J. Clive Ellory

Differential expression of volume-regulated anion channels during cell cycle progression of human cervical cancer cells

1 This study investigated the volume‐regulated anion channel (VRAC) of human cervical cancer SiHa cells under various culture conditions, testing the hypothesis that the progression of the cell cycle is accompanied by differential expression of VRAC activity. 2 Exponentially growing SiHa cells expressed VRACs, as indicated by the presence of large outwardly rectifying currents activated by hypotonic stress with the anion permeability sequence I− > Br− > Cl−. VRACs were potently inhibited by tamoxifen with an IC50 of 4.6 μm. 3 Fluorescence‐activated cell sorting (FACS) experiments showed that 59 ± 0.5, 5 ± 0.5 and 36 ± 1.1% of unsynchronized, exponentially growing cervical cancer SiHa cells were in G0/G1, S and G2/M stage, respectively. Treatment with aphidicolin (5 μm) arrested 88 ± 1.4% of cells at the G0/G1 stage. 4 Arrest of cell growth in the G0/G1 phase was accompanied by a significant decrease of VRAC activity. The normalized hypotonicity‐induced current decreased from 48 ± 5.2 pA pF−1 at +100 mV in unsynchronized cells to 15 ± 2.6 pA pF−1 at +100 mV in aphidicolin‐treated cells. After removal of aphidicolin, culturing in medium containing 10% fetal calf serum triggered a rapid re‐entry into the cell cycle and a concomitant recovery of VRAC density. 5 Pharmacological blockade of VRACs by tamoxifen or NPPB caused proliferating cervical cancer cells to arrest in the G0/G1 stage, suggesting that activity of this channel is critical for G1/S checkpoint progression. 6 This study provides new information on the functional significance of VRACs in the cell cycle clock of human cervical cancer cells.

A stretch-activated anion channel is up-regulated by the malaria parasite Plasmodium falciparum

A recent study on malaria‐infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host‐ or parasite‐derived and their molecular nature have not been resolved. Here we report a comparison of a malaria‐induced anion channel with an endogenous anion channel in Plasmodium falciparum‐infected human RBCs. Ion channel activity was measured using the whole‐cell, cell‐attached and excised inside‐out configurations of the patch‐clamp method. Parasitised RBCs were cultured in vitro, using co‐cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (∼15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I− > Br− > Cl−. In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (∼80 pS, outward conductance) was spontaneously active. The host membrane of malaria‐infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria‐infected RBCs were shown to have a low open‐state probability (Po) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole‐cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up‐regulated by the malaria parasite P. falciparum.

Diminished L-arginine bioavailability in hypertension

L-Arginine is the precursor of NO (nitric oxide), a key endogenous mediator involved in endothelium-dependent vascular relaxation and platelet function. Although the concentration of intracellular L-arginine is well above the Km for NO synthesis, in many cells and pathological conditions the transport of L-arginine is essential for NO production (L-arginine paradox). The present study was designed to investigate the modulation of L-arginine/NO pathway in systemic arterial hypertension. Transport of L-arginine into RBCs (red blood cells) and platelets, NOS (NO synthase) activity and amino acid profiles in plasma were analysed in hypertensive patients and in an animal model of hypertension. Influx of L-arginine into RBCs was mediated by the cationic amino acid transport systems y+ and y+L, whereas, in platelets, influx was mediated only via system y+L. Chromatographic analyses revealed higher plasma levels of L-arginine in hypertensive patients (175+/-19 micromol/l) compared with control subjects (137+/-8 micromol/l). L-Arginine transport via system y+L, but not y+, was significantly reduced in RBCs from hypertensive patients (60+/-7 micromol.l(-1).cells(-1).h(-1); n=16) compared with controls (90+/-17 micromol.l(-1).cells(-1).h(-1); n=18). In human platelets, the Vmax for L-arginine transport via system y+L was 86+/-17 pmol.10(9) cells(-1).min(-1) in controls compared with 36+/-9 pmol.10(9) cells(-1).min(-1) in hypertensive patients (n=10; P<0.05). Basal NOS activity was decreased in platelets from hypertensive patients (0.12+/-0.02 pmol/10(8) cells; n=8) compared with controls (0.22+/-0.01 pmol/10(8) cells; n=8; P<0.05). Studies with spontaneously hypertensive rats demonstrated that transport of L-arginine via system y+L was also inhibited in RBCs. Our findings provide the first evidence that hypertension is associated with an inhibition of L-arginine transport via system y+L in both humans and animals, with reduced availability of L-arginine limiting NO synthesis in blood cells.

