Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC310
Oscillatory changes of intracellular chloride concentration during cell cycle progression in SiHa cells
W. Wei1, J. Galvanovskis1, Y. Jacob2, M. Shen3, R. J. Wilkins1, J. C. Ellory1
1. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. 2. Department of Virology, Pasteur Institute, Paris, France. 3. Department of Pharmacology, National Cheng Kung University, Tainin, Taiwan.
KCl cotransporters (KCCs) play significant roles not only in ionic and osmotic homeostasis but also tumor biology. Overexpression of KCC3 has been shown to lower intracellular chloride concentration and correlates with human cervical carcinogenesis and enhances proliferation of cervical cancer cells (1). Furthermore, removal of the N-terminal 117 amino acids from KCC1 (ΔN117) produces a dominant-negative loss-of-function phenotype for KCl cotransport in human cervical cancer cells causing inhibition of cell proliferation and tumor growth (2). It has been shown that concentrations of intracellular ionic composition changes during cell cycle progression; with calcium, potassium and magnesium ions having particularly significant roles in regulating cell cycle progression (3,4). However, the role of chloride in cell cycle regulation still remains largely unknown. Therefore, in this study, we have investigated if changes of intracellular chloride concentration ([Cl-]i) occur during cell cycle progression of cervical cancer cells (SiHa). The genetically encoded Cl-sensor (5) was used as a tool for monitoring intracellular chloride concentration in SiHa cells. Cells were transiently transfected with Cl-sensor and then synchronized into different stages of cell cycle. G0 phase cells were prepared by a 72 h cultivation in serum-free medium, G1 phase cells by 5 μM aphidicolin treatment for 24 h, S phase cells were prepared by 5 μM aphidicolin treatment for 24 h followed by a 4 h release in fresh medium, and G2 phase cells by 5 μM aphidicolin treatment for 24 h followed by an 8 hr release in fresh medium. M phase cells were prepared by 10 μg/ml nocodazole treatment for 18 h and thereafter the shake-off cells were collected. Fluorescent images were acquired using an IonOptix system with excitation filter (440/480 nm) and emission filter (above 510 nm). The intensity ratios (F480/F440) were determined. Calibration was via nigericin/tributyltin chloride treatment. Our results show oscillatory changes of the [Cl-]i in SiHa cells as the cell cycle progresses. [Cl-]i rises sharply (from 10 to 40 mM) in S phase. This level is maintained in G2 but then decreases markedly to 20 mM in M phase. Similar trends in oscillatory changes of the [Cl-]i were also found in KCC3-overexpressed cells and ΔN117 mutant cells. [Cl-]i rises from 5 to 20 mM (KCC3-overexpressed cells) or 15 to 60 mM (ΔN117 mutant cells) in S and G2 phase and then decreases to 15 mM (KCC3-overexpressed cells) and 40 mM (ΔN117 mutant cells) in M phase (Fig.1). The present results not only imply that the regulation of intracellular chloride concentration is an important factor in controlling cell cycle progression but also provide a clue for a critical regulatory role for cation chloride cotransporters in the aetiology of epithelial cancers.
Where applicable, experiments conform with Society ethical requirements