The role
calmodulin plays in the growth and differentiation of nerve cells was assessed by altering the levels of
calmodulin in the PC12 rat pheochromocytoma cell line and determining the effects of altering these levels on cellular proliferation and differentiation.
Calmodulin levels in the PC12 cells were increased or decreased by transfecting the cells with a mammalian expression vector into which the rat
calmodulin gene I had been cloned in the sense or antisense orientation, respectively. The cells transfected with the
calmodulin sense gene showed increased levels of
calmodulin immunoreactivity and increased levels of
calmodulin messenger RNA as ascertained by immunocytochemistry and slot-blot analysis, respectively. Cells transfected with the
calmodulin antisense construct showed reduced levels of
calmodulin immunoreactivity. Reducing the levels of
calmodulin by expression of antisense
calmodulin messenger RNA resulted in a marked inhibition of cell growth, whereas increasing the levels of
calmodulin by overexpressing
calmodulin messenger RNA resulted in an acceleration of cell growth. Transfected PC12 cells having reduced levels of
calmodulin immunoreactivity exhibited spontaneous outgrowth of long, stable and highly branched neuritic processes. PC12 cells in which
calmodulin was overexpressed showed no apparent changes in cell morphology, but did show an altered response to the addition of
nerve growth factor. While
nerve growth factor slowed cellular proliferation and induced extensive neurite outgrowth, in parental PC12 cells
nerve growth factor induced little or no neurite outgrowth and little inhibition of cell proliferation in transfected cells overexpressing
calmodulin. These results indicate that
calmodulin is essential for the proliferation of nerve cells and for the morphological changes that nerve cells undergo during differentiation. The study also suggests the possibility that a
calmodulin antisense approach may be used to inhibit the proliferation of neuronal
tumors.