alpha-chymotrypsin and Melanoma

For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.

Topics: Cell Line, Transformed; Chymotrypsin; Cysteine Endopeptidases; Histocompatibility Antigens Class I; HLA Antigens; Humans; Interferon-gamma; Lymphocyte Activation; Melanoma; Multienzyme Complexes; Proteasome Endopeptidase Complex; Proteins; T-Lymphocytes, Cytotoxic; Trypsin; Tumor Cells, Cultured; Viral Matrix Proteins

Tumor cells avidly secrete various proteinases, and cascades of proteolytic activation occur around the cells. Therefore, cell surface receptors of tumor cells are under the constant influence of proteinases. In this study, the effects of serine proteinases on integrin-medicated cell-matrix interactions were studied in C32TG and Mewo human melanoma cells. These melanoma cells were pretreated with proteinases and their adhesive properties on various substrata were evaluated by cell adhesion assays. Paradoxically, appropriate cell surface proteolysis enhanced the RGD-sensitive cell adhesion on fibrinogen and vitronectin, but not the RGD-insensitive adhesion on type I collagen or laminin. Pretreatment of these cells with 0.1 to 1 microM of trypsin, chymotrypsin, or plasmin for 30 min at 37 degrees C increased the number of spread cells on fibrinogen and vitronectin by 200-300%. The enhancement of cell spreading was not accompanied by up-regulation of the relevant RGD-sensitive integrin expression. Analysis of the cell surface receptor by GRGDSPK-Sepharose affinity chromatography showed that trypsin treatment did not up-regulate alpha v beta 3 integrin, an RGD-sensitive receptor for fibrinogen and vitronectin in the melanoma cells, nor the induced appearance of novel receptors. Treatment of cells with 100 nM proteinases increased cell binding of both monoclonal and polyclonal antibodies against alpha v beta 3 integrin subunits by 70%, but not that of monoclonal antibody against alpha 2, alpha 3, or alpha 6 subunit, indicating that cell surface proteolysis exposed more alpha v beta 3 integrin on the cell surface. These results suggest that exposure of alpha v beta 3 integrin is a part of the mechanisms underlying the serine proteinase-induced enhancement of melanoma cell adhesion on fibrinogen and vitronectin.

Topics: Amino Acid Sequence; Cell Adhesion; Chymotrypsin; Extracellular Matrix Proteins; Fibrinogen; Fibrinolysin; Glycoproteins; Humans; Integrins; Laminin; Melanoma; Molecular Sequence Data; Oligopeptides; Receptors, Cytoadhesin; Receptors, Vitronectin; Serine Endopeptidases; Trypsin; Tumor Cells, Cultured; Up-Regulation; Vitronectin

The sensitivity to serine proteinases of cellular proteins involved in cell-matrix adhesion was investigated using C32 melanoma cells. Cells dissociated from monolayers by the metal chelator ethylenediaminetetraacetic acid were incubated with proteolytic enzymes, and then attachment was quantified by standard cell adhesion assays. The effect of proteinases was found to depend on the presence of Ca2+ in the incubations. Incubation with 100 nM trypsin or chymotrypsin for 1-2 h (37 degrees C) in the absence of Ca2+ reduced cell attachment to vitronectin (Vn), fibrinogen (Fb), laminin, and fibronectin by approximately 80, 80, 40, and 30%, respectively. Viability studies indicated that such treatment with proteinases was not cytotoxic. Inclusion of 0.1 nM CaCl2 in the incubations prevented the loss in attachment to all substrata. In the case of Fb, proteinase treatment in the presence of Ca2+ had an additional effect; it improved cell attachment to this substratum by about 50%. C32 cells have been shown to express the integrin alpha v beta 3 (Vn receptor) which mediates attachment to Vn and Fb in a GRGDS-sensitive manner. Attachment of C32 cells to Vn and Fb prior to proteinase treatment and after proteinase treatment in the presence of Ca2+ was 90% inhibited by the addition of GRGDS peptide to the attachment assays. These results suggest that the adhesion observed both before and after proteinase treatment was mediated by this integrin. Analysis of the Vn receptor from proteinase-treated cells by immunoblotting of cell extracts and by SDS gel electrophoresis of immunoprecipitated receptor revealed no detectable change in either the alpha v or beta 3 subunit that correlated with loss in attachment. Similarly proteinase treatment in the presence of Ca2+ did not produce detectable alterations in the subunits which might correlate with the improved attachment to Fb. Consistent with these results, an enzyme-linked immunoassay to quantify cell surface receptors revealed little difference in the amount of Vn receptor on cells treated with proteinase in the presence or absence of Ca2+. Degradation of the alpha v subunit was demonstrated, however, at proteinase concentrations higher than those required to affect cell attachment. Thus, treatment of cells with serine proteinases can affect integrin-mediated attachment to matrix proteins in a manner moderated by Ca2+, but the alterations in attachment do not appear to be accompanied by detectable proteolytic modification

Topics: Amino Acid Sequence; Calcium; Cations, Divalent; Cell Adhesion; Chymotrypsin; Extracellular Matrix; Fibrinogen; Fibronectins; Glycoproteins; Humans; In Vitro Techniques; Integrins; Laminin; Melanoma; Molecular Sequence Data; Molecular Weight; Oligopeptides; Tumor Cells, Cultured; Vitronectin

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.

