alpha-chymotrypsin and Mast-Cell-Sarcoma

Topics: Animals; Carboxypeptidases; Cats; Chymotrypsin; Dogs; Humans; Mast Cells; Mast-Cell Sarcoma; Peptide Hydrolases; Protease Inhibitors; Rats; Trypsin

Mast cell secretory granules contain unique tryptic and chymotryptic serine proteases that differ between species and tissues. Direct comparison of these proteases in single-cell types has been hindered by the difficulty of obtaining adequate numbers of pure mast cells. In this study, we were able to compare tryptic and chymotryptic enzyme activity in cells of presumed monoclonal origin, using two stable lines ('BR' and 'G') of dog mastocytomas. The gel-filtration profiles, inhibitor susceptibilities and substrate preferences of tryptic and chymotryptic mastocytoma protease activities established their close resemblance to the tryptases and chymases of human and rodent mast cells. Striking heterogeneity was observed in the amounts and solubilities of the tryptic and chymotryptic activity in the two different mastocytoma cell lines. Incubation of cells from both lines with calcium ionophore A23187 caused non-cytotoxic release of protease activity. In contrast to chymase from rat connective tissue mast cells, protease activity that was insoluble after extraction at low ionic strength became soluble following ionophore-stimulated release. Neither tryptic nor chymotryptic activity was activated during degranulation, suggesting the absence of inactive precursors. Cells of the 'BR' line released both tryptic and chymotryptic activity in parallel with the granule marker histamine; cells of the 'G' line released a much smaller proportion of tryptic activity than of either chymotryptic activity or histamine. These differences in release of granule constituents from cells of common origin could be explained by developmental variations in the production of performed mediators or by differential regulation of preformed mediator release. We conclude that the differences in protease content, solubility and release in these mastocytoma lines are useful in evaluating the potential pathophysiological significance of the contribution of proteases to mast cell heterogeneity.

Topics: Animals; Calcimycin; Chymases; Chymotrypsin; Cytoplasmic Granules; Dogs; Mast Cells; Mast-Cell Sarcoma; Peptide Hydrolases; Serine Endopeptidases; Substrate Specificity; Trypsin; Tumor Cells, Cultured

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.

Topics: Animals; Bronchoalveolar Lavage Fluid; Chymases; Chymotrypsin; Dogs; Histocytochemistry; Mast Cells; Mast-Cell Sarcoma; Peptide Hydrolases; Serine Endopeptidases; Skin Neoplasms; Trypsin; Tumor Cells, Cultured

Topics: Alkylation; Animals; Binding Sites; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Electrophoresis; Heparin; Indoles; Mast Cells; Mast-Cell Sarcoma; Mice; Molecular Weight; Neoplasms, Experimental; Peptide Hydrolases; Peritoneal Neoplasms; Protease Inhibitors; Protein Binding; Tritium; Trypsin