TARGETED NEUTRALIZATION OF CALMODULIN IN THE NUCLEUS BLOCKS DNA-SYNTHESIS AND CELL-CYCLE PROGRESSION

Calmodulin (CaM) is a major intracellular calcium binding protein whic h has been implicated in the regulation of cell proliferation. Previou s studies using chemically synthesized CaM antagonists and anti-sense RNA indicated that CaM is important for initiation of DNA synthesis an d cell cycle progression. However, these methods reduce total intracel lular CaM and globally interfering with all the CaM-dependent processe s. In order to explore the function of nuclear CaM during the cell cyc le, a CaM inhibitor peptide was targeted to the nucleus of intact mamm alian cells. Cell progression through S-phase was assessed by incorpor ation of the thymidine analogue, BrdU. Cells were transfected for 48 h with either the CaM inhibitor peptide gene or the control plasmid pri or to analysis. Approx. 70% of the control cells incorporated BrdU. In striking contrast, double immunofluorescent labeling demonstrated tha t none of the cells expressing the CaM inhibitor peptide entered S-pha se. This result indicates that neutralization of nuclear CaM by target ed expression of a CaM inhibitor peptide blocks DNA synthesis and cell cycle progression.