Comparison of basement membrane matrix degradation by purified proteases and by metastatic tumor cells.
We have examined the nature of biochemical degradation of an isolated basement membrane matrix (bovine lens capsule) using different methodologies. The first strategy was quantitation of the release of surface-bound 125I and a second the documentation by SDS-PAGE of the appearance of putative cleavage products and the loss of high-molecular-weight components from the matrix. Basement membrane matrix bands resolved on SDS-PAGE were identified by their protease sensitivities as well as by Western immunoblots using monoclonal antibodies developed for this study. Radioiodinated components were found predominantly at positions on the gel equivalent to 160-200 kd and 400 kd proteins. Since these labeled moieties were sensitive to bacterial collagenase digestion and stained with anticollagen type IV antibodies, they were determined to represent various configurations of collagen type IV. Several other lower-molecular-weight bands also stained with the anticollagen IV antibodies. Monoclonal antibodies reactive with laminin exhibited a complex staining pattern on the gels, which included the expected 200 and 400 kd components. We confirmed that lens capsule basement membrane contained only a single heparan sulfate glycosaminoglycan species, and tumor cell-induced glycosaminoglycan degradation within the basement membrane matrix was detected using cellulose acetate electrophoresis. Distinctive putative cleavage products were resolved on SDS-PAGE gels from matrices subjected to digestion by a variety of purified proteases as well as by metastatic tumor cells or their conditioned media. Tumor cells of different histiotypes produced different characteristic cleavage patterns, suggestive of the existence of several pathways of matrix degradation. Overall, primary tumor cells exhibited a greater degradative activity towards the basement membrane matrix than did long-term tissue culture-passaged cells. The same tumor cell line could exhibit considerably different patterns of both protein and glycosaminoglycan degradation depending on recent culture history. The relevance of these biochemical studies to the pathogenesis of malignant neoplasms is shown by: 1) the evaluation of degradative activities of B16 tumor cell populations exhibiting enhanced lung-colonizing phenotypes, and 2) the ability of a known antimetastatic moiety with antiprotease activity (Haementeria leech species salivary gland extract) to protect matrix components from degradation by tumor cell-conditioned medium.
Starkey
J R
JR
Department of Microbiology, Montana State University, Bozeman 59717.
Stanford
D R
DR
Magnuson
J A
JA
Hamner
S
S
Robertson
N P
NP
Gasic
G J
GJ
D000818:Animals; D001485:Basement Membrane / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D007903:Lens Capsule, Crystalline / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D009362:Neoplasm Metastasis / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D007797:Laminin; D010447:Peptide Hydrolases