In vitro differentiation of human osteosarcoma cells

组织病理学和电镜观察表明，骨肉瘤细胞的骨形成模式主要与正常钙化组织相似。利用从人骨肉瘤中培养的骨肉瘤细胞系（HOS），研究了(1)体外诱导HOS细胞分化和(2)经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，我们还研究了在成骨细胞中表达的骨连接基因。(1)分化标志物的表达。发现HOS细胞表达骨/肝/肾型碱性磷酸酶（B/K/L/ALPase）。发现ALPase的表达受生长速率和/或细胞接触的调节。在培养基中加入-甘油磷酸酯和抗坏血酸可诱导Hos细胞内钙沉积。电镜观察显示HOS细胞产生羟基磷灰石（HA）晶体，经Debye-Scherrer环和微x射线分析鉴定。两个钙化起始点为内部有透明质酸晶体的囊泡；一种与基质囊泡相似，直径0.3-1 μ m，另一种直径1-3 μ m。我们还观察到沿胶原纤维形成透明质酸晶体。这些结果表明，HOS细胞具有成骨潜能，因此可以诱导表达成骨细胞的典型分化标记。(2)分化标志物在KHOS细胞中的表达。ALPase的表达和钙沉积均受到抑制。KHOS细胞含有一份K-MSV前病毒整合DNA拷贝，从中转录RNA。因此，k-ras癌基因极有可能抑制分化标志物的表达。(3)分子克隆了人和牛的骨连接cDNA，并确定了核苷酸序列。

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.