Volume-sensitive KCl cotransport associated with human cervical carcinogenesis

This study investigates the volume-sensitive KCl cotransporter (KCC) in various types of human cervical epithelial cell, testing the hypothesis that cervical malignancy is accompanied by differential expression of volume-sensitive KCC. Normal human cervical epithelial cells have KCCs which are quiescent in normal physiological conditions and are relatively refractory to hypotonic stress. By contrast, cervical cancer cells have KCCs which are also nearly quiescent in normal physiological conditions but high transport rates are observed in response to hypotonic challenge. Using isoform-specific primers, mRNA transcripts of KCC1, KCC3 and KCC4 were identified by reverse transcriptase polymerase chain reaction (RT-PCR) in several types of cervical cell, and confirmed by digestion with specific restriction endonucleases. By semiquantitative RT-PCR with β-actin as the internal standard, the results indicate that cervical carcinogenesis is accompanied by the up-regulation of mRNA transcripts in KCC1, KCC3 and KCC4. [(Dihydroindenyl)oxy] alkanoic acid (DIOA), a KCC inhibitor, blocked both the regulatory volume decrease (RVD) process and volume-sensitive 86Rb+ efflux from cervical cancer cells in a dose-dependent manner. The volume-sensitive 86Rb+ efflux from cervical cancer cells was also blocked by two protein phosphatase inhibitors, calyculin A and okadaic acid, with IC50 values of 0.8 and 6xa0nM, respectively. Conversely, protein kinase inhibitors, chelerythrine and staurosporine, increased Cl–-dependent 86Rb+ efflux. NEM (1xa0mM) led to a fivefold stimulation of 86Rb+ efflux which was totally Cl– dependent in cervical cancer cells. Hypotonicity could not stimulate any further 86Rb+ efflux after NEM treatment. These results indicate that the volume-sensitive KCC in cervical cancer cells plays a significant role in volume regulation and that the activities are modulated by a phosphorylation cascade. Taken together with our previous studies, we suggest the volume-regulatory ion channels and the cotransport systems work synergistically for volume regulation in human cervical cancer cells.

Modulation of whole-cell currents in Plasmodium falciparum-infected human red blood cells by holding potential and serum

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum‐infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole‐cell configuration of the patch‐clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole‐cell recordings in malaria‐infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4‐fold and inward currents by approximately 2‐fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.

KCl cotransport is an important modulator of human cervical cancer growth and invasion

Cervical cancer is a major world health problem for women, but the pathophysiology of this disease has received scant attention. Here we show that the growth and invasion of cervical cancer cells are strongly linked the expression and activity of the KCl cotransporter (KCC), an important regulator of the ionic and cellular osmotic homeostasis. Functional assays of KCl cotransport activation by osmotic swelling, staurosporine, and N-ethylmaleimide indicate that removal of the N-terminal 117 amino acids from KCC1 produces a dominant-negative loss-of-function phenotype for KCl cotransport in human cervical cancer cells. The capability for regulatory volume decrease is much attenuated in the loss-of-function KCC mutant cervical cancer cells. The loss-of-function KCC mutant cervical cancer cells exhibit inhibited cell growth accompanied by decreased activity of the cell cycle gene products retinoblastoma and cdc2 kinase. Reduced cellular invasiveness is in parallel by reduced expression of αvβ3 and α6β4 integrins, accompanied by decreased activity of matrix metalloproteinase 2 and 9. Inhibition of tumor growth in SCID mice confirms the crucial role of KCC in promoting cervical cancer growth and invasion. Thus, blockade of KCl cotransport may be a useful therapeutic adjunctive strategy to retard or prevent cervical cancer invasion.

Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism

Hereditary forms of iron-deficiency anemia, including animal models, have taught us much about the normal physiologic control of iron metabolism. However, the discovery of new informative mutants is limited by the natural mutation frequency. To address this limitation, we have developed a screen for heritable abnormalities of red blood cell morphology in mice with single-nucleotide changes induced by the chemical mutagen ethylnitrosourea (ENU). We now describe the first strain, fragile-red, with hypochromic microcytic anemia resulting from a Y228H substitution in the ferrireductase Steap3 (Steap3(Y288H)). Analysis of the Steap3(Y288H) mutant identifies a conserved motif required for targeting Steap3 to internal compartments and highlights how phenotypic screens linked to mutagenesis can identify new functional variants in erythropoiesis and ascribe function to previously unidentified motifs.

Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease.

1 This study was aimed at identifying the signalling pathways involved in the activation of volume‐regulatory mechanisms of human cervical cancer cells. 2 Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3 The activation of swelling‐activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling‐activated Cl− channel. 4 Cell swelling triggered mitogen‐activated protein (MAP) kinase cascades leading to the activation of extracellular signal‐regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume‐responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl− channels, and taurine transport. However, the volume‐regulatory mechanism was independent of the activation of p38 MAP kinase. 5 The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up‐regulated by protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (PMA) and down‐regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen‐activated/ERK‐activating kinase (MEK) activity. 6 Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume‐regulatory mechanism of human cervical cancer cells.

KCl Cotransporter-3 Down-regulates E-Cadherin/β-Catenin Complex to Promote Epithelial-Mesenchymal Transition

The potassium chloride cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. Here, we investigate if KCC is involved in the regulation of epithelial-mesenchymal transition (EMT), a critical cellular event of malignancy. E-cadherin and beta-catenin colocalize in the cell-cell junctions, which becomes more obvious in a time-dependent manner by blockade of KCC activity in cervical cancer SiHa and CaSki cells. Real-time reverse transcription-PCR on the samples collected from the laser microdissection indicates that KCC3 is the most abundant KCC isoform in cervical carcinoma. The characteristics of EMT appear in KCC3-overexpressed, but not in KCC1- or KCC4-overexpressed cervical cancer cells, including the elongated cell shape, increased scattering, down-regulated epithelial markers (E-cadherin and beta-catenin), and up-regulated mesenchymal marker (vimentin). Some cellular functions are enhanced by KCC3 overexpression, such as increased invasiveness and proliferation, and weakened cell-cell association. KCC3 overexpression decreases mRNA level of E-cadherin. The promoter activity assays of various regulatory sequences confirm that KCC3 expression is a potent negative regulator for human E-cadherin gene expression. The proteosome inhibitor restores the decreased protein abundance of beta-catenin by KCC3 overexpression. In the surgical specimens of cervical carcinoma, the decreased E-cadherin amount was accompanied by the increased KCC3 abundance. Vimentin begins to appear at the invasive front and becomes significantly expressed in the tumor nest. In conclusion, KCC3 down-regulates E-cadherin/beta-catenin complex formation by inhibiting transcription of E-cadherin gene and accelerating proteosome-dependent degradation of beta-catenin protein. The disruption of E-cadherin/beta-catenin complex formation promotes EMT, thereby stimulating tumor progression.

Nitrendipine is a potent inhibitor of the Ca2*-activated K ÷ channel of human erythrocytes

Nitrendipine, a classical blocker of L‐type Ca2+ channels, is shown to be a potent inhibitor of the Ca2+‐activated K+ channel of human erythrocytes. In erythrocytes suspended in a solution with physiological Na+ and K+ concentrations and in which the channel was activated using the Ca2+ ionophore ionomycin, nitrendipene inhibited K+(80Rb+) influx with an I 50 of around 130 nM. Similar results were obtained for K+(80Rb+) efflux, and for K+(80Rb+) influx into cells suspended in a high‐K+ medium.