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

1.p53缺失小鼠成骨细胞样细胞系MMC2的建立。用p53基因敲除小鼠颅骨建立成骨样细胞系。MMC2表现出长生不老。然而，表现出锚定依赖性生长和接触抑制。因此没有转化。MMC2作为成骨细胞具有分化的表型，如ALP活性、型胶原蛋白和骨钙素基因的mRNA表达。在体外也产生了钙化基质。将HPV16E7基因导入MMC2构建转化细胞系。为了灭活RB蛋白的功能，将HPV16E7基因导入MMC2细胞，并接种于nu/nu小鼠。以1只小鼠的肿瘤组织为材料，建立了E7TC细胞系。E7TC细胞周期蛋白A2和胸苷激酶基因的表达不正常，表明E2F在该细胞系中的活性增加。另一方面，作为成骨细胞的分化表型丧失，E7TC产生的肿瘤更多地与骨肉瘤不相容，而是与未分化的肉瘤不相容。自发转化细胞系MMC2TC23的建立。1只小鼠在接种MMC2 8个月后出现皮肤肿瘤形成。从该肿瘤（TC23）建立了体外细胞系。TC23表现出与E7TC一样的全转化细胞系的多种表型，但同时具有分化成骨细胞的表型。TC23产生的肿瘤表现为典型的人骨肉瘤。因此，我们成功地从MMC2.4中建立了两种表型不同的肉瘤细胞系。差分显示法分离新基因。采用差异显示法分离MMC2向E7TC或TC23完全转化过程中表达改变的基因。目前已分离到两个新基因：一个在E7TC中表达增加，另一个在E7TC中表达缺失。目前正在克隆这两个基因的全长cdna。少

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