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Swelling-activated taurine and K+ transport in human cervical cancer cells: Association with cell cycle progression
Abstract
The aim of this study was to investigate swelling-activated taurine and K+ transport in human cervical cancer cells under various culture conditions, testing the hypothesis that the progression of cell cycle was accompanied by differential activities of swelling-activated transport pathways. Aphidicolin, an inhibitor of deoxyribonucleic acid (DNA) synthesis, was used to synchronize the cell cycle. The distribution of cell cycle stage was determined by fluorescence-activated cell sorting (FACS). Hypotonicity activated taurine efflux, which was sensitive to tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Cell swelling also induced both Cl- -dependent and -independent K+ (86Rb+) efflux, presumably mediated by KCl cotransport (KCC) and Ca2+ -activated K+ channels, respectively. Cell cycle arrest in G0/G1 was accompanied by a remarkable decrease in the rate constant for swelling-activated taurine efflux, from 0.20+/-0.007 to 0.026+/-0.002 min(-1) (n=6). The activity of swelling-activated taurine efflux recovered progressively on re-entry into the cell cycle. After removal of aphidicolin and culture with 10% fetal calf serum for 10 h, the rate constant increased significantly from 0.026+/-0.002 to 0.093+/-0.002 min(-1) (n=6). After 24 h release from aphidicolin, the efflux rate constant had increased further to 0.195+/-0.006 min(-1) (n=6), a value not significantly different from that in normally proliferating cells. The differential activities of swelling-activated taurine transport matched well with our previous study showing a volume-sensitive anion channel associated with cell cycle progression. In contrast to the differential activities of swelling-activated taurine transport, swelling-activated K+ (86Rb+) transport was independent of the progression of cell cycle. Most importantly, pharmacological blockade of swelling-activated taurine efflux by tamoxifen or NPPB caused proliferating cervical cancer cells to arrest in G0/G1, suggesting that the activity of this efflux was associated with G1/S checkpoint progression. This study provides new and important information on the functional significance of swelling-activated transport system in the regulation of cell cycle clock of human cervical cancer cells.
... Meng-Ru Shen * †, Cheng-Yang Chou †, Joseph A. Browning *, Robert J. Wilkins * and J. Clive Ellory * process of malignant transformation of human cervical epithelial cells (Shen et al. 1996(Shen et al. , 2000aChou et al. 1995Chou et al. , 1997. The cell cycle progression of cervical cancer cells is also accompanied by differential activities of the swelling-activated Cl _ channel and taurine transport (Shen et al. 2000b(Shen et al. , 2001. In addition, alterations in osmosensing signalling pathways are apparent during human cervical carcinogenesis (Shen et al. , 1999Chou et al. 1998). ...
... [ 3 H]Taurine efflux rate constants were estimated from the negative slope of the graph of ln[X i (t )/X i (t = 0)] vs. time (t ), where X i (t = 0) denotes the total amount of [ 3 H]taurine inside the cells at the beginning of the efflux time course and X i (t ) denotes the amount of [ 3 H]taurine inside the cells at the time point t. Following hypotonic shock, [ 3 H]taurine efflux occurs in two phases: an initial fast loss within 5 min and followed by a slower loss in the subsequent time course (Shen et al. 2001). Therefore, the [ 3 H]taurine efflux rate constant in the hypotonic solution was calculated from the negative slope of the semi-logarithm graph in the initial 5 min after exposure to hypotonicity. ...
... Exposure to the hypotonic solution markedly increased K + ( 86 Rb + ) efflux both in the Cl _ -containing medium and NO 3 _ -containing (Cl _ -free) medium (Fig. 5A). Analysis of the data confirmed that cell swelling induced both Cl _ -dependent and -independent K + ( 86 Rb + ) efflux, presumably mediated by K + -Cl _ cotransport and K + channels, respectively (Shen et al. 2000a(Shen et al. , 2001. Under hypotonic challenge, both the Cl _dependent and -independent K + ( 86 Rb + ) efflux were blocked significantly by inhibitors of tyrosine kinase, Figure 3. ...
This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells.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.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.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.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.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.
... [4][5][6][7] This study demonstrated the close relationship between the Ca 2+ -activated K + channel and cervical cancer. [8,9] This study utilized the K + -channel blocker clotrimazole (CLT) and small hairpin ribonucleic acid (RNA) to explore the role played by (and the effect of) IKCal in the proliferation of HeLa cells. ...
... [5] A clinical pathological evaluation indicated a significant correlation between hKCa3.1-expression and the tumor grade. In our previous studies, we have observed higher mRNA expression of IKCal in cervical cancer tissues compared to normal tissues; [8,9] the results of this study further indicated an obvious decrease in the IKCal mRNA expression in the transfection group (in which the IKCal gene was blocked or silenced), compared to the negative and control groups. This result indicated that the cell potential and energy transfer was affected after blockage of IKCal and silencing of the IKCal gene; in addition, we believe that the permeation pressure was also altered to influence the growth of cells so as to reduce cell count, or even induce cell necrosis (resulting from low nutrition). ...
Purpose:
To explore the influence of intermediate-conductance-Ca2+-activated K+channels. (IKCal) on HeLa cell proliferation.
Materials and methods:
An IKCal blocking agent (clotrimazole (CLT)) and small hairpin ribonucleic acid interference (RNAi) was used to block IKCal in HeLa cells; subsequently, cell growth was observed. Furthermore, the messenger ribonucleic acid (mRNA) expression of IKCal was detected by reverse transcriptase polymerase chain reaction (RT-PCR) after IKCal-blocking.
Results:
The obvious morphological changes in HeLa cells were observed 48 h after CLT-blocking. The PCR results indicated that CLT reduced the mRNA expression of IKCal in HeLa cells. HeLa cells were transfected with pGenesil via RNAi; the HeLa cells transfected with pGenesil-IK displayed obvious morphological changes 48 h after transfection. In addition, RT-PCR further demonstrated the reduced mRNA expression of IKCal in the pGenesil group.
Conclusion:
CLT and blocking of IKCal gene expression effectively inhibits HeLa cell proliferation; therefore, the use of a blocking agent and RNAi both effectively downregulated the mRNA expression of IKCal, which in turn mediated the proliferation of HeLa cells, producing an antitumor effect.
... During unhindered cancer proliferation lipidogenesis, nucleic acid formation and enhanced protein synthesis takes place along with osmoregulation and volume regulation. It is found that higher concentration of Taurine which acts as an osmolyte helps in regulating cell swelling induced by hypoxia, 19,21,22 thus also helps in maintaining the cell volume. Because of their uninterrupted proliferation of cells Taurine is also known as a free-radical scavenger and is the one which reduces the oxidative stress of the cells. ...
