Proffered Papers— Basic Science/Biology

MOLECULAR CLONING OF PUTATIVE ONCOGENE AND ANTIONCOGENE INVOLVED IN THE DEVELOPMENT OF OSTEOSARCOMA

Toguchida J, Murakami H, Nakamata T, Nakayama T. Nakamura T. (Institute for Frontier Medical Sciences and Department of Orthopaedic Surgery, Kyoto University, Kyoto, Japan)

Mutations of the Rb and p53 genes were found in approximately 60% of osteosarcomas, and hereditary mutations of either genes predispose individuals for the risk of osteosarcomas, suggesting the major role of these tumor suppressor genes in osteosarcoma. However, it is not yet clear whether mutations of both genes will be sufficient or other genetic alterations will be necessary for the development of osteosarcoma. To address this issue we have undertaken the in vitro transformation experiment. First, we have established an osteoblast-like cell line, MMC2, from the p53 (-/-) mice. MMC2 showed several phenotypes as differentiated osteoblasts such as the ability to produce calcified nodules in vitro. To inactivate the Rb gene in MMC2, HPV16E7 was introduced by the retrovirus vector and one cell line was established and designated as MMC2-E7. MMC2-E7 per se seemed to be a non-transformed cell line, because MMC2-E7 failed to make colonies in the soft agar and no constant tumor formation was observed in vivo. These results suggested that genetic alterations other than the mutation of the p53 and Rb genes will be involved in the development of osteosarcoma. After a prolonged latent period, however, MMC2-E7 produced tumors in vivo, and a cell line (MMC2-TC) was established from tumor tissues, which showed several phenotypes as a fully transformed cell. To isolate the genes responsible for the final transformation step, the differential display method was performed and two fragments, designated DDM23 and DDM36, were isolated. The expression of DDM23 was detected only in MMC2- TC, but not in MMC2 or MMC2-E7, and therefore it was considered to be a new oncogene involved in the process of malignant transformation. The expression of the other fragment, DDM36, was lost during the progression from MMC2-E7 to MMC2-TC, suggesting that it was a candidate for the new tumor suppressor gene in osteosarcoma. Functional and mutational analyses of these putative osteosarcoma-involved genes will provide the clue to understand the molecular mechanism of osteosarcoma development

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