In vitro differentiation of human osteosarcoma cells

组织病理学和电子显微镜观察表明，骨肉瘤细胞的骨形成模式与正常钙化组织的模式基本相似。以人骨肉瘤建立的骨肉瘤细胞系(HOS)为研究对象，探讨了(1)HOS细胞是否在体外被诱导分化；(2)KHOS细胞、Kirsten小鼠肉瘤病毒(K-MSV)转化的HOS细胞是否表达成骨细胞分化标志物。此外，我们还对成骨细胞中表达的成骨连接基因进行了研究。(1)分化标志物的表达。HOS细胞表达骨/肝/肾型碱性磷酸酶(B/K/L/ALPase)。ALPase的表达受生长速度和/或细胞接触的调节。在培养液中加入β-甘油磷酸和抗坏血酸，可诱导HOS细胞内钙沉积。电子显微镜观察表明，HOS细胞产生了羟基磷灰石(HA)晶体，德拜-谢勒环和显微X射线分析证实了这一点。观察到两种钙化起始点，一种类似于基质泡，直径0.3-1um，另一种为1-3um，内含HA晶体。我们还观察到HA晶体沿胶原纤维的生长。这些结果提示HOS细胞具有成骨潜能，可以被诱导表达典型的成骨细胞分化标志物。(2)KHOS细胞分化标志物的表达。ALPase表达和钙沉积均受到抑制。KHOS细胞含有单一拷贝的K-MSV前病毒整合DNA，RNA从该DNA转录而来。因此，k-ras癌基因很可能抑制了分化标记物的表达。(3)从人和牛的成骨细胞中克隆了成骨相关基因，并测定了其核苷酸序列。

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.