The diagnosis and confirmation of oral SCC (squamous cell carcinoma) is still dependent on histopathology report in spite of development of radiological investigations. It is, thus important to understand the underlying molecular mechanisms and how the alterations in metabolic pathways effect the tumor development and progression. The simultaneous and comprehensive information about the presence and absence of small molecule metabolites and their relative concentrations has been provided by ¹H HR-MAS NMR spectroscopy on tissue specimens. In this paper a unique case study was presented in order to correlate histological and NMR spectroscopic findings. The patient’s initially lesion was found to be non-malignant in nature based on histological findings but its periodic localized recurrence even after laser ablation prompted us to perform HR-MAS based analysis and its role in identifying the metabolic alterations in known pathways occurring during its progressions. Thus it was confirmed after analysis that HR-MAS NMR can also be used as an analytical tool which is reliable in order to distinguish between malignant and non-malignant tissues, in combination with histopathology.
... It is one of the biomarker which also used as in vivo MRS in clinical practices (Bolan et al. 2003). Taurine, important in osmoregulation and volume regulation process, also helps in protecting cells from swelling and free radical under hypoxic and oxidative stress conditions (Griffin and Shockcor 2004;Shen et al. 2001). Taurine reported as a potential diagnostic biomarker in differentiation of malignant from benign and control tissues and its level was found to be increased (Sitter et al. 2010;Wang et al. 2010;Srivastava et al. 2011). ...
Gall bladder tissue specimens obtained from 112 patients were examined by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Fifty one metabolites were identified by combination of one and two-dimensional NMR spectra. To our knowledge, this is the first report on metabolic profiling of gall bladder tissues using HR-MAS NMR spectroscopy. Metabolic profiles were evaluated for differentiation between benign Chronic Cholecystitis (CC, n = 66) and xantho-granulomatous cholecystitis (XGC, n = 21) and malignant gall bladder cancer (GBC, n = 25). Increase in choline containing compounds, amino acids, taurine, nucleotides and lactate as common metabolites were observed in malignant tissues whereas lipid content was found low as compared to benign tissues. Principal component analysis obtained from the NMR data showed clear distinction between CC and GBC tissue specimens; however, 27 % of XGC tissues were classified with GBC. The partial least square discriminant analysis (PLS-DA) multivariate analysis between benign (CC, XGC) and malignant (GBC) on the training data set (CC; n = 51, XGC; n = 15, GBC; n = 19 tissues specimens) provided 100 % sensitivity and 94.12 % specificity. This PLS-DA model when executed on the spectra of unknown tissue specimens (CC; n = 15, XGC; n = 6, GBC; n = 6) classified them into the three histological categories with more than 95 % of diagnostic accuracy. Non-invasive in vivo MRS technique may be used in future to differentiate between benign (CC and XGC) and malignant (GBC) gall bladder diseases.
... 2-4), and retained cells at G0/G1 phase (i.e., inhibiting entry into S phase, Figs. 3 and 6); furthermore, the effect is related to the regulation of the cell cycle regulatory proteins cyclin D1 and cyclin E (Fig. 7). This finding supports the notion that I Cl.vol channels in human cardiac fibroblasts, as observed in cancer cells [39,40], and mouse MSCs [13], mediate the cell cycle progression. ...
Our previous study demonstrated that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts, including a large-conductance Ca(2+)-activated K(+) current (BKCa), a volume-sensitive chloride current (I(Cl.vol)), and voltage-gated sodium currents (I(Na)). The present study was designed to examine the possible involvement of these ion channels in proliferation of cultured human cardiac fibroblasts using approaches of cell proliferation assay, whole-cell patch voltage-clamp, siRNA and Western blot analysis. It was found that the blockade of BKCa with paxilline (1-3μM) or I(Cl.vol) with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium (DIDS, 100-200μM), but not I(Na) with tetrodotoxin (0.1-10μM), remarkably suppressed proliferation in human cardiac fibroblasts. Knockdown of KCa1.1 or Clcn3 with specific siRNAs significantly reduced BKCa or I(Cl.vol) current, mRNA and channel protein levels, and inhibited growth of human cardiac fibroblasts. Flow cytometry analysis showed accumulation of cardiac fibroblasts at G0/G1 phase and reduced cell number in S phase after inhibition of BKCa or I(Cl.vol) with channel blockers or knock down of the corresponding channels with specific siRNAs; these effects were accompanied by a decreased expression of cyclin D1 and cyclin E. The present results demonstrate the novel information that BKCa and I(Cl.vol) channels, but not I(Na) channels, are involved in the regulation of proliferation in cultured human cardiac fibroblasts by promoting cell cycle progression via modulating cyclin D1 and cyclin E expression.
... To further examine the dynamic changes in Cx43 localization through mitosis we performed time-lapse microscopy on MDCK cells stably expressing Cx43 with a Green Fluorescent Protein and tetracysteine tag at the C-terminus (Cx43-GFP-4C). In order to image several cells progressing through mitosis, we synchronized cells using a combination of serum starvation and application of aphidicolin, an inhibitor of DNA synthesis (31), to create an enriched population of cells undergoing mitosis suitable for imaging (diagrammed in Figure 3A). Specifically, confluent cells were trypsinized and grown for about 20 hours in the presence of minimal serum and aphidicolin, resulting in a G1/S block. ...
During the cell cycle, gap junction communication, morphology and distribution of connexin43 (Cx43)-containing structures change dramatically. As cells round up in mitosis, Cx43 labeling is mostly intracellular and intercellular coupling is reduced. We investigated Cx43 distributions during mitosis both in endogenous and exogenous expressing cells using optical pulse-chase labeling, correlated light and electron microscopy, immunocytochemistry and biochemical analysis. Time-lapse imaging of green fluorescent protein (GFP)/tetracysteine tagged Cx43 (Cx43-GFP-4C) expressing cells revealed an early disappearance of gap junctions, progressive accumulation of Cx43 in cytoplasmic structures, and an unexpected subset pool of protein concentrated in the plasma membrane surrounding the midbody region in telophase followed by rapid reappearance of punctate plaques upon mitotic exit. These distributions were also observed in immuno-labeled endogenous Cx43-expressing cells. Photo-oxidation of ReAsH-labeled Cx43-GFP-4C cells in telophase confirmed that Cx43 is distributed in the plasma membrane surrounding the midbody as apparent connexons and in cytoplasmic vesicles. We performed optical pulse-chase labeling and single label time-lapse imaging of synchronized cells stably expressing Cx43 with internal tetracysteine domains through mitosis. In late telophase, older Cx43 is segregated mainly to the plasma membrane while newer Cx43 is intracellular. This older population nucleates new gap junctions permitting rapid resumption of communication upon mitotic exit.