Topics: Amino Acid Sequence; Animals; Cell Line; Chromatography, High Pressure Liquid; Chymotrypsin; Epidermal Growth Factor; Fucose; Glycosylation; Humans; Melanoma; Molecular Sequence Data; Peptide Fragments; Peptide Mapping; Recombinant Proteins; Threonine; Tissue Plasminogen Activator; Trypsin

The isoenzyme profiles of tyrosinase isolated from melanosomal and cytoplasmic fractions of hamster melanoma were investigated. The liberation of the enzyme from the melanosomal membranes was achieved by their treatment with Triton X-100 or alpha-chymotrypsin. The isoenzyme spectrum of tyrosinase liberated by use of Triton X-100 from melanosomes differing in the degree of melanization is identical to that of the cytoplasmic enzyme and gives three peaks on a densitogram. The liberation of melanosomal tyrosinase by alpha-chymotrypsin results in the appearance of an additional isoenzyme due to proteolytic degradation. The role of tyrosinase isoenzymes in the synthesis of melanin is discussed.

Topics: Animals; Catechol Oxidase; Chymotrypsin; Cricetinae; Electrophoresis, Polyacrylamide Gel; Isoenzymes; Melanocytes; Melanoma; Monophenol Monooxygenase; Neoplasm Transplantation; Octoxynol; Polyethylene Glycols

The murine monoclonal antibody 791T/36 cross-reacts with cells other than the immunizing osteogenic sarcoma cell line 791T, upon which the 791T/36-defined epitope is expressed on a protein of apparent molecular weight 72,000. An investigation was performed to determine whether the epitope occurred on similar molecules on other cell lines. Radiolabelled immunoprecipitates, prepared with the 791T/36 antibody, from three osteogenic sarcoma cell lines (2 OS, 788T and 278T), the prostate carcinoma EB33 and the colon carcinoma HcLo each contained a protein with a molecular weight of 72,000 as the major constituent, together with, in some cases, material of lower molecular weight. This heterogeneity was shown by neuraminidase treatment of the immune precipitates to be due to variations in sialic acid content of the antigens since, in five of the six cell lines tested, such treatment produced homogeneous material of apparent molecular weight 55,000. Chymotrypsin treatment of the immune precipitates produced in each instance a major polypeptide of molecular weight 47,000 which displayed no microheterogeneity. Immunoadsorbent-purified antigen from 791T cells was shown to bind strongly to Sepharose-wheat-germ agglutinin and less to Sepharose-concanavalin A, confirming the glycoprotein nature of this antigen. These studies demonstrate that molecules expressing the 791T/36-defined epitopes on different tumour cell lines are glycoproteins which display heterogeneity with respect to apparent molecular weight that is attributable to varying degrees of sialylation. No apparent differences were detected in the polypeptide "backbone" of these antigenic molecules.

Topics: Antigens, Neoplasm; Antigens, Surface; Carcinoma; Cell Line; Chymotrypsin; Humans; Lectins; Melanoma; Molecular Weight; Osteosarcoma; Papain

A tissue culture line of a human malignant melanoma, SEKI, induced cachexia in nude mice (BALB/c-nu/nu) (Kondo et al., Cancer Res., 41: 2912-2916, 1981). During the investigation of the cause of the cachexia, the melanoma was found to produce a protein immunologically identical to human alpha 1-antichymotrypsin (alpha 1-Ach). Tissues of the SEKI melanoma contained the protein immunologically equivalent to 0.29 +/- 0.11 (S.D.) mg of human alpha 1-Ach per g of wet tissue, while the other six human malignant tumors transplanted into nude mice did not contain a detectable amount of it. In the serum of nude mice bearing the melanoma, this protein appeared soon after the tumor growth occurred and gradually increased up to the level equivalent to 5 mg of human alpha 1-Ach per dl. Removal of the tumor resulted in a rapid decrease of the protein in the serum to an undetectable level within 1 day. This problem was never detected in the serum of nude mice bearing the other 27 human malignant tumors or controls. Purification of this protein was carried out by the column chromatography using DE-52, Blue-Sepharose, and SP-Sephadex. The elution patterns were the same as those of alpha 1-Ach in human serum, and the molecular weight of the protein was estimated as 69,000 by Sephadex G-100 column chromatography and 65,000 by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. This purified protein, however, did not exhibit inhibitory activity against chymotrypsin. These results show that this melanoma produced a protein immunologically identical and physicochemically very similar to human alpha 1-Ach. This melanoma-nude mouse system may provide a useful model for investigating the synthesis of human alpha 1-Ach and analysis of its physiological roles.

