxicity was assessed in a 60-hr cytotoxicity assay against [3H]proline-prelabeled human melanoma target cells. More than 20% cytotoxicity was considered to be significant. Suppressor activity was measured by determining whether admixing PAMs at various ratios with autologous or allogeneic mononuclear cells could suppress concanavalin A-induced blastogenesis by T-lymphocytes. At least 50% suppression was considered to be significant. Of the 20 specimens evaluated, 13 were cytotoxic and 5 of these exhibited suppressor activity. None of the 7 noncytotoxic PAM preparations had suppressor activity. Suppression was nonspecific and not HLA restricted, since autologous and allogeneic mononuclear cells were inhibited to a similar extent. Suppression was probably not due to prostaglandin production by the PAMs since assays were performed under optimal conditions and required extremely high concentrations of prostaglandins. A significant correlation between suppressor and cytotoxic activity was found. Suppression was observed only with PAM specimens that were also highly cytotoxic to tumors, but not all cytotoxic PAM specimens were suppressive. Whether these actions reflect different levels of activation of PAMs or are the properties of different macrophage subsets remains to be clarified.

Topics: Concanavalin A; Cytotoxicity, Immunologic; DNA Replication; Humans; Immune Tolerance; Lung; Lung Diseases; Lymphocyte Activation; Macrophages; Melanoma; Monocytes; T-Lymphocytes

The structural alteration of carbohydrate moieties of tyrosinases associated with the depigmentation process induced by glycosylation inhibition has been investigated by using concanavalin A (Con A) affinity chromatography. Con A affinity chromatography of deoxycholate-solubilized large and small granule fractions shows that while all tyrosinases found in control B-16 cells exhibit affinity to Con A lectin, there is an emergence on non-Con A binding tyrosinases in the unpigmented cells induced by glycosylation inhibitors, such as tunicamycin and glucosamine. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, control tyrosinase activity forms 2 distinct bands consisting of T1 and T3. But tyrosinases from the unpigmented cells lose T3 tyrosinase and are resolved into a few different molecular weight components, one of which is Con A affinitive T1 tyrosinase and the others are non-Con A affinitive tyrosinases with smaller molecular weights than the T1 tyrosinase. These findings suggest that altered structures of carbohydrate moiety in tyrosinase molecules play a role in the induction of loss of membrane-binding capacity of tyrosinases, resulting in the loss of melanization in pigment cells.

Topics: Animals; Carbohydrates; Catechol Oxidase; Chromatography, Affinity; Concanavalin A; Cycloheximide; Electrophoresis, Polyacrylamide Gel; Melanins; Melanoma; Mice; Monophenol Monooxygenase; Tunicamycin

Cloned lines from 4 different families of B-16 melanoma cells were heated repeatedly in tissue culture at 45 degrees C with step-up time intervals. These lines included the partially heat-resistant lines previously selected at 43 degrees C(HR 43 degrees) from wheat-germ-agglutinin-resistant mutant (WGAR, HR43 degrees), concanavalin-resistant mutant (conR-HR 43 degrees), ricin-resistant mutant (ricinR, HR 43 degrees), and parental B-16 (B-16, HR 43 degrees) cells. The heating cycles were repeated 7 to 11 times at 45 degrees C increasing from 45 min to 150 min with 3 weeks of culturing at 37 degrees C between cycles. Heat resistance in most cases increased progressively with each heating step. An extensive library of mutants was thus generated, varying in the degree of heat resistance and the apparent stability. HR variants from the WGAR family appeared to be the most resistant and the most stable. The heat-resistant phenotype was expressed not only by increased survival after a normally lethal heat dose, but also by protection against heat-mediated suppression of proteins and DNA syntheses. Protein synthesis in the heat-resistant cells was not only less suppressed by heat shock, but also recovered more rapidly after removal of shock. Clinical implications of these results and the potential usefulness of the mutant lines for genetic studies are discussed.

Topics: Adaptation, Physiological; Animals; Cell Line; Cell Survival; Concanavalin A; Drug Resistance; Hot Temperature; Melanoma; Mice; Mutation; Phenotype; Ricin; Time Factors

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.