... In the present study, we demonstrated that blockade of I Cl.vol with NPPB or downregulation of Clcn3 with the specific siRNAs significantly reduced cell proliferation (Figs. 1-3) and accumulated cells at G 0 /G 1 phase (Fig. 5); this finding is consistent with recent observations in cancer cells and fibroblasts (6,34,45). Our results demonstrated that this effect is related to the downregulation of the cell cycle regulatory proteins cyclin D1 and cyclin E (Fig. 6). ...
Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine; however, their cellular physiology is not fully understood. The present study aimed at exploring the potential roles of the two dominant functional ion channels, intermediate-conductance Ca(2+)-activated potassium (IK(Ca)) and volume-sensitive chloride (I(Cl.vol)) channels, in regulating proliferation of mouse MSCs. We found that inhibition of IK(Ca) with clotrimazole and I(Cl.vol) with 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) reduced cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific short interference (si)RNAs significantly reduced IK(Ca) or I(Cl.vol) density and channel protein and produced a remarkable suppression of cell proliferation (by 24.4 +/- 9.6% and 29.5 +/- 7.2%, respectively, P < 0.05 vs. controls). Flow cytometry analysis showed that mouse MSCs retained at G(0)/G(1) phase (control: 51.65 +/- 3.43%) by inhibiting IK(Ca) or I(Cl.vol) using clotrimazole (2 microM: 64.45 +/- 2.20%, P < 0.05) or NPPB (200 microM: 82.89 +/- 2.49%, P < 0.05) or the specific siRNAs, meanwhile distribution of cells in S phase was decreased. Western blot analysis revealed a reduced expression of the cell cycle regulatory proteins cyclin D1 and cyclin E. Collectively, our results have demonstrated that IK(Ca) and I(Cl.vol) channels regulate cell cycle progression and proliferation of mouse MSCs by modulating cyclin D1 and cyclin E expression.
... 3,9 Beyond its possible housekeeping function, the role of KCC1 in volume regulation and transepithelial transport in renal cells, in vascular reactivity, in cervical cancer, and in the genesis of arrhythmias after myocardial ischemia are some of the functions under investigation. 2,[64][65][66][67][68] The identification of a TATA-less KCC1 gene promoter containing functional InR and DPE elements is an unusual finding in a mammalian promoter. In Drosophila, approximately half of all core promoters contain a TATA box and an InR at the transcription initiation site. ...
Most K-Cl cotransport in the erythrocyte is attributed to potassium chloride cotransporter 1 (KCC1). K-Cl cotransport is elevated in sickle erythrocytes, and the KCC1 gene has been proposed as a modifier gene in sickle cell disease. To provide insight into our understanding of the regulation of the human KCC1 gene, we mapped the 5' end of the KCC1 cDNA, cloned the corresponding genomic DNA, and identified the KCC1 gene promoter. The core promoter lacks a TATA box and is composed of an initiator element (InR) and a downstream promoter element (DPE), a combination found primarily in Drosophila gene promoters and rarely observed in mammalian gene promoters. Mutational analyses demonstrated that both the InR and DPE sites were critical for full promoter activity. In vitro DNase I footprinting, electrophoretic mobility shift assays, and reporter gene assays identified functional AP-2 and Sp1 sites in this region. The KCC1 promoter was transactivated by forced expression of AP-2 in heterologous cells. Sequences encoding the InR, DPE, AP-2, and Sp1 sites were 100% conserved between human and murine KCC1 genes. In vivo studies using chromatin immunoprecipitation assays with antihistone H3 and antihistone H4 antibodies demonstrated hyperacetylation of this core promoter region.
... As shown earlier, in activated HBLs, all these mechanisms of the early pump regulation are independent of new protein synthesis [15]. For the last few years, several authors have reported a sustained , cell cycle-dependent activation both of ion transporters and of ion channels in various cell types35363738 . In most studies , a direct correlation between the activity of ion-transporting pathways and the cell cycle progression have been demonstrated , thus, indicating a possible involvement of ion transport in cell cycle regulation. ...
The present study provides the first evidence that the abundance of catalytic alpha1-subunit of Na,K-ATPase increases in the course of T cell blast transformation. Immunodepressant cyclosporin A at anti-proliferative doses diminished the induction of alpha1 protein in activated lymphocytes. Furthermore, in competent T cells, IL-2 increases both the transport activity of Na/K pump and the content of Na,K-ATPase alpha1 protein in a time-dependent manner. A correlation was found between the long-term elevation in ouabain-sensitive Rb influxes and the increase in alpha1 protein content in late activated T cells. These results suggest that (1) the increased expression of Na,K-ATPase proteins underlie the cell cycle-dependent upregulation of ion pump during T cell transformation, and (2) IL-2 is involved in the regulated expression of Na,K-ATPase in human lymphocytes.
... In many cell types, especially when intracellular Cl Ϫ concentration is not high, organic osmolyte effluxes also occur through yet undefined channels [11,41]. In addition to blocking volumesensitive Cl Ϫ channels, the effective RVD blockers presently found (DIDS, NPPB, and tamoxifen) can also inhibit the efflux of amino acids [42,43] and other organic osmolytes such as myo-inositol during RVD [11,41]. Some of these organic osmolytes are found in high concentrations in the epididymal lumen, including myo-inositol, glutamate, and taurine [44]. ...
As with other mammalian species, human spermatozoa experience a decrease in extracellular osmolarity in cervical mucus upon ejaculation, which requires the efflux of osmolytes and water to counteract swelling that hinders mucus penetration. Recent evidence for the operation of K+ channels in the process of volume regulation suggests parallel involvement of Cl-/anion channels for electro-neutrality as in somatic cells. This was studied using ejaculated spermatozoa washed at seminal osmolality and incubated for 30 min in a medium of mucus osmolality in the presence of Cl- channel blockers. Increases in cell size measured as laser forward-scatter by flow cytometry were detected in the presence of 100 microM 5-nitro-2(3-phenylpropylamino) benzoic acid, 400 microM diisothiocyanato-stilbene-2,2'-disulphonic acid, and 20 microM tamoxifen. No volume changes were found with 400 microM 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid, 200 microM verapamil, or niflumic acid, whereas 1 mM niflumic acid induced shrinkage. Among the candidate channel proteins, Western blotting revealed the presence of ClC-3 (CLCN3) at 87 kDa, but the absence of ClC-2 (CLCN2) from sperm proteins in all samples tested. ICln (CLNS1A) was found in only one of eight samples. Immunocytochemistry localized CLCN3 to the sperm tail. To confirm molecular identities, sperm mRNA was extracted and checked for quality by the presence of protamine 2 transcripts and the absence of sperm DNA and leukocyte mRNA using reverse transcription-polymerase chain reaction. Transcripts of Clcn3 were found in all samples and that of Clns1a in some but not all samples. Clcn3 was therefore considered the most likely candidate of Cl- channel involved in volume regulation of human sperm.