Topics: alpha 1-Antichymotrypsin; Animals; Chymotrypsin; Female; Humans; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Protease Inhibitors

Media conditioned by tumor cells were studied for the presence of factor(s) that increase the rate of random migration (chemokinesis) of Corynebacterium parvum-activated macrophages. A capillary tube assay was developed and utilized to expediently monitor the chemokinetic activity of macrophages incubated in whole and fractionated media. Media conditioned by six different syngeneic and allogeneic mouse tumor cell lines demonstrated significantly higher chemokinetic activity than unconditioned or normal fibroblast conditioned media. The chemokinetically active component of the Lewis Lung conditioned media was found to be a trypsin sensitive, heat stable, high molecular weight (300,000-480,000 dalton range) factor that had no chemotactic (directional migration) activity. Pyran-activated macrophages also responded chemokinetically to the Lewis Lung factor while oyster glycogen and thioglycolate-elicited macrophages did not. The similarity and differences between the physical properties of the chemokinetic factor, other migration stimulating factors, and tumor-associated proteins are discussed.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Line; Cell Movement; Chemotactic Factors; Chemotaxis; Chymotrypsin; Fibrosarcoma; Lung Neoplasms; Macrophages; Male; Mammary Neoplasms, Experimental; Melanoma; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Trypsin

The tyrosinase activity in two sucrose gradient isolated melanosome fractions from a melanotic hamster melanoma was found to increase after alpha-chymotrypsin treatment. The enhancement in tyrosinase activity had its maximum at a concentration of 1 mg/ml alpha-chymotrypsin after 120 min incubation at 37 degrees C. No direct activating effect of alpha-chymotrypsin was found either on the soluble tyrosinase fraction from freshly prepared untreaed whole-tumor homogenate or on purified mushroom tyrosinase. The activating effect of alpha-chymotrypsin upon the melanosome tyrosinase is believed to be due to the endopeptidic hydrolysis of the--CO--NH--bound existing between tyrosinase and tyrosine and phenylalanine residues in the melanin molecule. Although alternative interpretations are not excluded, the observed enhancement in tyrosinase activity after alpha-chymotrypsin treatment of melanosomes might indicate the existence of an "enzyme liberating" mechanism in the melanosomes.

Topics: Animals; Catechol Oxidase; Chymotrypsin; Cricetinae; Enzyme Activation; Melanins; Melanocytes; Melanoma; Monophenol Monooxygenase; Neoplasms, Experimental; Time Factors

Mechanical and enzymatic methods of disaggregating tumors were studied with the goals of (1) minimizing cell losses while (2) maintaining functional and surface membrane markers needed to objectively identify inflammatory cells (IC)1 in resultant suspensions. Application of the principles and methods described makes accurate estimation of the percentage of each IC type present in neoplasms possible for the first time. Compared to purely mechanical means of disaggregating tumors, all enzyme mixtures tested markedly increased yields of viable cells/g neoplasm. Best results were obtained with a combination of collagenase and a protease of broader substrate range (alpha chymotrypsin, papain, pronase or trypsin). The combination of enzymes that gave the highest yields with the least effect on inflammatory cell markers was trypsin, collagenase and DNAse (TCD). Because mechanical injury appeared to be the greatest single cause of cell loss (the enzymes themselves had little direct effect), potential sources were identified and either eliminated or minimized. With TCD, depending on the tumor system, cell recovery (measured as DNA recovered in cell suspensions) was as high as 50% and yields were as much as 6.9 X 10(8) viable cells/g tumor. Complete disaggregation was not required to obtain representative IC populations from tumor fragments. Neutrophils, eosinophils and mast cells from disaggregated neoplasms were counted in Giemsa stained cytocentrifuge preparations based on their unique morphologic appearances. Macrophages were identified by their capacity to phagocytose zymosan, a function which proved highly resistant to the effect of enzymes. Flourescent microscopic identification of brain associated thymus antigen (BATA) allowed quantification of T lymphocytes, since this marker was virtually unchanged by enzyme exposure. Surface immunoglobulin (Ig) was stripped from B lymphocytes most rapidly by pronase and chymotrypsin, slowly by trypsin and papain, and not at all by collagenase. Ig positive cells therefore could be quantified in suspensions generated by collagenase or very short (20 min) exposure of fragments to trypsin.

Topics: Animals; Ascitic Fluid; Cell Count; Cell Membrane; Chymotrypsin; Deoxyribonucleases; DNA, Neoplasm; Fluorescent Antibody Technique; Leukocytes; Lymphocytes; Macrophages; Male; Mast Cells; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microbial Collagenase; Moloney murine leukemia virus; Neoplasms, Experimental; Phagocytosis; Pronase; Sarcoma, Experimental; Spleen; Trypsin

Topics: Amino Acids; Animals; Catechol Oxidase; Cattle; Chymotrypsin; Dihydroxyphenylalanine; Kinetics; Melanins; Melanoma; Mice; Mice, Inbred Strains; NAD; Neoplasms, Experimental; Oxidation-Reduction; Pepsin A; Peptide Hydrolases; Peptides; Phosphoproteins; Plants; Serum Albumin, Bovine; Time Factors; Trypsin

Topics: Animals; Cell Division; Cells, Cultured; Chromatography, Gel; Chymotrypsin; Drug Stability; Epinephrine; Glycoproteins; Growth Inhibitors; Hot Temperature; Humans; Hydrocortisone; Melanocytes; Melanoma; Molecular Weight; Swine; Time Factors; Trypsin