Takahashi Mitsugu, Chernin Mitchell I, Yamamoto Osamu, Tonzetich John, Kinsey Conan G, Novak Josef F
Department of Orthopaedics, Chitose General Hospital, Japan.
Anticancer Res. 2002 Mar-Apr;22(2A):585-98.
A continuous cell line, MC3T3-E1 cells, originally derived from murine calvaria bones, loses its osteogenic properties as a result of extended passage number under stress conditions. These aged/stressed MC3T3-S cells, although nontumorigenic, do not display some of the osteogenic properties characteristic of the MC3T3-E1 cells. Altered properties include low expression of alkaline phosphatase, diminished collagen synthesis and inability to form mineralized nodules in vitro. We attempted to reactivate these osteogenic properties by transfections with a pSV2neo plasmid containing the TGFbeta1 gene. During these experiments we found that transfected MC3T3-S cells not only acquired high alkaline phosphatase activity and a potent mineralization potential, but also properties akin to the transformed state, such as ability to grow in soft agar and ability to produce tumors in immunodeficient animals. Further analysis showed that the TGFbeta1 gene is not required and that the changes can be introduced by transfections with pSV2neo alone. In contrast, MC3T3-S cells transfected with pcDNA3 (a plasmid containing only the SV40 origin of replication, early promoter, enhancer and polyadenylation signals) or mock-transfected MC3T3-S cells did not show any transformation traits. The results identify two additional SV40 fragments present in pSV2neo (SV40 virus sequence; Genbank accession number: NC_001669: 4100-4191 and 2668-2774) as functional elements contributing to the transformation of aged/stressed and immortalized osteoblastic cells. These findings are analogous to earlier reports describing the cell modifying potential of pSV2neo. We conclude that stressed and aged MC3T3-S can be transformed by transfection with pSV2neo and that such cells acquire not only the tumorigenic potential but exhibit also some of the osteogenic properties characteristic of the parent MC3T3-E1 cells.
一种连续细胞系MC3T3-E1细胞最初源自小鼠颅骨，在应激条件下由于传代次数增加而丧失其成骨特性。这些老化/应激的MC3T3-S细胞虽然不具有致瘤性，但不具备MC3T3-E1细胞某些典型的成骨特性。改变的特性包括碱性磷酸酶表达降低、胶原蛋白合成减少以及在体外无法形成矿化结节。我们试图通过用含有TGFβ1基因的pSV2neo质粒转染来重新激活这些成骨特性。在这些实验中，我们发现转染的MC3T3-S细胞不仅获得了高碱性磷酸酶活性和强大的矿化潜力，还获得了类似于转化状态的特性，如在软琼脂中生长的能力以及在免疫缺陷动物中产生肿瘤的能力。进一步分析表明，TGFβ1基因并非必需，仅用pSV2neo转染就能引发这些变化。相比之下，用pcDNA3（一种仅含有SV40复制起点、早期启动子、增强子和聚腺苷酸化信号的质粒）转染的MC3T3-S细胞或mock转染的MC3T3-S细胞未显示出任何转化特征。结果确定了pSV2neo中存在的另外两个SV40片段（SV40病毒序列；Genbank登录号：NC_001669: 4100-4191和2668-2774）是导致老化/应激和永生化成骨细胞转化的功能元件。这些发现与早期描述pSV2neo细胞修饰潜力的报告相似。我们得出结论，应激和老化的MC3T3-S细胞可通过用pSV2neo转染而转化，并且这些细胞不仅获得了致瘤潜力，还表现出一些亲本MC3T3-E1细胞典型的成骨特性。