... Many studies have shown that hyposmoticallyinduced taurine efflux can be inhibited by DIDS and/or tamoxifen (see e.g. [22,38,43,90,92,[101][102][103]. As discussed above, inhibition by either compound is commonly cited as evidence for taurine transport via volume-activated anion channels. ...
Cells have to regulate their volume in order to survive. Moreover, it is now evident that cell volume per se and the membrane transport processes which regulate it, comprise an important signalling unit. For example, macromolecular synthesis, apoptosis, cell growth and hormone secretion are all influenced by the cellular hydration state. Therefore, a thorough understanding of volume-activated transport processes could lead to new strategies being developed to control the function and growth of both normal and cancerous cells. Cell swelling stimulates the release of ions such as K(+) and Cl(-) together with organic osmolytes, especially the beta-amino acid taurine. Despite being the subject of intense research interest, the nature of the volume-activated taurine efflux pathway is still a matter of controversy. On the one hand it has been suggested that osmosensitive taurine efflux utilizes volume-sensitive anion channels whereas on the other it has been proposed that the band 3 anion-exchanger is a swelling-induced taurine efflux pathway. This article reviews the evidence for and against a role of anion channels and exchangers in osmosensitive taurine transport. Furthermore, the distinct possibility that neither pathway is involved in taurine transport is highlighted. The putative relationship between swelling-induced taurine transport and volume-activated anionic amino acid, alpha-neutral amino acid and K(+) transport is also examined.
Previous studies have demonstrated that cancer cells harbor unique metabolic characteristics relative to healthy counterparts. The current study is a prospective ex-vivo HR-MAS NMR analysis of malignant colorectal cancer (CRC) tissue specimens and its corresponding benign tissues.
To assess the HR-MAS Spectroscope qualitatively & to analyze significant difference between the normal, benign and malignant intestinal mucosa.
Between November 2013and January 2016, 36 consecutive patients with confirmed CRC were recruited to a prospective observational study. Fresh tissue samples were obtained from center of tumor and 5 cm from tumor margin from surgical resection specimens. Samples were run in duplicate where tissue volume permitted to compensate for anticipated sample heterogeneity. Typically, the sample was packed into a 4 mm ZrO2 rotor of 50 μl capacity; a volume of 20μl of D2O having 0.03% TSP was used as a chemical shift reference. The sample-rotor-setup was then transferred into the HR-MAS NMR probe for analysis.
A total of 36 spectra were acquired (center of tumor, n = 18; 5 cm from tumor margin, n = 18). The malignant clustering occurs due to increased Val (0.90ppm), Lac (1.34ppm), Ala(1.48ppm) levels of acetate (1.90ppm), glutamate (2.35ppm), taurine (3.23 ppm), choline containing compounds (3.20-3.22ppm), glycine (3.56ppm), lactate (4.12ppm) and α-H of Leu, Ileu, Val, Lys, Ala (3.76-3.79ppm . In addition unique metabolic profiles were observed for tumors of differing T-stage. The information gathered from clustering in PCA had highly suggested that malignancy induces metabolic perturbations at cellular levels.
HR-MAS NMR profiling demonstrates cancer-specific metabolic signatures in CRC and reveals metabolic differences between benign and malignant tumors. In addition, this approach reveals that tumor metabolism undergoes modification during local tumor advancement, offering potential in future staging and therapeutic approaches.
Aim and Objective
Mechanisms in etiopathogenesis of nasal polyposis; particularly, it is not clear whether the amino acids contribute in a causal way to the development of the disease. The aim of this study was to determine the plasma-free amino acid profile in patients with nasal polyposis and to compare the results with the healthy control group.
Materials and Methods
This is a prospective controlled study of the Ear-Nose-Throat Department of the Our University Faculty of Medicine between April 2017 and April 2018. The study included 25 patients with nasal polyposis and 20 healthy control groups. Plasma-free amino acid profiles levels were studied in serum samples taken from the patient group and the healthy control group.
Results
In patients with nasal polyposis; 2-Methyl histidine (2-MHIS), arginine (arg), asparagine (asn), citrulline (cit), cystine (cys), glutamic acid (glu), serine (ser), taurine (tau), tryptophan (trp) , lysine (Lys), ornithine, homocitrulline, norvaline, argininosuccinic acid plasma concentrations were significantly higher than healthy control group (p <0.05). Also, Gamma-aminobutyric acid (Gaba) plasma concentration was significantly lower (p <0.03).
Conclusion
To reveal the interactions and mechanisms between nasal polyposis and plasma-free amino acid profiles may help us to understand the complex etiopathogenesis of nasal polyposis. It is important to determine the possibility of using the information obtained to prevent recurrence and to develop effective treatment strategies. Due to its pathophysiological significance, our result may shed light on the development of potential drug therapies and individual diet programs targeting the imbalance in plasma free amino acid profiles.
We present the results that showcase a novel technique that can be used to increase the specificity of treatment evaluation procedures by means of differentiating the Magnetoencephalographic (MEG) signals received from patients with malignant central nervous system (CNS) lesions, during treatment using non-linear analysis. We present recordings from 2 patients diagnosed with malignant CNS lesions. A 122-channel SQUID biomagnetometer in an appropriate shielded room was used to record the MEG signals and the Grassberger-Procaccia method of phase space reconstruction was applied to the recorded signal from each patient. By applying the Grassberger-Procaccia method of phase space reconstruction to the MEG of patients with malignant CNS lesions we found clear evidence linking the embedding dimension m in which we have the onset of saturation with the progress of the treatment. These 2 case reports provide a clear indication that the pretreatment MEG signals of CNS malignant lesions present strong qualitative and quantitative differences from the after-treatment MEG signals of the same lesions and that non-linear analysis of MEG signals can be used for the purpose of treatment monitoring during radiotherapy. Further research is being conducted in order to ascertain possible quantitative thresholds in the value of the embedding dimension m above which saturation occurs and to confirm the physiological mechanism which would explain our findings.
