The effects of altered cellular calmodulin expression on the growth and viability of C6 glioblastoma cells.

Calmodulin (CaM) is involved in cellular processes that are vital to cell proliferation and viability. Elevated CaM content is seen in transformed cells. Anti-CaM compounds alone are cytotoxic to tumor cells and are synergistic with certain cancer chemotherapeutic agents. However, all known CaM antagonists are nonselective, complicating interpretation of these studies. To more rigorously analyze the relationship between CaM protein expression and the behavior of cancer cells, tumor-derived cell lines were engineered such that CaM concentration could be manipulated by overexpressing CaM RNA. A full-length rat CaM I cDNA was inserted into the mammalian expression vector pMTCB6+ so that either CaM mRNA (sense) or antisense RNA was expressed under the control of an inducible metallothionein promoter. Constructs were introduced into C6 glioblastoma cells by liposome-mediated transfection and colonies were selected in G418. Significantly fewer clones were recovered from transfections with antisense vectors compared to CaM sense RNA or control (empty) vector alone. This difference was attributed to the cytotoxic effects of antisense CaM RNA as opposed to differences in transfection efficiencies. CaM expression was analyzed at the RNA level by Northern blotting and CaM protein concentrations were quantitated by immunofluorescence. Clones were identified in which CaM protein could be increased or decreased following exposure to zinc ions. Changes in CaM mRNA preceded changes in CaM protein by several hours. Overexpression of CaM had no significant effects on the growth of C6 cells. However, reductions in CaM lead to decreased growth rates of C6 cells and lowered cell viability.