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 microg/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.

Estradiol Represses Human T-cell Leukemia Virus Type 1 Tax Activation of Tumor Necrosis Factor-α Gene Transcription

“…2B). E 2 also repressed Tax activation of the TNF-RE in an adult human osteoblastic (AHTO) cell line (40) that is immortalized by the SV-40 large T oncogene (Fig. 2C), demonstrating that the effect of E 2 also occurs in bone cells.…”

Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in Saethre-Chotzen syndrome

“…Both calvaria cell populations obtained from the mutant and normal subjects were immortalized in order to determine further the cellular phenotype induced by the mutation. Immortalization was performed similarly in the two cell types using a plasmid containing the SV-40 large T antigen, as described (28). All immortalized normal (Nl) and mutant Twist (M-Tw) cells showed genomic functional insertion of the large T antigen as determined by immunocytochemistry and RT-PCR analysis (data not shown).…”

Age‐related intrinsic changes in human bone‐marrow‐derived mesenchymal stem cells and their differentiation to osteoblasts

“…One-tenth or one-twentieth of the cDNA was used in each 50 μL PCR reaction (30-40 cycles of 94 °C for 1 min, 55-60 °C for 1 min, and 72 °C for 2 min) as described (Zhou et al, 2005b). The gene-specific primers for human p53 (Vakifahmetoglu et al, 2006); p21 (Lohr et al, 2003); BAX (Tirado et al, 2005); Cbfa1/RUNX2, Osterix, and bone sialoprotein (D'Ippolito et al, 2006); AlkP (Winn et al, 1999), COL I, and Osteocalcin (Lomri et al, 1999) were used for amplification. Gene expression levels were measured by semiquantitative PCR.…”