Bechberger J F, Khoo N S, Naus C C
Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada.
Cell Growth Differ. 1996 Oct;7(10):1403-13.
Transfection of C6 glioma cells with connexin43 (Cx43) cDNA under a constitutive promoter resulted in expression of Cx43 protein, an increase in functional gap junctions, and reduced growth under in vitro and in vivo conditions (D. Zhu et al., Proc. Natl. Acad. Sci. USA, 88: 1883-1887, 1991). To allow for precise temporal and quantitative control of Cx43 gene expression, the Cx43 cDNA was inserted into an expression vector [pSV2M(2)6] containing a modified metallothionein promoter. Upon transfection of this vector into C6 cells, clones were isolated that expressed increased levels of inducible Cx43 protein and dye coupling. The level of induction of Cx43 expression increased with increasing concentration of Zn2+, thus enabling the use of the same clone with different levels of gap junctions present. Although we observed no change in cell growth under in vitro conditions following exposure to Zn2+ or Cd2+, clones with inducible expression of Cx43 were characterized by reduced growth in vivo. Within tumors, the level of expression of Cx43 mRNA and protein corresponded to that seen in vitro following the addition of Zn2+. The suppression of tumor growth in vivo correlated with the level of induced Cx43 expression.
在组成型启动子的调控下，用连接蛋白43（Cx43）cDNA转染C6胶质瘤细胞，结果导致Cx43蛋白表达、功能性缝隙连接增加以及在体外和体内条件下生长减缓（D.朱等人，《美国国家科学院院刊》，88: 1883 - 1887, 1991）。为了实现对Cx43基因表达的精确时间和定量控制，将Cx43 cDNA插入到一个含有修饰金属硫蛋白启动子的表达载体[pSV2M(2)6]中。将该载体转染到C6细胞后，分离出了表达可诱导Cx43蛋白水平增加和染料偶联增加的克隆。Cx43表达的诱导水平随着Zn2+浓度的增加而升高，因此能够使用具有不同水平缝隙连接的同一克隆。尽管我们观察到在体外条件下暴露于Zn2+或Cd2+后细胞生长没有变化，但具有可诱导Cx43表达的克隆在体内的特征是生长减缓。在肿瘤内部，Cx43 mRNA和蛋白的表达水平与添加Zn2+后在体外观察到的水平相对应。体内肿瘤生长的抑制与诱导的Cx43表达水平相关。