This chapter deals with the anion channels and their multiple functions. Anion channels (ACs) are present both in the plasma membrane and in the membranes of intracellular organelles. Animal cells express a large variety of anion channels in their plasma membrane. ACs are involved in a wide range of functions, such as inhibitory synaptic transmission through plasma membrane hyperpolarization, epithelial Cl transport, as well as transport of other organic anions such as glutamate and anionic forms of ATP. In contrast to cation channels, ACs are not directly involved in the initiation and termination of action potentials in nerves and muscles. In neurons and other cell types, membrane potential is determined by the relative electromotive forces and the conductances of each ion permeation pathway.
The metabolic activitiesactivity of articular chondrocytes areis influenced by osmotic alterations inthat occur in articular cartilage occurs secondary to mechanical loads. The mechanismskey membrane transporter that sense and transduce mechanical signals fromthe cell swelling and initiatemediate volume regulatory ion isare poorly understood. The purpose of this study was to investigate how the expression of two putativepotential osmolyte channels (TRPV4 and BKCa) in chondrocytes is modulated in different osmotic conditions and totheir interaction examine a potential role for with MAPKss in this processpathways. Isolated equine articular chondrocytes were subjected to anisosmotic conditions and and the expression of the TRPV4 and, BKCa channels and also ERK1/2 and p38 MAPKs protein phosphorylations were investigated using western blotting. Results indicated that This study indicated to the contribution of TRPV4 contributes toin the early stages of hypo-osmotic stress, while BKCa is involved in providing a feedback to the responses to elevatedion of intracellular Ca(2+) and mediated mediating regulatory volume decrease (the RVD) response. ERK1/2 is phosphorylated by hypo-osmotic stress (P<0.001), and p38 MAPK is phosphorylated by hyper-osmotic stress (P<0.001). In addition this study showeddemonstrated the importance of endogenous ERK1/2 phosphorylation in the expression of TRPV4 channel where blocking ERK1/2 by specific inhibitor (PD89059) significantly decreased the endogenous level of the TRPV4 channel in cells exposed to at hypo-osmotic stress and below endogenous levels in at iso-osmotic conditionsstress (P<0.001). It also showed the importance of ERK1/2 phosphorylation in the expression of endogenous levels of TRPV4 channel.
Cell motility is dependent on a coordinated reorganization of the cytoskeleton, membrane recycling, and focal adhesion to the extracellular matrix. Each of these cellular processes involves re-distribution of cell water, which is facilitated by the transport of inorganic ions (with obligatory water movement). Scratch-wound healing assays of Wistar C6 glioblastoma cells demonstrated cell motility in advance of cell proliferation. Although bumetanide inhibition of Na-K-2Cl cotransport activity did not affect cell motility, treatment of glioma cells with furosemide to inhibit K-Cl cotransport activity prevented ~75% of wound closure in a reversible reaction. Genetic silencing of KCC3 with short hairpin interfering RNA reduced protein expression by 40 - 60%, K(+) influx by ~50%, and cell motility by ~50%. Appearance of KCC1 mRNA and KCC3 mRNA at 25 PCR cycles versus KCC4 mRNA at 35 PCR cycles, suggests more KCC1/KCC3 expression in both primary rat astrocytes and C6 glioma cells. Altogether, these experiments suggest that the presence/function of multiple isoforms of the Na(+-)independent K-Cl cotransporter may have a role in glioma cell motility.
Oral squamous cell carcinoma (SCC) represents more than 90% of all head and neck cancers as reported by Hermans (Cancer Imaging, 5(Spec No A), S52-S57, 2005), which draws attention of investigative research for novel predictive metabolic biomarkers to understand the malignancy induced biochemical perturbations occurring at molecular level. In the present work, proton HR-MAS NMR spectroscopic studies have been performed on resected human oral SCC tumor tissues, its neighboring margins and bed tissues (n = 159), obtained from 36 patients (n = 27 training set; n = 9 unknown test set), for the identification of metabolic fingerprints. The proton NMR spectra were then subjected to chemometric unsupervised PCA and supervised OSC-filtered PCA and PLS-DA multivariate analysis. Application of PLS-DA on orthogonally signal corrected training data-set (n = 120 tissue specimens; 27 patients) allowed >95% correct classification of malignant tissues from benign samples with >98% specificity and sensitivity. The OSC-PLS-DA model thus constructed was used to predict the class membership of unknown tissue specimens (n = 39) obtained from nine patients. These tissue samples were correctly predicted in its respective histological categories with 97.4% diagnostic accuracy. The regression coefficients obtained from OSC-filtered PLS-DA model indicated that malignant tissues had higher levels of glutamate, choline, phosphocholine, lactate, acetate, taurine, glycine, leucine, lysine, isoleucine and alanine, and lower levels of creatine and PUFA, representing altered metabolic processes (lipidogenesis, protein synthesis, and volume regulation) during tumor progression. Thus proton HR-MAS MR spectroscopy could efficiently identify the metabolic perturbations of malignant tumor from non-malignant bed and margins tissue specimens, which may be helpful in understanding the extent of tumor penetration in neighboring tissues.
Maintaining cell volume is critical for cellular function yet shift in cell volume is a prerequisite for mitosis and apoptosis. The ubiquitously and evolutionary conserved serine/threonine kinase CK2 promotes cell survival and suppresses apoptosis. The present review describes how mammalian cells regulate the cellular content of the major cellular organic osmolyte, taurine with emphasis on CK2 mediated regulation of active taurine uptake and volume-sensitive taurine release. Furthermore, we discuss how CK2-mediated regulation of taurine homeostasis is potentially involved in cellular functions such as proliferation and survival.
Urinary bladder cancer is a major epidemiological problem that continues to grow each year. It opens avenues for investigative research for the identification of new disease markers and diagnostic techniques. In this pilot study, utility of non-invasive (1)H NMR spectroscopy has been evaluated for probing the metabolic perturbations occurring in non-muscle invasive urinary bladder cancer. (1)H NMR spectra of urine of bladder cancer patients and controls (healthy and urinary tract infection/bladder stone) (n = 103) were acquired at 400MHz. The non-overlapping resonances of citrate, dimethylamine, phenylalanine, taurine and hippurate were first identified and then quantitated by (1)H NMR spectra, with respect to an external reference sodium-3-trimethylsilylpropionate (TSP). The concentrations of these metabolites were then statistically analyzed. The cancer patients showed significant (p < 0.05) variations in concentration of hippurate and citrate as compared with healthy controls and benign controls. The significant elevation in concentration of taurine was observed in urine of bladder cancer patients, which was below the sensitivity limit of 400MHz in control cases. However, stages Ta, T1 and carcinoma in situ (CIS) cannot be differentiated on the basis of altered metabolite indices but their composition may reflect the biochemical alterations in metabolism of cancer cells.