Topics: Adult; Animals; Antibodies, Monoclonal; Antigens, Surface; Cell Line; Complement System Proteins; Concanavalin A; Cytotoxicity, Immunologic; Epitopes; Female; HeLa Cells; Humans; Iritis; Killer Cells, Natural; Kinetics; Lymphocytes; Male; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Middle Aged; Rabbits; Syndrome; T-Lymphocytes, Cytotoxic; Uveitis

Concanavalin A (con A) is a plant-derived lectin that has the capability of agglutinating malignant cells in vitro. We studied the binding of fluorescein isothiocyanate-conjugated con A to formalin-fixed, paraffin-embedded malignant melanomas and nevocellular nevi. Both malignant melanoma cells and nevus cells emitted partial or circumferential, cytoplasmic rim, apple green fluorescence. There was no demonstrable difference between fluorescence distribution or intensity between the two groups. Control, unstained tissue specimens yielded a brilliant nuclear and nucleolar yellow-green autofluorescence, which is peculiar to melanoma cells and rare to absent in nevus cells. Fluorescein isothiocyanate-conjugated con A provided no clear differentiation between malignant melanomas and nevocellular nevi in fixed tissue. However, characteristic melanoma cell autofluorescence may prove to be of benefit for differentiating malignant melanocytic from benign nevocytic lesions.

Topics: Binding Sites; Concanavalin A; Diagnosis, Differential; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Histocytochemistry; Humans; Melanoma; Nevus, Pigmented; Skin Neoplasms

Cell surface carbohydrate differences were observed between two human cell lines initiated from primary melanoma and metastasis of the same patient. Although total sialic acid content was similar in both cell lines, neuraminidase-released sialic acid was twice as high in metastatic cells than that of primary cells. One class of Concanavalin A binding sites with similar affinity constant was found in untreated and neuraminidase-treated cells in both cell lines. Before surface sialic acid release, the primary cell line expressed two classes of Ricinus lectin binding sites with high and low affinity; the cell line of metastatic origin had only one class of Ricinus lectin binding sites with low affinity. After neuraminidase treatment, the number of Ricinus lectin binding sites with low affinity increased two- or three-fold in both cell lines, whereas the high-affinity binding sites were not observed in primary cells. The present data indicated that differences in surface sialic acid level modified the Ricinus lectin binding in two human melanoma cell lines. However, the ability of the cells to bind Concanavalin A was not changed.

Topics: Adult; Binding Sites; Cell Line; Concanavalin A; Galactose; Humans; Lectins; Male; Melanoma; Neuraminidase; Plant Lectins; Sialic Acids

Cellular glycoprotein carbohydrate chains of B16 melanoma sublines of various metastatic colonization capacities were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and direct lectin staining, combined with chemical modification of carbohydrate chains in situ. For these studies, we utilized B16 sublines selected for low (B16-F1) or high lung (B16-F10), high brain (B16-B15b), or high ovary (B16-O13) colonization properties, or high tissue invasiveness in vitro (B16-BL6). The major B16 cell surface sialoglycoproteins were of Mr approximately 115,000, approximately 90,000, approximately 82,000, and 60,000 to 65,000, and were detectable by periodate NaB3H4 labeling and binding of 125I-wheat germ agglutinin (WGA). Terminal sialic acid residues in the carbohydrate chains were responsible for WGA binding, since chemical removal of sialic acid prevented WGA labeling of the glycoproteins. However, removal of sialic acid residues followed by Smith degradation resulted in reappearance of WGA-binding sites on these sialoglycoproteins, indicating that the carbohydrate chains possessed at least one branching point at an outer alpha-mannosyl residue. This structural feature was further indicated by the failure of 125I-Lens culinaris hemagglutinin to bind to these sialoglycoproteins. The fact that the carbohydrate residues of the Mr approximately 115,000, approximately 90,000, and approximately 82,000 sialoglycoproteins were of the complex type was confirmed by their reactivity with 125I-Ricinus communis agglutinin I, which preferentially binds to Gal leads to GlcNAc sequences after removal of sialic acid in situ. In contrast, 125I-peanut (Arachis hypogaea) agglutinin, specific for Gal leads to GalNAc sequences, failed to bind to the major WGA-reactive sialoglycoproteins, but strongly interacted after removal of sialic acid with Mr approximately 51,000 and approximately 56,000 glycoproteins from sublines B16-F1, -F10, and -BL6 and with a Mr approximately 63,000 glycoprotein from sublines B16-F10, -BL6, -O13, and -B15b. Thus, the small, mucin-type carbohydrate chains were expressed almost exclusively on these lower Mr sialoglycoproteins, and very little on the Mr approximately 82,000, approximately 90,000, and approximately 115,000 sialoglycoproteins. Differences in lectin binding to glycoproteins were observed with different sublines. These glycoproteins included: (a) a WGA-binding Mr 60,000 to 75,000 sialoglycoprotein prominent

Topics: Animals; Carbohydrates; Cell Line; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Lectins; Melanoma; Mice; Neoplasm Metastasis; Peanut Agglutinin; Plant Lectins

