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-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.
Ichikawa
J
J
Kyoto Institute of Technology, Department of Applied Biology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Kiyohara
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006873:Hydroquinones / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology*; D007150:Immunohistochemistry; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D011247:Pregnancy; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D007093:Imidazoles; D018809:Proliferating Cell Nuclear Antigen; C019359:2,5-di-tert-butylhydroquinone; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium; D014544:Uridine Triphosphate