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

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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.