Cell surface glycoconjugate patterns of human epidermal cells and of melanoma cells (MC) in primary culture derived from 11 primary and metastatic melanomas were investigated using fluorescent and horseradish peroxidase conjugated lectins for visualization at the light and electron microscopic level. The lectin labeling profiles of human melanocytes (M) and MC were found to be identical. According to their binding patterns, the lectins tested were grouped into three categories: (1) lectins binding to both keratinocytes (K) and M/MC, irrespective of neuraminidase pretreatment (concanavalin-A, wheatgerm agglutinin, succinylated wheatgerm agglutinin); (2) lectins binding to K but not to M/MC, irrespective of neuraminidase pretreatment (Ulex europaeus agglutinin I); (3) lectins binding to K, but to M/MC only after neuraminidase pretreatment (soybean, Helix pomatia, and peanut agglutinins). Untreated M were reactive for soybean and peanut agglutinins only at contact sites with K. Since the lectins from soybean, Helix, and peanut bind specifically to D-galactose and N-acetyl-D-galactosamine residues, we conclude that these particular glycoconjugates are normally masked by sialic acid on M/MC surfaces and can be unmasked by neuraminidase. These features, which have been previously observed in guinea pig M, appear to be interspecies surface markers of melanocytic cells which remain unaltered in the course of malignant transformation.

Topics: Animals; Concanavalin A; Epidermal Cells; Epidermis; Guinea Pigs; Humans; Immunologic Techniques; Keratins; Lectins; Melanins; Melanoma; Receptors, Mitogen; Wheat Germ Agglutinins

The suppressor activity induced by Concanavalin A (Con A) was evaluated in peripheral lymphocytes from 20 patients with solid malignant tumors of different origin, that is: 9 lung epidermoid carcinomas; 6 breast adenocarcinomas and 5 melanomas. Simultaneously 10 normal control subjects were studied. No significant differences in the percentage of suppression were found in patients bearing breast adenocarcinoma and lung epidermoid carcinoma as compared to normal subjects. Melanoma patients, on the contrary, showed significant differences on the same test. On the other hand, the Con A lymphoproliferative response was found to be only significantly increased in the melanoma patients compared to normal controls. No differences were found in the percentage of peripheral blood lymphocytes between patients and controls.

Topics: Adenocarcinoma; Adult; Aged; Breast Neoplasms; Carcinoma, Squamous Cell; Concanavalin A; Humans; In Vitro Techniques; Lung Neoplasms; Lymphocyte Activation; Lymphocytes; Melanoma; Middle Aged; T-Lymphocytes, Regulatory

Topics: Adhesiveness; Alkaloids; Amino Acids; Animals; Carbohydrate Metabolism; Cell Division; Cell Line; Concanavalin A; Cricetinae; Dogs; Dose-Response Relationship, Drug; Escherichia coli; Female; Glycoproteins; Intestines; Kidney; Lectins; Melanoma; Membrane Proteins; Mice; Ovary; Ricin; Swainsonine; Vacuoles; Wheat Germ Agglutinins

When the tumor-bearing leg of C57BL/6J mice was amputated 16 days after SC inoculation of 10(6)B16 melanoma cells, all the amputated mice died of pulmonary metastases. Transfer of lungs from the amputated to normal syngeneic mice revealed tumor cells in the lungs just after amputation. Repeated weekly injections of BCG and irradiated tumor cells, beginning 24 h after amputation of the tumor-bearing limb, prolonged the survival only of mice presensitized to BCG. Injections of BCG or irradiated melanoma cells alone, or neuraminidase- and mitomycin C-treated tumor cells or of Levamisole had no effect, but injections of ConA-coated tumor cells slightly prolonged the survival of the amputated mice. Both BCG and B16 cells induced humoral and cell-mediated immunity but there was no cross-reactivity between BCG and B16 cells.

Topics: Animals; Antibody Formation; BCG Vaccine; Concanavalin A; Disease Models, Animal; Immunity, Cellular; Immunization; Immunotherapy; Levamisole; Lung Neoplasms; Melanoma; Mice; Neoplasm Metastasis; Neoplasms, Experimental; Neuraminidase

