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.
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.
Lomri
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INSERM Unit 349, Cellular & Molecular Biology of Bone and Cartilage, Lariboisiere Hosp. 6, rue Guy Patin, 75475 Paris Cedex 10, France.
Fromigué
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O
Hott
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Marie
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PJ
D000368:Aged; D000469:Alkaline Phosphatase / Q000378:metabolism; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001205:Ascorbic Acid / Q000494:pharmacology; D001842:Bone and Bones / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D002113:Calcification, Physiologic; D002454:Cell Differentiation; D002455:Cell Division; D002472:Cell Transformation, Viral / Q000235:genetics*; D002762:Cholecalciferol / Q000494:pharmacology; D003094:Collagen / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis; D003907:Dexamethasone / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D008297:Male; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D015675:Osteocalcin / Q000378:metabolism; D010641:Phenotype; D010710:Phosphates / Q000494:pharmacology; D012313:RNA / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013539:Simian virus 40 / Q000276:immunology*
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D004279:DNA, Viral; D010710:Phosphates; D015675:Osteocalcin; D002762:Cholecalciferol; D012313:RNA; D003907:Dexamethasone; D003094:Collagen; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid