Development and characterization of a conditionally transformed adult human osteoblastic cell line

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Abstract
Many osteoblastic cell lines are currently in use, but these have limitations either in terms of their relevance to adult human biology and disease or in terms of their suitability for biochemical and molecular analyses. Consequently, we undertook the development of conditionally transformed adult human osteoblastic cell lines. Osteoblasts were obtained from a normal explant cancellous bone chip culture. These cells were infected with adenovirus-ori− SV40 tsA 209, which encodes a temperature-sensitive large T-antigen mutant. Cells immortalized with this virus express a transformed phenotype at the permissive temperature of 34°C but revert to a normal phenotype at the nonpermissive temperature of 40°C. Using this approach, we have isolated several cell clones and describe the characterization of one that was designated HOB-02-C1. Immunocytochemistry revealed that >95% of the cells express the large T-antigen at both temperatures. These cells exponentially proliferate at 34°C with a doubling time of ˜2 days but irreversibly stop dividing at 40°C. However, cell volume increases >2-fold when the cells are maintained for 6 days at the higher temperature. This clone expresses α1 type (I) procollagen mRNA and secretes type I procollagen C-peptide at both temperatures, although the levels were slightly elevated at 40°C. The cell line expresses alkaline phosphatase activity at 34°C, and the basal level of this enzyme increases 2- to 6-fold at 40°C. Alkaline phosphatase activity is induced 4- to 8-fold by 1α,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures, but transforming growth factor-β1 (TGF-β1) suppresses enzyme expression >90% at 40°C. Vitamin D3 also induces a 10-fold increase in osteocalcin secretion when the clone is maintained at 34°C, and this induction is enhanced > 8-fold at 40°C. Parathyroid hormone and forskolin stimulate a 4- to 6-fold increase in the production of intracellular cyclic AMP (cAMP) by the cells at 34°C, and this stimulation is enhanced 2- to 4-fold at 40°C. In contrast, prostaglandin E2 stimulates a 7- to 8-fold increase in cAMP only when the cells are maintained at 34°C. This cell line secretes TGF-β1 and interleukin-6 (IL-6) at 34°C, but only the basal secretion of IL-6 increases 70% at 40°C. Finally, alizarin red-S histochemical staining demonstrates that these cells produce mineralized nodules at both temperatures. In summary, the results of this study indicate that the HOB-02-C1 cells have a mature osteoblastic phenotype. Consequently, this new cell line and others obtained in a similar fashion should be valuable in vitro tools for cellular, biochemical, and molecular studies of adult human osteoblast biology.