Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, PC175

Poster Communications

Effects of altered KCl cotransporters expression on intracellular chloride levels and pH in cervical cancer cells

W. Wei1, J. Davis1, M. Shen2, R. J. Wilkins1, J. C. Ellory1

1. Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, United Kingdom. 2. Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan.

  • Figure 1 (A) Intracellular chloride concentration measured by MEQ. (B) Intracellular pH measured by BCECF-AM. (C) Expression of membrane transporters measured by RT-PCR.

KCl cotransporters (KCCs) play significant roles not only in ionic and osmotic homeostasis but also tumor biology. Overexpression of KCC1, KCC3 and KCC4, with KCC3 the most abundant isoform, has been shown to correlate with human cervical carcinogenesis (1). KCC1 and KCC4 are osmotically-sensitive and involved in volume regulation (2) while KCC3 contributes to cell proliferation (3). KCC3 overexpression enhances proliferation, migration and invasiveness of cervical cancer cells. 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 which exhibit inhibited cell proliferation, tumor growth and invasiveness (4). We have shown previously that expression levels of KCC3 correlate inversely with intracellular Cl- levels ([Cl-]i) estimated by 36Cl- equilibrium. There is no significant difference in cell volume between wild-type, KCC3-overexpressed, and ΔN117 mutant cervical cancer cells (4). To maintain electroneutrality, changes in [Cl-]i associated with altered KCC activity must result in alterations to the levels of other ions, most likely HCO3-. In the present study, the properties of these KCC transfectants in regulating their intracellular pH (pHi) with changes in [Cl-]i have been investigated. 6-Methoxy-N-ethylquinolinium chloride (MEQ) was used to determine [Cl-]i in the three cell lines. [Cl-]i estimated by MEQ was 73.80±15.82 mM (n=5) in wild-type cervical cancer cells, 45.63±1.65 mM (n=3) in KCC3-overexpressed cervical cancer cells and 104.01±6.66mM (n=3) in ΔN117 mutant cells (Fig. 1A). The resting pHi was determined by cuvette fluorimetry using BCECF-AM (10 μM for 15 min at 37°C). As shown in figure 1B, there are no significant differences in resting pHi between KCC3-overexpressed cells and ΔN117 mutant cells in HEPES-buffered solution (HBS). However, ΔN117 mutant cells have a more acidic pHi than KCC3-overexpressed cells in bicarbonate-buffered solution (BBS). A number of Na-dependent and acid extruding transporters were assessed by RT-PCR. Only the expression of NHE3 correlated with [Cl-]i (Fig. 1C). The present results suggest that bicarbonate can replace Cl- as the intracellular anion during changes of KCC transport activity.

Where applicable, experiments conform with Society ethical requirements