To research the alternation effect of insulin-like growth factors-2 (IGF-II) on the expression of KC1 co-transport-1 (KCC1) in the SiHa cells of cervical cancer, and to explore the activation of ERK1/2MAPK and PI3K/AKT signal transduction pathways during the expression process.
To apply semi-quantitative RT-PCR and Western blot analysis to measure changes in mRNA and protein expression of KCC1 after exposure to different concentrations of IGF-II for different time durations in the SiHa cells of cervical cancer. The change in protein expression of the ERK1/2 and AKT pathways is also measured. Furthermore, the protein expression variation in ERK1/2, AKT, and KCC1 is observed after the addition of a specific pathway blocker for the ERK1/2MAPK and PI3K/AKT pathways.
The mRNA and protein expression of KCC1 increases dramatically after the application of IGF-II on the SiHa cells, and shows a definite dosage-time dependence relationship. The protein phosphorylation is enhanced in the ERK1/2 and AKT pathways, where the protein activity increases. By adding a specific pathway blocker, the protein activity and phosphorylation of the two pathways are no longer promoted even under the effect of IGF-II.
IGF-II can enhance KCC1 gene expression in cervical cancer cells through the ERK1/2MAPK and PI3K/AKT signal transduction pathways.
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.
Perturbations of cell hydration as provoked by changes in ambient osmolarity or under isoosmotic conditions by hormones, second messengers, intracellular substrate accumulation, or reactive oxygen intermediates critically contribute to the physiological regulation of cell function. In general an increase in cell hydration stimulates anabolic metabolism and proliferation and provides cytoprotection, whereas cellular dehydration leads to a catabolic situation and sensitizes cells to apoptotic stimuli. Insulin produces cell swelling by inducing a net K+ and Na+ accumulation inside the cell, which results from a concerted activation of Na+/H+ exchange, Na+/K+/2Cl- symport, and the Na+/K(+)-ATPase. In the liver, insulin-induced cell swelling is critical for stimulation of glycogen and protein synthesis as well as inhibition of autophagic proteolysis. These insulin effects can largely be mimicked by hypoosmotic cell swelling, pointing to a role of cell swelling as a trigger of signal transduction. This article discusses insulin-induced signal transduction upstream of swelling and introduces the hypothesis that cell swelling as a signal amplifyer represents an essential component in insulin signaling, which contributes to the full response to insulin at the level of signal transduction and function. Cellular dehydration impairs insulin signaling and may be a major cause of insulin resistance, which develops in systemic hyperosmolarity, nutrient deprivation, uremia, oxidative challenges, and unbalanced production of insulin-counteracting hormones. Hydration changes affect cell functions at multiple levels (such as transcriptom, proteom, phosphoproteom, and the metabolom) and a system biological approach may allow us to develop a more holistic view on the hydration dependence of insulin signaling in the future.
FACS analysis and [14C]-taurine efflux were used to determine whether activation of the volume-sensitive organic osmolyte/anion channel plays a role in cell cycle progression. This was achieved by examining the effects of a collection of (i) H(1) antagonists and tricyclic antidepressants with a known inhibitory effect on cell cycle progression, and (ii) antidepressants and oestrogen receptor modulators with molecular structures likely to confer inhibition of the volume-sensitive organic osmolyte/anion channel. Of the 13 compounds examined in this study, the following showed no cytotoxicity following a 48-h exposure, and specifically inhibited osmosensitive taurine efflux (over lactate transport and anion exchange) with IC(50) values of (in microM): fluoxetine, approximately 14; fluvoxamine, approximately 24; amitriptyline, approximately 32; imipramine, approximately 32; mianserin, approximately 40. A 48-h application of these compounds at these concentrations significantly increased arrest in the G0/1 stage of the cell cycle by approximately 10%. The uniformity and specificity of the response elicited by these compounds strongly reinforces a correlation between cell cycle progression and osmosensitive taurine efflux activation.
This study characterized the membrane permeability to cAMP in a cell line derived from the rat colon (CC531(mdr+)) by comparison of fluxes of 3H-cAMP, 3H-8-bromo-cAMP, 3H-taurine, 3H-adenosine and 3H-5'AMP under various experimental conditions including cell membrane depolarization and hypotonic cell swelling. Cell volume was modified by changing the osmolality and composition of the extracellular medium. Incubation in iso- and hypotonic KCl media induced graded increases in cell volume and stable activation of volume-sensitive channels that was reflected in an increased efflux of 3H-taurine. Incubation in hypotonic KCl solution also enhanced the efflux of 3H-8-Br-cAMP (a non-hydrolysable analogue of cAMP). Both the efflux of 3H-taurine and of 3H-8-Br-cAMP were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 100 microM) suggesting the involvement of volume-sensitive anion channels. To gain further insight into the route mediating cAMP permeability, the uptakes of 3H-cAMP, 3H-8-Br-cAMP and 3H-taurine were determined over short (5-min) periods. Uptakes of these substrates demonstrated close similarities: comparable increases were observed that correlated with the increases in cell volume in iso- and hypoosmotic KCl media; they were inhibited strongly by NPPB (100 microM) and metabolic inhibitors (deoxyglucose, 20 mM together with the mitochondrial uncoupler carbonylcyanide p-(trifluoromethoxy)phenylhydrazone, FCCP, 10 microM) while barely reduced by dipyridamole (100 microM) and they were not affected by adenosine (1 mM). In contrast, the uptakes of 3H-adenosine and 3H-5'AMP had strikingly different properties; they were insensitive to cell swelling; barely inhibited by NPPB (100 microM) and metabolic inhibitors (deoxyglucose and FCCP) while strongly reduced by dipyridamole (100 micro M). Unlike the uptakes of 3H-cAMP, 3H-8-Br-cAMP and 3H-taurine, the uptakes of 3H-adenosine and 3H-5'AMP were reduced in Na(+)-free media, suggesting the presence in this cell line of two different adenosine carriers, one sodium-dependent and one sodium-independent. Taken together the present data show that in this rat colonic cell line, cAMP permeability is increased by cell swelling in hypotonic KCl medium and inhibited by NPPB and metabolic inhibitors. The similarity of these characteristics to those of taurine permeability suggests the involvement of a volume-sensitive anion pathway.
