Studies on the mechanism of endochondral ossification using a clonal chondrocyte like cell line newly established from a human chondrosarcoma - approach using the methods of cellular and molecular biology

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1. HCS-2/8 cells produced both IGF-I and IGF-II and expressed mRNA of these growth factors. The cells had types I and II receptors for IGF. Both IGF-I and II stimulated proteoglycan synthesis. These findings suggest that both IGFs act as autocrine differentiation factors in maintaining a high level of proteoglycan synthesis in HCS-2/8 cells. In contrast to previous findings using other cell lines, we found that IGF-type II receptor on HCS-2/8 cells play an important role in signal transduction by IGF-II-stimulated proteoglycan synthesis.2. Basic FGF stimulated the proliferation of HCS-2/8 cells only in sparse culture and the cells produced large amount of bFGF, suggesting that overexpression of bFGF is involved in permanent growth of the cell line. TGF-beta stimulated DNA and proteoglycan syntheses in the cells. The factor supports the proliferation and differentiation of HCS-2/8 cells in serum-free medium.3. Using northern blotting and RT-PCR, we found that HCS-2/8 cells express many genes related to chondrocyte differentiation such as aggrecan core protein, collagen types II, X and XI, IGF-I and IGF-II and these receptors and vitamin D_3 receptor. Ascorbic acid induced alkaline phosphatase activity and its mRNA expression. It also induced hypertrophy of the cells.4. There were no detectable transcripts for the following proto-oncogenes: c-sis, c-met, c-src, c-lyn, c-fgr, c-ros, c-pim, and Blym. However, transcripts of 12 other proto-oncogenes (int-2, erbB, c-abl, c-raf-1, c-fyn, K-ras, H-ras, c-mos, c-myc, c-myb, c-fos, and c-jun) are readily detectable by Northern analysis. Two proto-oncogenes, c-fos and c-raf-1 had an elevated levels of transcripts as high as 10 and 3 folds, respectively, at the overconfluent phase in comparison with the earlier phases of culture, suggesting relation to hypertrophy.