Purpose: Chondrosarcoma is the second most common malignancy in bone and results from unregulated growth of mesenchymal cells.. Chondrosarcoma does not appear to respond either to chemotherapy or radiation. Currently chondrosarcoma has no effective treatment and a new approach to adjuvant therapy for this tumor is urgently needed. We have previously reported that Matrix metalloproteinase -1 (MMP-1)gene expression is an independent predictor of survival in chondrosarcoma.(Scully, 2000). We have reported also that the expression of the large TNC splice variant may correlate with malignancy and poor clinical outcome in human chondrosarcoma and be implicated in the process of basement membrane invasion. The aim of the current study was to confirm that Tenascin-C large splice variant(TNC320) stimulates matrix metalloproteinase-1(MMP-1)expression and invasive potential and to elucidate molecular mechanisms underlying this activation.

Method: The chondrosarcoma cell line was recultured in alginate beads. (Guo et al, 1989)The beads contained exogenous proteins according to design. Alginate beads were dissociated later by chelation and the cells were pelleted by centrifugation. Gel electrophoresis and Western blotting were performed to demonstrate the expression of MMP-1 protein in the cells cultured with treatments. MMP-1 protein was detected by mouse antiMMP-1 monoclonal antibody from Chemicon. QPCR and RT-PCR were used to detect MMP-1mRNA expression with different treatments Transactivation of MMP-1 luc construct was detected in transiently transfected JJ012 human chondrosarcoma cultured cells. Reporter gene, Collagenase, Cell invasion assays and MMP-ELISA were performed for this study.

Results: The analysis of gene expression in cultured cells grown under different condition indicated significant increases of MMP-1mRNA steady-state levels in the cells with TNC 320 treatment. Gel electrophoresis demonstrated augmented MMP-1 protein in cells cultured with TNC 320. The result was confirmed by examining MMP-1 promoter transactivation of 30fold in comparison with control and other treatments. Both invasion and collagenase assays demonstrated 3 fold difference in the cells treated with TNC 320. Experiments with constitutively active expression kinases indicated that MMP-1 expression induced by TN320 was associated with MAPK cascade activation.

Conclusion: Preliminary results presented demonstrate that Tenascin-C 320kda splice variant stimulates MMP-1 expression and involves MAPK pathway. We hypothesize that Tenascin-C stimulates invasive potential of human chondrosarcoma cells via upregulation of MMP-1 expression.