Suppression of EGF-induced cell proliferation by the blockade of Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-in duced cell proliferation was investigated in mouse mammary epithelial cells . We have previously demonstrated that EGF enhances Ca2+ mobilization (rele ase of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry cor related with cell proliferation in mouse mammary epithelial cells. To confi rm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butyl-hydroquinone (DBHQ), a reversible inhibitor of the Ca 2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacita tive Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was e xamined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhib ited capacitative Ca2+ entry in a dose-dependent manner (IC50: 1-5 muM). SK &F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration IC50: 1-5 muM). DBHQ suppressed [Ca2+](i) response to UTP an d thus depleted completely Ca2+ stores at 5 muM. DBHQ also inhibited EGF-in duced cell proliferation at an IC50 value of similar to 10 muM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium b romide, no dead cells were detected in any of the cultures. These results i ndicate that the inhibitory effects of SK&F 96365 and DBHQ on cell prolifer ation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobili zation suggesting the importance of capacitative Ca2+ entry and Ca2+ mobili zation in the control of EGF-induced cell cycle progression in mouse mammar y epithelial cells. Copyright (C) 2001 John Wiley & Sons, Ltd.