Key elements of tumor development include proliferation, migration, invasiveness, and angiogenesis. Activation of the volume-sensitive organic osmolyte/anion channel (VSOAC) has been suggested to play a role in all of these processes. VSOACs may therefore represent an important therapeutic target in the etiology of cancer. However, pharmacological inhibitors of VSOAC are nonselective and of low potency, highlighting the importance of identifying novel regulators of the channel. The use of electrophysiological methods coupled with techniques such as pull-down assays, yeast 2-hybrid, and functional protein arrays have already proved valuable in studying protein-protein interactions in a variety of systems. Some of these methods have been used to identify small molecules that modulate the function of other types of ion channels. Given that several proteins have already been identified as putative modulators of VSOACs, proteomics technologies may prove useful in elucidating the molecular identity of VSOACs and helpful in identifying novel modulators of channel function. In this paper, we review the involvement of VSOACs in tumor development processes and its regulation by pharmacological agents and cellular proteins. Proteomic approaches to study protein-protein interactions and how such approaches may be used to study VSOACs are also discussed. We speculate on how modulation of protein-protein interactions may result in the identification of a novel class of compounds for modulating VSOACs.
Redistribution or translocation of plasma free amino acids (PFAAs) to support visceral or tumor protein synthesis is an essential feature in cancer patients. An abnormal PFAA profile might be presented via the total reflection of cancer-induced protein metabolism in tumors, skeletal muscle and the liver in cancer patients. Clinical data from 13 studies have demonstrated a cancer-related PFAA profile, especially in digestive organ cancers. The PFAA profile can differ between the early and late stages of cancer. The profile is also affected by the type of cancer. Therefore, it is postulated that a detailed analysis of the PFAA profile may serve as one of the biological markers for cancer patients.
The K+-Cl- cotransport (COT) regulatory pathways recently uncovered in our laboratory and their implication in disease state are reviewed. Three mechanisms of K+-Cl- COT regulation can be identified in vascular cells: (1) the Li+-sensitive pathway, (2) the platelet-derived growth factor (PDGF)-sensitive pathway and (3) the nitric oxide (NO)-dependent pathway. Ion fluxes, Western blotting, semi-quantitative RT-PCR, immunofluorescence and confocal microscopy were used. Li+, used in the treatment of manic depression, stimulates volume-sensitive K+-Cl- COT of low K+ sheep red blood cells at cellular concentrations <1 mM and inhibits at >3 mM, causes cell swelling, and appears to regulate K+-Cl- COT through a protein kinase C-dependent pathway. PDGF, a potent serum mitogen for vascular smooth muscle cells (VSMCs), regulates membrane transport and is involved in atherosclerosis. PDGF stimulates VSM K+-Cl- COT in a time- and concentration-dependent manner, both acutely and chronically, through the PDGF receptor. The acute effect occurs at the post-translational level whereas the chronic effect may involve regulation through gene expression. Regulation by PDGF involves the signalling molecules phosphoinositides 3-kinase and protein phosphatase-1. Finally, the NO/cGMP/protein kinase G pathway, involved in vasodilation and hence cardiovascular disease, regulates K+-Cl- COT in VSMCs at the mRNA expression and transport levels. A complex and diverse array of mechanisms and effectors regulate K+-Cl- COT and thus cell volume homeostasis, setting the stage for abnormalities at the genetic and/or regulatory level thus effecting or being affected by various pathological conditions.
Streptomycin is a common antibiotic used in culture media. It is also a known blocker of stretch-activated and mechanosensitive ion channels in neurons and cardiac myocytes. But very little information is available on its effect in the regulation of epithelial ion channels. Osmotic swelling is a kind of mechanical stretch. The opening of stretch-activated Ca(2+) channels contributes to hypotonicity-induced Ca(2+) influx which is necessary for the activation of volume-regulated Cl(-) channels in human cervical cancer cells. This study aimed to investigate the role of streptomycin in cell volume regulation. Treatment of cervical cancer SiHa cells with streptomycin and its analogues (gentamicin and netilmicin) did not affect the basal cytosolic Ca(2+) ([Ca(2+)](i)) level. But it attenuated the hypotonicity-stimulated increase of [Ca(2+)](i) in a dose-dependent manner with half-maximal inhibitory concentrations (IC(50)) of 25, 90 and 200 microM for streptomycin, gentamicin and netilmicin, respectively, when measured at room temperature. In contrast, under free extracellular Ca(2+) condition, hypotonic stress only induced a small, progressive increase of [Ca(2+)](i), while 500 microM streptomycin did not affect this Ca(2+) signaling. Streptomycin and its analogues (gentamicin and netilmicin) also inhibited the activation of volume-regulated Cl(-) channels in a dose-dependent manner with IC(50) of 30, 95 and 250 microM at room temperature, respectively. Chronic culture with 50 microM streptomycin downregulates the activity of volume-regulated Cl(-) channels and retards the process of regulatory volume decrease in SiHa cells and MDCK cells. We suggest that using cells chronically cultured with streptomycin to study epithelial ion channels risks studying cellular and molecular pathology rather than physiology.
Cell swelling is associated with the activation of an increase in the osmosensitive taurine release (OTR) rate, which serves to decrease cell volume as part of a process known as regulatory volume decrease. OTR, which is sensitive to many pharmacological agents including anion channel blockers and signalling pathway modulators, has also been suggested to play a role in cell cycle progression. At non-cytotoxic concentrations, the anion channel blocker NPPB (25 microM), the extra-cellular signal-regulated kinase inhibitor PD98059 (50 microM), and the c-Jun NH2-terminal kinase inhibitor SP 600125 (5 microM) each decreased the OTR rate by > or =50%, decreased cell proliferation, and increased G0/G1 cell cycle arrest.
We have investigated the volume-activated transport of organic solutes in flounder erythrocytes. Osmotic swelling of cells suspended in a Na(+)-free medium led to increased membrane transport of taurine, glucose, and uridine. For each compound there was a significant lag period (1-2 min at 10 degrees C) between cell swelling and activation of the flux. The volume-activated fluxes of each of the substrates increased in parallel with increasing cell volume, and those of taurine and uridine increased linearly with concentration (up to 19 mM). The volume-activated fluxes of each of the three compounds showed similar sensitivities to a number of anion-selective channel blockers (5-nitro-2-(3-phenylpropylamino)benzoic acid > 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid approximately MK-196 > niflumic acid > furosemide); the IC50 for the inhibition of the volume-activated fluxes by NPPB was around 12 microM. The results are consistent with the hypothesis that the volume-activated transport of organic osmolytes is via a pathway with the characteristics of a volume-activated "chloride channel." This raises the question of whether the transport of organic substrates might represent a physiological role for such channels in other cell types.
Inhibitors of DNA replication in mammalian cells are of great interest because of their potential use In chemotherapy and
in cell synchronizing protocols in the laboratory. We have used a combination of Isotopic labelling protocols and a two-dimensional
gel replicon mapping procedure to determine the specific effects of five different replication Inhibitors in cultured cells.
