2002 CTOS Annual Meeting Posters — Medical Oncology

IN VITRO TRANSFORMATION OF THE RB(-/-) MURINE OSTEOBLAST
[Abstract ID: 44]

Category: Medical Oncology

Authors: Takeshi Okamoto¹, Hiroshi Yamamoto¹, Tomoki Aoyama¹, Koichi Nishijo¹, Takeharu Nakamata¹, Taisuke Hosaka¹, Tomitaka Nakayama¹, Takashi Nakamura¹, Junya Toguchida²

Author Institutions: ¹Department of Orthopaedic Surgery Kyoto University, Japan; ²Institiute for Frontier Medical Science Kyoto Univeresity, Japan

Presenter: Takeshi Okamoto
tokmt@frontier.kyoto-u.ac.jp

Correspondent: Junya Toguchida
togjun@frontier.kyoto-u.ac.jp
Kyoto city Japan 606-8507
Ph: +81-75-751-4142
Fax: +81-75-751-4144

Objectives: Objective: It is well known that the retinoblastoma (Rb) gene plays an important role in the development of osteosarcoma. Precise sequential events after the inactivation of the Rb gene, however, remains to be investigated. Here we have performed in vitro transformation experiments using Rb-/- murine osteoblasts as a starting material.

Methods: Methods: Rb-/- osteoblast-like cells were isolated from long bone of neonatal chimeric mice composed of wild-type and Rb-/- cells, and using the ALP activity as an ostoblast marker, one clonal cell line (ΔRbOB1) was established.

Results: Results: ΔRbOB1 expressed several marker genes as osteoblast (type I collagen, osteocalcin, and cbfa1/runx2), and showed vigrous growth in vitro, but neither anchorage-independent growth nor in vivo tumorigenesity was observed. To inactivate the p53 gene in the ΔRbOB1, p53DD, the truncated dominant-negative form of human p53 gene, was introduced by the retrovirus vector, and a clonal cell line expressing p53DD was established (ΔRbOB1p53DD). Induction of p53 gene by actinomycin D was observed in the ΔRbOB1, but not in the ΔRbOB1p53DD, suggesting that the wild-type p53 was inhibited inΔRbOB1p53DD. However, neither anchorage-independent growth nor in vivo tumorigenecity was observed in the ΔRbOB1p53DD. Finally, trasfection of the oncogenic H-ras gene endowedΔRbOB1p53DD with the activity as completely transformed cells, but tumors developed in nude mice failed to form osteoid.

Conclusions: Conclusion: The inactivation of Rb and p53 in osteoblast wasn’t enough for the development of osteosarcoma. Therefore, we still missed the last piece to develop osteosarcomas from osteoblasts.