Chloride Ion Modulates Cell Proliferation of Human Androgen-independent Prostatic Cancer Cell

Abstract

In the present study, we investigated if the intracellular Cl^- affects cell growth and cell cycle progression of androgen-independent prostate cancer PC3 cells. PC3 cells cultured in a medium containing 113 mM Cl^- for 96 h grew up 9-fold in cell number, while PC3 cells cultured in an 8 mM-Cl^--containing culture medium showed complete arrest of cell growth even after culture for 96 h. Exposure of cells to the 8 mM-Cl^- culture medium diminished phosphorylation levels of Rb and cdc2, which are respectively key accelerators of transition from G₁ to S phase and G₂ to M phase in cell cycle progression. Culturing cells in the 8 mM-Cl^--containing culture medium upregulated the protein expression level of p21 (a CDK inhibitor) inhibiting transition of G₁ to S phase, and diminished the incorporation of 5-ethynyl-2′-deoxyuridine (EdU; a thymidine analogue) into DNA. These results suggest that cells cultured in the low Cl^- medium prolonged the duration of all phases of the cell cycle (G₁, S, and G₂/M), thereby abolishing overall cell cycle progression. Effects of culturing cells in the low Cl^- culture medium on cell cycle progression would be mediated via a change in the intracellular Cl^- concentration ([Cl^-]_i), since [Cl^-]_i was decreased under a low Cl^- culture medium. To clarify this possibility, we studied effects of furosemide and bumetanide, Na⁺/K⁺/2Cl^- cotransporter (NKCC) inhibitors, on proliferation of PC3 cells. Furosemide and bumetanide decreased [Cl^-]_i and cell growth of PC3 cells. These results suggest that a change in [Cl^-]_i would play a critical role in this growth mechanism.