In vitro differentiation of human osteosarcoma cells

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Histopathological and electron microscopical observations suggest that the pattern of bone formation in osteosarcoma cells is principally similar to that of normally calcifying tissue. Using osteosarcoma cell line (HOS) established from human osteosarcoma, in the present work we asked questions whether or not (1) HOS cells is induced to differentiate in vitro and (2)KHOS cells, HOS cells transformed by Kirsten murine sarcoma virus (K-MSV) express differentiation markers of osteogenic cells. In addition, we studied on osteonection gene which is expressed in osteogenic cells.(1) The expression of differentiation markers. HOS cells were found to express the bone/liver/kidney-type alkaline phosphatase(B/K/L/ALPase). The expression of ALPase was found to be regulated by growth rate and/or cellular contact. The addition of beta-glycerophosphate and ascorbic acid to the culture medium induced Hos cells to deposition of calcium inside. The electron microscopic observation showed that HOS cells produced hydroxyapatite (HA) crystals, which was identified by Debye-Scherrer ring and micro X-ray analyses. Two initiation sites for calcification were observed as vesicles with HA crystals inside; one type similar to matrix vesicle was 0.3-1 um in diameter and the other 1-3 um. We also observed development of HA crystals along collagen fibers. These results suggest that HOS cells possess an osteogenic potential and thus can be induced to express differentiation markers typical of osteogenic cells.(2) The expression of differentiation markers in KHOS cells. The expression of both ALPase and deposition of calcium was suppressed. KHOS cells contained a single copy of K-MSV proviral integrated DNA from which RNA was transcribed. Therefore, it is highly likely that k-ras oncogene suppressed the expression of differentiation markers.(3) We molecularly cloned osteonection cDNA from human and bovine and determined nucleotide sequences.