MMP-9 secretion and MMP-2 activation distinguish invasive and metastatic sublines of a mouse mammary carcinoma system showing epithelial-mesenchymal transition traits.

We have investigated the gelatinase profiles and invasiveness of clonal tumour sublines derived from a spontaneously arising mammary tumour in a Balb/cfC3H mouse. The 67NR. 66c14 and 4T1.2 sublines have low, intermediate and high metastatic potential respectively. In Boyden chamber studies, Matrigel invasion was seen to be progressively higher in the more metastatic lines 4T1.2>66c14>67NR, consistent with MMP-2 activation potential, MMP-9 secretion, and migration over either type I or IV collagen, which were low in both 67NR and 66c14 cells compared to 4T1.2 cells. These attributes are consistent with those seen in human breast cancer cell lines which appear to have undergone an epithelial-mesenchymal transition (EMT) as indicated by vimentin expression. We were, however, surprised to find vimentin expression, MT1-MMP expression and stellate Matrigel outgrowth in the non-invasive, non-metastatic 67NR cells. indicating that they had undergone an EMT despite not being invasive. We conclude that the EMT is manifested to differing degrees in these three clonal cell lines, and that the 67NR cells have either undergone a partial EMT or have since lost certain important attributes of the EMT-derived phenotype. This model should prove useful in further characterizing the regulation of MTI-MMP mediated MMP-2 activation and delineating the EMT in breast cancer progression.

Metabolic processing of newly synthesized link protein in bovine articular cartilage explant cultures.

In explant cultures of articular cartilage from cattle of different ages radiolabeled leucine was shown to be incorporated into link proteins 1, 2 and 3. The newly synthesized link proteins were incorporated into and lost from the cartilage extracellular matrix with time. The levels of radiolabeled link proteins 1 and 2 remaining in the matrix declined over the culture period, but there was an initial increase in the amount of radiolabeled link protein 3, before its level declined. The turnover time of the radiolabeled link proteins 1 and 2 were similar, indicating that neither link protein was preferentially processed to generate link protein 3, nor lost from the extracellular matrix. The majority of the radiolabeled link protein lost from the cartilage matrix could not be recovered from the culture medium, suggesting that turnover of the radiolabeled aggrecan complexes involves the newly synthesized link protein being internalized by the chondrocytes. Inclusion of cytotoxic proteinase inhibitors to the culture medium resulted in a marked decrease in the rate of loss of link protein from the cartilage, suggesting that the catabolism of link protein is cell-mediated and dependent on metabolically active cells.

Characterization of a large chondroitin sulfate proteoglycan present in bovine collateral ligament.

Bovine collateral ligament synthesized a 35S-labeled large proteoglycan species which eluted with a Kav of approximately 0.27 on Sepharose CL-2B and contained only chondroitin sulfate chains with a molecular mass of approximately 32 kDa. Fluorography of the 35S-labeled core proteins derived from the large ligament proteoglycan revealed a broad range of molecular masses above approximately 200 kDa, which was of comparable size to the four major endogenous core protein bands derived from this proteoglycan detected with 5/6/3-B-3, an antibody directed against terminal unsaturated chondroitin-6-sulfate disaccharides. The core proteins derived from the large ligament proteoglycan exhibited immunoreactivity of 12/21/1-C-6, an antibody specific for a peptide epitope common to both the G1 and G2 domains of aggrecan. Four major core protein bands with molecular masses greater than approximately 200 kDa derived from the large ligament proteoglycan, were detected using the antibodies raised against versican from bovine aorta or human fibroblasts. Compared with aggrecan, the 35S-labeled large ligament proteoglycan was distributed over a broader range of buoyant densities in an associative caesium chloride density gradient. This polydispersity may be indicative of differences in the degree of glycosylation as well as heterogeneity in the size of the large ligament proteoglycan core proteins. The 35S-labeled large ligament proteoglycan also demonstrated the ability to form complexes with an aggrecan aggregate preparation, the majority of which could not be dissociated by the presence of HA10-50. These findings indicate that the large chondrotin sulfate proteoglycan synthesized by bovine collateral ligament may be a versican-like proteoglycan which exhibited the potential to form like protein-stabilized complexes.