Indomethacin significantly enhances the depressed levels of lymphocyte proliferation to the mitogens phytohemagglutinin and concanavalin A in melanoma patients. We postulated that these results were related to an abnormality in prostaglandin E2 (PGE2)-mediated suppression, since this mechanism has previously has previously been demonstrated in patients with Hodgkin's lymphoma and with head and neck carcinoma. However, the results of three experimental approaches did not support this hypothesis. First, in vitro PGE2 production by cultured blood mononuclear cells was the same in 16 melanoma patients as in 45 normal controls (4.9 versus 4.7 ng/ml). Second, lymphocyte sensitivity to PGE2 for melanoma patients was essentially the same as that for normal controls, since exogenous doses of PGE2 inhibited the mitogen responses to the same degree. Third, another prostaglandin synthetase inhibitor (RO-205720), which is structurally unrelated to indomethacin, did not augment the mitogen response in these patients. Thus PGE2 cannot be implicated as a mediator of immunosuppression in melanoma patients. To further examine the immunomodulatory mechanism of indomethacin, we preincubated the drug with purified populations of either lymphocytes or monocytes, which were then recombined and tested for mitogen response. The results suggested that indomethacin had a direct effect on the responding T lymphocytes rather than an indirect effect on monocytes. These are the first studies demonstrating that indomethacin can act directly as a modulator of cellular immune function, independent of PGE2-mediated suppression.

Topics: Adult; Aged; Carbazoles; Concanavalin A; Cyclic AMP; Humans; Immunity; Immunosuppression Therapy; Indomethacin; Lymphocytes; Melanoma; Middle Aged; Phytohemagglutinins; Prostaglandins E

Topics: Adult; Aged; Concanavalin A; Female; Humans; Immunity, Cellular; Leukocyte Count; Levamisole; Lymphocyte Activation; Male; Melanoma; Middle Aged; Phytohemagglutinins; Pokeweed Mitogens; Rosette Formation; T-Lymphocytes

From C57BL mouse melanoma B-16 cells, variant clones were selected in vitro which were resistant to the lectins wheat-germ agglutinin and ricin. Cells were also selected which survived toxic concentrations of concanavalin A. Four different in vivo assays using intradermal, intravenous, intraperitoneal and intramuscular injections were used to assess the tumorigenicity and metastasizing capacity of these lectin-resistant variants. It was concluded that to obtain a complete picture of the malignant properties of a given cell line or clone, all four assays have to be carried out. In comparison with the parental cells, the WGA-resistant cells showed a most dramatic decrease in metastasizing capacity through both lymphatic and vascular channels. Tumorigenicity was also reduced. The ricin-resistant cells showed a defective development into lung tumors and thus displayed a reduction in metastasis through the hematogenous route. Since this line did not change its capacity to metastasize via the lymphatic route, and the tumorigenicity was not significantly altered, it will be a good model for studies seeking to dissociate these two properties. The Con-A-selected cells, when injected intravenously, developed tumor nodules in the liver in addition to those in the lungs, while no striking alterations in tumorigenicity or metastasizing capacity could be detected in this line.

Topics: Animals; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Concanavalin A; Drug Resistance; Lectins; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental

The relative proportions of T, B and T gamma suppressor cells were determined sequentially in peripheral blood of melanoma patients before and after surgery. Concanavalin A (Con A)-induced suppression against lymphocyte mitogenesis of melanoma patients and age matched controls was measured concurrently. The mean percentage of T gamma cells was significantly higher (p less than 0.001) in melanoma patients before surgery (21.8 +/- 5.4) compared to a group of age-matched controls (14.9 +/- 5.4). There was a tendency for the proportion of T gamma cells in patients to decrease after surgery, although the relative levels of T gamma were still significantly elevated (p less than 0.05) 6-8 weeks after surgery when compared to normal controls. The mean percentage of T and B cells in melanoma patients before and after surgery was comparable to that observed in normal controls. The degree of Con-A-induced suppression in patients increased significantly after surgery particularly at 6-8 weeks (p less than 0.02). No difference in con-A-induced suppressor cell activity was observed between melanoma patients and controls before or after surgery. An inverse relationship was found between the amount of Con-A-induced suppression and percentage of T gamma cells in melanoma patients before surgery. Similar associations were not apparent in patients after surgery or in normal control populations. The inverse correlation of Con-A-induced suppression with T gamma cell numbers suggests that the former may measure potential suppressor cell activity whereas the T gamma cells may indicate active suppressor cells. The significance of these findings for the monitoring of suppressor cell activity in vivo and their role in suppression of immune responses in melanoma patients is discussed.

