Investigation of the molecular mechanisms for the development of osteosarcoma using the retinoblastoma gene chimeric mice.

使用视网膜母细胞瘤基因嵌合小鼠研究骨肉瘤发生的分子机制。

• 批准号: 10470306
• 负责人: TOGUCHIDA Junya
• 金额: $ 6.66万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470306/
• 关键词: osteosarcoma; retinoblastoma gene; chimeric mice; transformation

To investigate the role of the retinobastoma (Rb) gene in the process of oncogenesis, we have performed the in vitro transformation experiments using the Rb(-/-) osteoblast as a starting material. First, we have created the Rb chimeric mice consisted of Rb(+/+) and Rb(-/-) cells by using Rb(-/-) ES cells. The Rb(-/-) osteoblasts were isolated from Calvary and long bones from newborn chimeric mice, and several clones were established. The genotypes of these clonal cells were analyzed by the allelespecific PCR and Rb(-/-) clones were further studied. The level of alkaline phosphatase (ALP) and osteocalcin, which are considered to be markers for differentiated-osteoblasts, were relatively low, whereas the expression the cbfal and BMP2 genes were almost equivalent with those of the Rb(+/+) osteoblastic cells.By the differentiation-induction experiments, these cells showed increased ALP activity and produced mineralized matrix, suggesting that the Rb deficiency has no remarkable effects for the differentiaotn process of osteoblasts. We found that these cells showed marked reduction of growth ability when the generation number was over 90, which is thought to be the life span of normal rodent cells, indicating that the Rb deficiency has no remarkable effects for the immortality. We are currently creating the tetracycline-regulated Rb expression system in these cells to further investigate the phenotypic differences of Rb(+/+) and Rb(-/-) osteoblasts, such as the response for growth factors.On the other hand, to obtain fully transformed cells, the dominant negative mutant of the human p53 gene was transfected into these cells. Transformants showed persistent growth ability even after the 90ィイD1thィエD1 generations, suggesting that the mutant p53 confer the immortality to these cells. We are currently analyzing the in vitro and in vivo phenotypes as fully neoplastic cells of these transfectants.

为了研究视网膜母细胞瘤（retinobastoma，Rb）基因在肿瘤发生过程中的作用，我们以Rb（-/-）成骨细胞为起始材料进行了体外转化实验。首先，我们利用Rb（-/-）ES细胞建立了由Rb（+/+）和Rb（-/-）细胞组成的Rb嵌合小鼠。从新生嵌合体小鼠的跟骨和长骨中分离Rb（-/-）成骨细胞，并建立了几个克隆。用等位基因特异性PCR分析克隆细胞的基因型，并进一步研究Rb（-/-）克隆。分化成骨细胞的标志物碱性磷酸酶（ALP）和骨钙素水平较低，而cbfal和BMP 2基因的表达与Rb（+/+）成骨细胞相当，诱导分化实验显示，这些细胞ALP活性增强，并产生矿化基质，提示Rb缺乏对成骨细胞的分化过程无明显影响。我们发现，当细胞的世代数超过90代时，细胞的生长能力明显下降，这被认为是正常啮齿动物细胞的寿命，表明Rb缺乏对永生性没有明显影响。为了进一步研究Rb（+/+）和Rb（-/-）成骨细胞表型的差异，如对生长因子的反应，我们目前正在这些细胞中建立四环素调控的Rb表达系统。另一方面，为了获得完全转化的细胞，将人p53基因的显性负突变体转染这些细胞。即使在第90代イD1 D1代之后，转化体仍表现出持续的生长能力，这表明突变型p53赋予这些细胞永生性。我们目前正在分析在体外和体内的表型作为完全肿瘤细胞的这些转染。

项目成果

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Kanoe, H., et al.: "Charactreristics of genomic breakpoints in TLS-CHOP translocations in liposarcomas suggest the imvolvement of Translin and topoisererase II in the process of translocation" Oncogene. 18(3). 721-729 (1999)

Kanoe, H. 等人：“脂肪肉瘤中 TLS-CHOP 易位基因组断点的特征表明 Translin 和拓扑酶 II 在易位过程中的参与”Oncogene。

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Yamamoto,H., et al.: "High incidence of SV40・like sequprces detedion in tumour and peripheral blood cells of Japanese osteosarcome patients"Br. J. Cancer. (in press).

Yamamoto, H. 等人：“日本骨肉瘤患者的肿瘤和外周血细胞中 SV40 样序列检测的高发生率”Br. J. Cancer（出版中）。