Diversity of melanoma plasma membrane proteinases: inhibition of collagenolytic and cytolytic activities by minocycline.

The tissue-destructive proteinases of B16-BL6 melanoma cells from C57BL/6 mice and subcellular fractions were examined. Cancer cell organelles were isolated following nitrogen cavitation with the use of sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed with the use of radiolabeled proteins and synthetic substrates. Tumor-induced red blood cell lysis was quantitated by measurement of the release of isotope from 59Fe-labeled red blood cells (RBC) cocultivated with melanoma cells; the RBC were from Wistar rats. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact melanoma cells, cell organelles, and cytosol contain proteinases that can degrade collagen and gelatin and lyse normal RBC. Melanoma plasma membranes are highly enriched in collagenase, gelatinase, cysteine proteinase, plasminogen activator, and cytolytic activity. The inhibition of tumor collagenolytic, gelatinolytic, and cytolytic activities by EDTA and 1,10-phenanthroline but not by diisopropyl fluorophosphate and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates that metalloproteinases are the active enzymes in these assays. Minocycline, a synthetic tetracycline with demonstrable inhibitory activity with other mammalian collagenases, also inhibited melanoma collagenolytic and cytolytic activities.

Diversity of human pancreatic cancer cell proteinases: role of cell membrane metalloproteinases in collagenolysis and cytolysis.

In this study we have examined the tissue-destructive proteinases of human pancreatic ductal cancer cell lines derived initially from xenogenic transplants. Cancer cell organelles were isolated following nitrogen cavitation using sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed using radiolabeled protein and synthetic substrates. Tumor-induced RBC lysis was quantitated by measuring the release of isotope from 59Fe-labeled RBCs co-cultivated with tumor cells or subcellular fractions. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact pancreatic cancer cells (RWP-1 and RWP-2 cell lines), cell homogenate, and cytosol contain proteinases which were able to degrade [3H]collagen (type I) and [3H]gelatin and lyse normal RBCs. Cancer cell membrane fractions were enriched in collagenolytic, gelatinolytic, and cytolytic activities which could be abrogated by EDTA but not by inhibitors of serine or cysteine proteinases, which indicates that metalloproteinases are the active enzymes in these assays. Although plasminogen activator and cysteine proteinases were also enriched in the tumor cell membranes, these activities were not required for collagen degradation or cytolysis. We conclude that human cancer cell membrane proteinases are advantageously situated to facilitate damage to surrounding normal tissues.

Metastatic mouse melanoma cells release collagen-gelatin degrading metalloproteinases as components of shed membrane vesicles.

The purpose of this study has been to compare collagen-gelatin degrading enzymes isolated from cancer cell organelles and cytosol to the metalloproteinases released by cancer cells. To this end, metastatic mouse melanoma cell organelles were isolated by sucrose density gradient centrifugation and metalloproteinases were assayed using native and denatured [methyl-3H]collagen substrates. Solubilized proteinases were purified by ammonium sulfate precipitation, anion exchange, concanavalin A affinity and gel-filtration column chromatographic procedures and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conclusions were as follows: malignant melanoma cells have a metalloproteinase (Mr = 59,000) which is shed from cells into conditioned medium as a component of intact membrane vesicles rather than as a soluble enzyme; storage of tumor-conditioned medium leads to the generation of autoactivated soluble metalloproteinases of lower molecular weight; purification of these metalloproteinase species yielded variant collagenases that have considerable gelatinolytic activity and a cleavage preference site for the Gly-Ile bond in a collagen-like synthetic octapeptide substrate which is typical for collagenase-type metalloproteinases. It is proposed that localization of potent proteinases to the surface of cancer cells facilitates the local breakdown of connective tissues during the invasive process.