Topics: B-Lymphocytes; Concanavalin A; Female; Humans; Lymphocyte Activation; Male; Melanoma; Skin Neoplasms; T-Lymphocytes; T-Lymphocytes, Regulatory

Lymphocyte responses to the mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) are important in vitro parameters of immunocompetence in cancer patients. The tempo and intensity of this response is regulated by monocytes. We found that the blood lymphocyte response to one or both of these mitogens was significantly depressed in 32 of 33 melanoma patients compared to 29 normal control subjects. However, these responses were significantly enhanced when the drug indomethacin, a prostaglandin synthetase inhibitor, was added to cultures of peripheral blood mononuclear cells (PBMCs) from the melanoma patients, but not from the normal control subjects. For example, at a 1 microgram/ml dose of Con A, the mean response of melanoma patients increased from 56% to 81% of the normal mean response, whereas at 20 micrograms/ml PHA the mitogen response increased from 57% to 77% of normal when PMBC were incubated with indomethacin (P less than 0.001). Overall, indomethacin enhanced the mitogen responses of melanoma patients by 35% to 85%, whereas indomethacin increased the response in normal subjects by only 5% to 15%. The mitogen response of monocyte-depleted ER+ lymphocytes for melanoma patients was equivalent to that of normal control subjects, and there was no enhancement of this response in the presence of indomethacin. This suggests that the abnormality was due to an altered function of immunoregulatory monocytes. The enhancement by indomethacin in melanoma patients was not significantly influenced by their stage of disease, age, or the proportion of blood monocytes. The decreased levels of cellular immunocompetence in these melanoma patients, as measured by their lymphocyte proliferative responses to mitogens, therefore appears to be associated with an abnormality in monocyte function that is partially corrected by indomethacin.

Topics: Adult; Age Factors; Aged; Concanavalin A; Humans; Immunocompetence; Indomethacin; Lymphocyte Activation; Melanoma; Middle Aged; Phytohemagglutinins; Prostaglandins E; Rosette Formation; T-Lymphocytes

Radioimmunoassay, using radiolabeled melanoma-associated antigens (MAA), rabbit anti-B16 melanoma serum and Staphylococcus aureus Cowan I, was developed for the detection of MAA in tumor tissues as well as in sera of B16 melanoma-bearing C57BL/6 mice. MAA were partially purified from 3 M KC1 extract of murine B16 melanoma tissue by affinity column chromatography using anti-B16 serum and concanavalin A. The content of MAA was high in B16 melanoma extract, much less in allogeneic Harding Passey melanoma, and very small in fetal and normal tissues including skin and brain. The amount of circulating MAA released from the tumor into the blood of B16 tumor-bearing C57BL-6 mice was linearly proportional to the size (cube root of weight) of the growing tumor, providing a rational basis for immunodiagnosis of malignant melanoma. Immunochemical data suggest that MAA which are extractable with 3M KC1 from B16 melanoma are glycoproteins with molecular weights of 54,000, 68,000 and 94,000 daltons.

Topics: Animals; Antigens, Neoplasm; Chromatography, Affinity; Concanavalin A; Glycoproteins; Immune Sera; Melanoma; Mice; Mice, Inbred ICR; Mice, Inbred Strains; Neoplasms, Experimental; Rabbits; Radioimmunoassay

The agglutination of cells isolated from solid melanotic and amelanotic malanomas in hamsters by Concanavalin A was studied. It has been found that the agglutination intensity of the two kinds of melanoma cells was different and depended on the concentration of the lectin. It has been suggested that the different susceptibility of the cells to Concanavalin A is related to changes of surface glycoproteins, resulting from a spontaneous alteration of the melanotic melanoma into amelanotic one.

Topics: Agglutination; Animals; Cell Membrane; Cells, Cultured; Concanavalin A; Cricetinae; Glycoproteins; Melanoma; Membrane Proteins; Neoplasms, Experimental; Receptors, Concanavalin A; Skin Neoplasms; Time Factors

Lymphocyte blast transformation of 23 melanoma patients was compared to that of 22 healthy persons after stimulation with the mitogens PHA, ConA, and PWM. Transformation rate of lymphocytes in microcultures was measured following 3H-Thymidin uptake in a Liquid-Scintillation-Counter. Calculations were based on analyses of variance. There was no significant difference in blastogenetic response between the 2 groups (patients and controls), but there were differences between the used mitogens.

