Investigation of the molecular process in the development of osteosarcomas by in vitro transformation.

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1.Establishment of osteoblast-like cell line, MMC2, from p53 deficient mice.Osteoblast-like cell line was established from calvarie of p53 knock-out mice. MMC2 showed growth immortalized. However, showed anchorage-dependent growth, and contact inhibition. There fore not transformed. MMC2 was found to possess differentiated phenotypes as osteoblasts, such as ALP activity, mRNA expression of type collagen and osteocalcin genes. Also calcified matrix was produced in vitro.2.Establishment of a transformed cell line by the introduction of HPV16E7 gene into MMC2.To inactivate the function of RB protein, HPV16E7 gene was introduced into MMC2 cells, and cells were inoculated onto nu/nu mice. A cell line (E7TC) was established from tumor tissue developed in one mouse. E7TC showed deregulated expression of cyclin A2 and thymidine kinase genes, suggesting the increased activity of E2F in this cell line. On the hand, differentiated phenotypes as osteoblasts were lost, and pathological findings of … More tumor produced by E7TC were not compatible with osteosarcoma, rather undifferentiated sarcomas.3.Establishment of spontaneous transformed cell line (MMC2TC23).Suncutaneous tumor formation was noticed in one mouse 8 month after the inoculation of MMC2. In vitro cell line was established from this tumor (TC23). TC23 showed several phenotypes as a full transformed cell line like E7TC,but at the same time possessed phenotypes as differentiated osteoblasts. Tumor produced by TC23 showed typical findings as human osteosarcomas. Therefore we have succeed to establish two phenotypically different sarcoma cell lines from MMC2.4.Isolation of novel genes by differential display method.Differential display method was used to isolate genes which has changed the expression during the course of full transformation from MMC2 to either E7TC or TC23. So far two novel genes were isolated : one showed increased expression in E7TC,and the other lost expression in E7TC.Full length cDNAs of these two genes are currently under cloning. Less