We recently reported that calcitonin (CT) can profoundly inhibit the growth of HEK-293 cells transfected with the human calcitonin receptor (hCTR). We also obtained preliminary evidence that suggested a role for CT in cell survival, and in the present study we have investigated the pro-apoptotic action of CT, which we observe in conditions of low serum concentration. Under these conditions, we have found that CT treatment of HEK-293 cells stably transfected with the insert-negative form of the human CTR (HR12 cells) caused a time-dependent decrease in cell number associated with loss of cellular attachment. Loss of cellular adherence in CT-treated cultures caused programmed cell death, as shown by Annexin V staining of cells, failure of cells to exclude Trypan Blue dye, condensation and cleavage of nuclear DNA, and appearance of hypodiploid cells in fluorescence-activated cell sorting (FACS) analysis. The accumulation of non-adherent cells and cell death was concomitant with increased intracellular activity of caspase-3. However, inhibition of caspase activation in HR12 cells did not prevent CT-mediated loss of attachment and did not maintain the viability of non-adherent cells, indicating that caspase activation accompanied, but was probably not the cause of, the loss of cell viability. Neither the effects of CT on cell survival nor the activation of caspase-3 were observed in serum-replete conditions, suggesting that serum-derived factors provide protection of cells from CT-induced apoptosis. The inhibitory effects of CT on cell growth were found previously to be related to activation of Erk1/2 MAP kinase. In the present experiments, it was found that the Erk1/2 inhibitor, PD 98059, inhibited the CT-induced loss of cellular adherence and the consequent reduction in cell numbers. These results demonstrate that CT can negatively affect cell survival and they identify roles for cell adherence and MAP kinase activation in this process.

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