Topics: Adolescent; Adult; Aged; Concanavalin A; Female; Humans; Lymphocyte Activation; Lymphocytes; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Phytohemagglutinins; Pokeweed Mitogens; Skin Neoplasms

Topics: Antibodies, Neoplasm; Antigens, Neoplasm; Chromatography, Affinity; Chromatography, Gel; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immune Sera; Immunosorbent Techniques; Leukocytes; Male; Melanoma; Sodium Dodecyl Sulfate

A series of 42 patients with malignant melanoma treated with BCG adjuvant immunotherapy were studied for sequential changes in cellular immune reactivity to non-specific mitogens. Lymphocyte preparations were made monthly and stored in a viable condition in liquid nitrogen. After 6 months of treatment, all lymphocyte samples from an individual were recovered and tested for DNA synthesis after stimulation with PHA, PWM, Con A, PPD and MLC. The responses to the mitogens in the blastogenesis test were stable during the course of therapy. The MLC response did not increase significantly in patients treated with tumor-cell vaccines, and declined sharply in the six patients who subsequently relapsed and died. The in vitro PPD response increased 1 to 3 months after initiation of BCG in patients who were initially unresponsive to PPD in vitro. However, PPD-positive patients did not show any significant alteration of the PPD response. The PPD response did increase less sharply in patients whose disease eventually recurred than in those who remained without evidence of clinical disease. BCG therapy does not appear to correct lymphocyte proliferative defects in melanoma patients. Of the assays employed, the MLC and PPD tests appear to be the most useful as monitors of clinical status and response to therapy.

Topics: BCG Vaccine; Concanavalin A; DNA; Female; Follow-Up Studies; Humans; Lectins; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Lymphocytes; Male; Melanoma; Mitogens; Skin Tests; Tuberculin

Topics: Antigens, Bacterial; BCG Vaccine; Breast Neoplasms; Concanavalin A; Cross Reactions; Cytotoxicity Tests, Immunologic; Escherichia coli; HLA Antigens; Humans; Immunity, Cellular; Immunization; In Vitro Techniques; Lectins; Listeria monocytogenes; Lymphocyte Activation; Lymphocytes; Melanoma; Mycobacterium bovis; Staphylococcus aureus

Macrophage-activating factor (MAF) was obtained from cultures of normal F344 rat lymphocytes incubated with insoluble concanavalin A. The MAF rendered macrophages from normal C57BL/6 mice cytotoxic against the syngeneic B16 melanoma and the allogeneic AC 15091. At the same time, normal syngeneic or allogeneic embryo cells were unharmed, even in the presence of susceptible tumor cells. Optimal MAF levels followed incubation of lymphocytes for 48 hr with Sepharose-bound concanavalin A. A 2-hr incubation of macrophages with MAF was sufficient to initiate activation, providing that 46 hr were allowed to elapse before tumor cells were added. The MAF activity was enhanced after heating the supernatant to 199 degrees. Control experiments largely excluded the possibility that residual unbound concanavalin A caused the observed macrophage-mediated tumoricidal effects.

Topics: Adenocarcinoma; Animals; Cells, Cultured; Concanavalin A; Cytotoxicity Tests, Immunologic; Lymphocytes; Macrophages; Melanoma; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Rats; Rats, Inbred F344; Time Factors

Quantitative assessment of the agglutination of 51Cr labelled canine cell suspensions to canine kidney cell monolayers has been performed over a range of concanavalin A concentrations. Agglutination was observed with all cell cultures tested, comprising four spontaneous canine melanomas, two canine mammary carcinomas, a benign mammary tumour and a contact-inhibited kidney cell line. The melanomas tested showed strong specific inhibition of concanavalin A agglutination by 10(-2)M alpha-methyl-D-glucopyranoside. Inhibition of agglutination of mammary tumour and kidney cells was weaker and less specific. Agglutination was inhibited at 4degrees C. Reduced agglutination to glutaraldehyde-fixed mono-layers was observed in the case of mammary tumours but was absent when contact-inhibited kidney cells were tested. The specificity of the reaction for transformed cells and the parameters involved are discussed.

Topics: Agglutination; Agglutination Tests; Animals; Cell Line; Chromium Radioisotopes; Concanavalin A; Dogs; Dose-Response Relationship, Drug; Mammary Neoplasms, Experimental; Melanoma; Neoplasms, Experimental; Temperature; Trypsin

The mitogens concanavalin A (Con A) and bacterial lipopolysaccharide (LPS) stimulated normal spleen cells of DBA/2J, CBA/J, and BALB/c mice about equally in the presence of either isologous or homologous serum. This system revealed that sera from mice with five different methylcholanthrene-induced rhabdomyosarcomas inhibited mitogen stimulation of normal spleen cells. Sera from mice with a mammaryadenocarcinoma and spontaneous rhabdomyosarcoma were similarly suppressive. In contrast, sera from mice with melanoma were not inhibitory and often enhanced stimulation. Sera from tumor-bearing animals had the same effects both qualitatively and quantitatively on cells from the strain carrying the tumor and on cells from the other two strains. The mixed lymphocyte response of CBA/J times BALB/c spleen cells was affected exactly as were the responses to mitogen by the various sera. Stimulation by mitogen of mouse lymph-node cells and spleen cells with macrophages removed, as well as that of guinea pig spleen cells, was also inhibited by sera from mice with rhabdomyosarcoma and mammary adenocarcinoma.

