Martens I, Ramqvist T, Dalianis T, Linder S
Department of Medical Virology, Uppsala University Biomedical Centre, Sweden.
Exp Cell Res. 1990 Mar;187(1):143-9. doi: 10.1016/0014-4827(90)90128-w.
The tumorigenicity of secondary rat embryo fibroblasts transfected with a plasmid harboring a replication origin-defective polyomavirus was found to increase during in vitro propagation. Thus, polyomavirus-transfected cells were found to be more than 10,000-fold more tumorigenic when injected into syngenic rats at 3 months after transfection compared to those injected at an earlier time point. Furthermore, most clones of polyomavirus-transfected cells did not grow in semisolid medium at 52 days after transfection but did grow at 95 days. Addition of glucocorticoid hormones, but not of 25% fetal calf serum, to the growth medium of the early passage cells resulted in limited anchorage-independent growth. An altered level of expression of a number of proteins was found in cells analyzed at different times after transfection. Notably, the expression of a component of the actin filament system, tropomyosin 2, was shown to decrease during growth in vitro. The development of a more fully transformed phenotype at late passages correlated with loss of the requirement for large T-antigen for growth. Thus, cells transfected with a polyomavirus mutant encoding a thermolabile large T-antigen did not grow at the restrictive temperature at 6 weeks after transfection, but grew well at 5 months after transfection. We suggest that these phenomena may be explained by assuming that establishment of rodent fibroblasts, and thereby sensitivity to transformation by middle T-antigen, is not an immediate consequence of expression of large T-antigen but occurs after a period of growth in vitro.
用携带复制起点缺陷型多瘤病毒的质粒转染的二代大鼠胚胎成纤维细胞的致瘤性在体外传代过程中被发现有所增加。因此，与在较早时间点注射的细胞相比，多瘤病毒转染的细胞在转染后3个月注射到同基因大鼠体内时，其致瘤性高出10000倍以上。此外，多瘤病毒转染细胞的大多数克隆在转染后52天不能在半固体培养基中生长，但在95天时可以生长。向早期传代细胞的生长培养基中添加糖皮质激素而非25%胎牛血清，会导致有限的不依赖贴壁生长。在转染后不同时间分析的细胞中发现了多种蛋白质表达水平的改变。值得注意的是，肌动蛋白丝系统的一个组分原肌球蛋白2的表达在体外生长过程中显示下降。后期传代时更完全转化表型的发展与生长对大T抗原需求的丧失相关。因此，用编码热不稳定大T抗原的多瘤病毒突变体转染的细胞在转染后6周的限制温度下不能生长，但在转染后5个月生长良好。我们认为，这些现象可以通过假设啮齿动物成纤维细胞的建立以及因此对中T抗原转化的敏感性不是大T抗原表达的直接结果，而是在体外生长一段时间后发生来解释。
