Analysis of connexin43 expression under the control of a metallothionein promoter

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.