Topics: Adenocarcinoma; Animals; Blood; Concanavalin A; Guinea Pigs; Lipopolysaccharides; Lymph Nodes; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Lymphocytes; Mammary Neoplasms, Experimental; Melanoma; Methylcholanthrene; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Mice, Inbred DBA; Mitogens; Neoplasms, Experimental; Polysaccharides, Bacterial; Rhabdomyosarcoma; Sarcoma, Experimental; Spleen

Viable frozen lymphocytes displayed activity in blastogenesis assays that was indistinguishable from freshly prepared lymphoid cells. Similarly, cytotoxic activity of lymphocytes against melanoma target cells from melanoma patients was only slightly affected by the freezing procedure. Frozen lymphocytes provided a highly reproducible source of cells in these assays. The use of viable frozen peripheral blood lymphoid cells for the retrospective analysis of a cancer patient's immune response is described.

Topics: Adult; Blood Preservation; Cell Separation; Cell Survival; Cells, Cultured; Concanavalin A; Cytotoxicity Tests, Immunologic; Freezing; Histocompatibility Antigens; Humans; Lectins; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Lymphocytes; Male; Melanoma; Mitomycins; Retrospective Studies; Streptodornase and Streptokinase; Thymidine; Tritium; Tuberculin

A bromodeoxyuridine(BUdr) dependent cell line, called B4, Which requires BUdr not only for optimal growth but also for the maintenance of the non-contact inhibited state was described previously. We have now shown that contact inhibition in the B4 cells in the absence of BUdr is associated with a marked decrease in the percent of cells synthesizing DNA. The transition to the contact inhibited state in the absence of BUdr does not seem to be due to changes in cyclic AMP levels. It has also been shown that several but not all of the characteristics which distinguish transformed from untransformed cells also distinguish B4 cells grown in the presence of BUdr from B4 cells grown in the absence of BUdr. In addition to being contact inhibited, B4 cells grown in the absence of BUdr have a higher serum requirement, grow less well in soft agar, and are less agglutinable by wheat germ agglutinin than B4 cells grown in the presence of BUdr. Agglutinability by concanavalin A, however, is the same for B4 cells grown in the presence and absence of BUdr. Dependent cells maintained in the presence of BUdr do not form tumors and it is not yet clear how the transformed characteristics of the dependent cells are related to malignancy.

Topics: Agglutination Tests; Animals; Blood Proteins; Bromodeoxyuridine; Cattle; Cell Line; Cell Transformation, Neoplastic; Concanavalin A; Contact Inhibition; Cricetinae; Culture Media; Cyclic AMP; DNA, Neoplasm; Female; Lectins; Melanoma; Mutation; Neoplasm Transplantation; Plant Lectins; Triticum

Blocking factors are small polypeptide molecules that may appear in the serum of patients with cancer. These factors block the transformation of lymphocytes in culture to nonspecific mitogens such as phytohemagglutinin or concanavalin A and, therefore, may reflect changes in the immunocompetence of the patient. Blocking factors were monitored during the clinical course of thirty-five patients with cancer. These factors did not develop in patients with response to therapy whereas they did develop in patients without response. A third group of patients without response to therapy after a previous remission showed an absence of lymphocyte responsiveness in culture that was not due to blocking factors, suggesting that immune clone consumption had occurred. Dermal responsiveness to tumor antigen correlated with a favorable clinical course and was usually absent when serum blocking factors were present.

Topics: Antibody Formation; Antigens, Neoplasm; Carcinoma, Squamous Cell; Concanavalin A; Humans; Immunotherapy; Lectins; Leiomyosarcoma; Lymphocyte Activation; Lymphocytes; Melanoma; Neoplasms; Osteosarcoma; Peptides; Pharyngeal Neoplasms; Rhabdomyosarcoma; Skin Tests

Maintenance of remission solely by repeated BCG vaccinations in seven patients with non-Hodgkin's lymphoma who had achieved a complete clinical remission with initial standard therapy has provided sufficient encouragement to begin a randomized clinical trial. In vitro lymphocyte responses to mitogens and PPD used as parameters of cell-mediated immunity have not proved to be of value in predicting early or late recurrence in six pre-trial and trial patients. Eight out of twenty-one patients with malignant melanoma have shown a satisfactory clinical response (10-34 months) to immunotherapy. Those who respond must show immunological reactivity to the stimulating agent, however the best clinical responses were not associated with the highest degrees of in vivo and in vitro sensitization. The skin reactivity and the in vitro lymphocyte response to PPD as well as a 2-3-fold increase in the appearance of colony-forming units in the peripheral blood following the intratumour injection of BCG or PPD are helpful in prognosis and management of these patients. All patients with malignant melanoma who presented with a PHA response less than 40% of normal made a poor response to immunotherapy. Autopsies performed on seven patients dying with extensive melanocarcinomatous disease failed to show any serious adverse toxic reactions or infections from oral and intratumour injections of BCG.

