Genomic Insertion of the SV-40 Large T Oncogene in Normal Adult Human Trabecular Osteoblastic Cells Induces Cell Growth Without Loss of the Differentiated Phenotype

Abstract.

In the present study, we established a new adult human trabecular osteoblastic (AHTO) cell line, immortalized by SV-40 Large T (LT) oncogene. From seven proliferative colonies identified, we selected clone 7 with high alkaline phosphatase (ALP) activity for further analysis. AHTO₋₇ cells were able to grow for at least 8 months and 25 passages, with a doubling time of about 22 hours. Immunocytochemistry staining and RT-PCR analysis indicated that the extended life-span of AHTO₋₇ cells results in genomic insertion of SV-40 LT oncogene. The cells responded to PTH and PGE₂ in terms of cAMP accumulation. The time course study, in the presence of 10⁻⁸ M vitamin D₃ (vit D₃) showed a marked increase (fourfold) in ALP activity with a peak at day 3. Furthermore, in the presence of ascorbic acid (50 μg/ml) and inorganic phosphate (3 mM), AHTO₋₇ cells produced abundant calcified extracellular matrix, as examined by the von Kossa staining after 2 weeks of culture. Molecular analysis of mRNAs for phenotypic osteoblast markers at day 15 showed the expression of ALP, osteocalcin (OC), and collagen type I (Col I) mRNAs constitutively. Col I expression was inhibited by vit D₃ and dexamethasone treatment. In contrast, treatment with vit D₃ induced a marked increase of ALP and OC transcripts. Therefore, the immortalized AHTO₋₇ cells express osteoblast markers that are induced by calciotropic hormones, and constitute a suitable model for identifying specific osteoblastic genes and their regulation during human osteoblast differentiation.