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

1. p53基因敲除小鼠成骨样细胞系MMC 2的建立MMC 2表现出永生化生长。然而，表现出锚定依赖性生长，和接触抑制。因此没有改变。MMC 2具有成骨细胞的分化表型，如ALP活性、型胶原和骨钙素基因的mRNA表达。将HPV 16 E7基因导入MMC 2细胞，建立了转化细胞系。为了验证RB蛋白的功能，将HPV 16 E7基因导入MMC 2细胞，并接种nu/nu小鼠。从在一只小鼠中发育的肿瘤组织建立细胞系（E7 TC）。E7 TC细胞周期蛋白A2和胸苷激酶基因表达下调，表明E2 F在该细胞系中的活性增加。另一方面，成骨细胞的分化表型丢失， ...更多信息 E7 TC产生的肿瘤与骨肉瘤不相容，而是未分化的肉瘤。3.自发转化细胞系（MMC 2 TC 23）的建立。从该肿瘤（TC 23）建立体外细胞系。TC 23与E7 TC一样具有完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。TC 23产生的肿瘤表现为典型的人骨肉瘤表现。4.差异显示技术分离新基因本研究利用差异显示技术分离了在MMC 2向E7 TC或TC 23转化过程中表达发生改变的基因。目前已分离到两个新基因，一个在E7 TC中表达增强，另一个在E7 TC中表达缺失，这两个基因的全长cDNA正在克隆中。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