Topics: Administration, Oral; Adult; BCG Vaccine; Concanavalin A; DNA; Female; Hematopoietic Stem Cells; Humans; Immunoglobulins; Immunotherapy; Injections, Intradermal; Lectins; Lymphocyte Activation; Lymphoma; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Non-Hodgkin; Melanoma; Skin Neoplasms; Tuberculin; Vaccinia virus

A strain-specific transplantable melanoma (S-91) growing progressively in DBA/1 mice and metastasizing selectively to the lungs was maintained for 16 days in organ culture before being grafted to syngeneic (DBA/1) and allogeneic (BALB/c and C57BL/6) recipients. The cultured S-91 grew progressively in the syngeneic mice and to a moderate degree in the allogeneic strains; it showed an increased tendency to metastasize in both the DBA/1 and C57BL/6 recipients. Heterophilic cytoagglutination assays of cultured S-91 were less apt to aggregate in the presence of concanavalin A than were their noncultured counterparts, which suggested alteration of the plasma membrane. Organ culture explantation appeared to alter phenotypically the cell-surface membrane and thus increase the cell's ability to metastasize while possibly reducing the immunogenicity of the cultured tumor cells.

Topics: Agglutination Tests; Animals; Cells, Cultured; Concanavalin A; Lung Neoplasms; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasm Metastasis; Neoplasms, Experimental

Topics: Animals; Antigens, Neoplasm; Antineoplastic Agents; Bleomycin; Carmustine; Cells, Cultured; Chickens; Clone Cells; Concanavalin A; Culture Techniques; Cytarabine; Cytological Techniques; Drug Resistance; Drug Synergism; Drug Therapy, Combination; Humans; Immunity, Cellular; Immunotherapy; Lectins; Lipopolysaccharides; Lymphocytes; Melanoma; Mice; Mitosis; Neoplasms; Neoplasms, Experimental; Thymidine

Topics: Adenocarcinoma; Animals; Antigens, Neoplasm; Cell Membrane; Concanavalin A; Epitopes; Female; Fibrosarcoma; Immunotherapy; Mammary Neoplasms, Experimental; Melanoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms, Experimental; Neuraminidase; Sarcoma, Experimental; Transplantation, Homologous; Vibrio cholerae

Topics: Animals; Binding Sites; Calcium; Cell Aggregation; Cell Line; Concanavalin A; Contact Inhibition; Cycloheximide; Dactinomycin; Dinitrophenols; Drug Resistance; Edetic Acid; Hyaluronoglucosaminidase; Kinetics; Magnesium; Mannose; Melanoma; Methylglycosides; Mice; Neuraminidase; Pronase; Temperature; Time Factors; Trypsin

Topics: Adenocarcinoma; Concanavalin A; Humans; Hypersensitivity, Delayed; Immunologic Memory; Lectins; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Melanoma; Neoplasms; Nitrobenzenes; Sarcoma; Skin Tests

Topics: Adenocarcinoma; Animals; Antigens, Neoplasm; Concanavalin A; Female; Fibrosarcoma; Immunotherapy; Mammary Neoplasms, Experimental; Melanoma; Methylcholanthrene; Mice; Mice, Inbred Strains; Mitomycins; Neoplasm Transplantation; Neuraminidase; Sarcoma, Experimental; Vibrio cholerae

Topics: Animals; Antibodies, Neoplasm; Antibody Formation; Antigen-Antibody Reactions; Antigens, Neoplasm; Antilymphocyte Serum; Autoantibodies; Concanavalin A; Cytotoxicity Tests, Immunologic; Fibrosarcoma; Histocompatibility Antigens; Humans; Immunotherapy; Lymphocytes; Mammary Neoplasms, Experimental; Melanoma; Methylcholanthrene; Mice; Neoplasm Transplantation; Neoplasms; Neuraminidase; Rats; Sarcoma, Experimental; Vibrio cholerae

Topics: Animals; Cells, Cultured; Concanavalin A; Cytotoxicity Tests, Immunologic; Histocompatibility Antigens; Immunity, Cellular; Lymphocytes; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Transplantation, Homologous