Utilizing this protocol, we show that hydroxyurea, aphidicolin, and cytosine arablnoside, three known chain elongation inhibitors,
are rather Ineffective at preventing fork progression even at relatively high concentrations. In contrast, two related compounds
that have been suggested to be G1/S Inhibitors (mimosine and clclopyrox olamine [CPX]) actually appear to inhibit Initiation at origins. One of these agents
(CPX) appears also to Inhibit replication in yeast, opening the possibility that the gene encoding the target (Initiator?)
protein can first be identified in yeast by genetic approaches and can then be used to Isolate the mammalian homologue.
The maintenance of a constant volume in the face of extracellular and intracellular osmotic perturbation is essential for the normal function and survival of animal cells. Osmotically swollen cells restore their volume, exhibiting a regulatory volume decrease by releasing intracellular K+, Cl-, organic solutes, and obligated water. In many cell types, the volume regulatory effluxes of Cl- and some organic osmolytes are known to be induced by swelling-induced activation of anion channels that are characterized by their moderate outward rectification, cytosolic ATP dependency, and intermediate unitary conductance (10-100 pS). Recently, simultaneous measurements of cell size by light microscopy and whole cell Cl- current have shown that the Cl- current density is proportionally increased with an increase in the outer surface area, which is mainly achieved through unfolding of membrane invaginations by volume expansion. Thus this anion channel can somehow sense volume expansion and can be called the volume expansion-sensing outwardly rectifying (VSOR) anion channel. Its molecular identity and activation mechanism are yet to be elucidated. Three cloned proteins, ClC-2, P-glycoprotein, and pIcln, have been proposed as candidates for the VSOR anion channel. The unitary conductance, voltage dependency, anion selectivity, pH dependency, and pharmacology of the VSOR anion channel are distinct from the ClC-2 Cl- channel, which is also known to be sensitive to volume changes. Recent patch-clamp studies in combination with molecular biological techniques have shown that P-glycoprotein is not itself the channel protein but is a regulator of its volume sensitivity. Although there is still debate about another candidate protein, pIcln, the most recent study has suggested that this is likely to be a regulator of some other distinct Cl- channel. Identification of the VSOR anion channel protein per se, its volume-sensing mechanism, and its accessory/regulatory proteins at the molecular level is currently a subject of utmost physiological importance.
We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie,
J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651–11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed inXenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation,
inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell
and unitary currents sensitive to low nm concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low
nm concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression
conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes
increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast
to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood
stem cells in liquid culture.
Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4-AP, or Gd3+. 86Rb efflux decreased in Cl(-)-depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl- and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]0, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling-activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+-sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+-dependence, or pharmacological sensitivity. We conclude that the swelling-activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+-sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage-gated K+ channels.
Human T lymphocytes express a Ca2+-activated K+ current (IK), whose roles and regulation are poorly understood. We amplified hSK4 cDNA from human T lymphoblasts, and we showed that its biophysical and pharmacological properties when stably expressed in Chinese hamster ovary cells were essentially identical to the native IK current. In activated lymphoblasts, hSK4 mRNA increased 14.6-fold (Kv1.3 mRNA increased 1.3-fold), with functional consequences. Proliferation was inhibited when Kv1.3 and IK were blocked in naive T cells, but IK block alone inhibited re-stimulated lymphoblasts. IK and Kv1.3 were involved in volume regulation, but IK was more important, particularly in lymphoblasts. hSK4 lacks known Ca2+-binding sites; however, we mapped a Ca2+-dependent calmodulin (CaM)-binding site to the proximal C terminus (Ct1) of hSK4. Full-length hSK4 produced a highly negative membrane potential (Vm) in Chinese hamster ovary cells, whereas the channels did not function when either Ct1 or the distal C terminus was deleted (Vm approximately 0 mV). Native IK (but not expressed hSK4) current was inhibited by CaM and CaM kinase antagonists at physiological Vm values, suggesting modulation by an accessory molecule in native cells. Our results provide evidence for increased roles for IK/hSK4 in activated T cell functions; thus hSK4 may be a promising therapeutic target for disorders involving the secondary immune response.
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 #-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 6 nM, respectively. Conversely, protein kinase inhibitors, chelerythrine and staurosporine, increased Cl--dependent 86Rb+ efflux. NEM (1 mM) 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.
Previous studies show that the regulatory volume decrease (RVD) in human cervical cells with different tumour potential may be mediated by different ion channels. The signalling events involved in regulating these channel activities are not clear. To screen the possible mechanisms involved in cell volume regulation in these cells, we examine intracellular mechanisms and second messengers listed as follows: phospholipase C (PLC), phospholipase A2 (PLA2), tyrosine kinase (TK), protein kinase C (PKC), protein kinase A (PKA), and cAMP. The involvement of G-protein was also studied. Our results showed that PLC signalling with downstream activation of PKC was involved in the cell volume regulation of cervical cancer cells. On the other hand, different PKC isoforms that were not related to upstream PLC regulation were involved in the RVD of human papillomavirus (HPV)-immortalised and normal cervical epithelia. Furthermore, GTP-gamma S facilitated the process of RVD in cervical cancer cells, while pertussis toxin retarded this process. In contrast, neither GTP-gamma S nor pertussis toxin showed effect on the RVD responses of HPV-immortalised and normal cervical cells.
Hypotonicity activates volume-sensitive Cl- currents, which are implicated in the regulatory volume decrease (RVD) responses and transport of taurine in human cervical cancer HT-3 cells. In this study, the role of cytoskeleton in the regulation of volume-sensitive Cl- channels and RVD responses in HT-3 cells was studied. Cells were incubated with various compounds, which depolymerized or polymerized cytoskeletal elements, i.e. actin filaments and microtubules. The hypotonicity-induced changes in Cl- conductance and in cell volume were measured by whole-cell voltage clamping and cell size monitoring, respectively. Our results show that in HT-3 cells hypotonicity activated an outward rectified Cl- current that was abrogated by Cl- channel blockers. Cytochalasin B, an actin-depolymerizing compound, induced a substantial increase in Cl- conductance under isotonic condition and potentiated the expression of Cl- currents in hypotonic stress. Phorbol 12-myristate 13-acetate (PMA) significantly inhibited the cytochalasin B-induced activation of Cl- conductance under isotonic condition. On the other hand, treatment with cytochalasin B significantly prolonged the RVD responses. Phalloidin, a stabilizer of actin polymerization, did not change the basal currents under isotonic condition, but completely abolished the increase in whole-cell Cl- conductance elicited by hypotonicity and retarded the cell volume recovery. Colchicine, a microtubule-assembly inhibitor, had no effect on either basal Cl- conductance or volume-sensitive Cl- current and was unable to inhibit the RVD responses. Taxol, a microtubule-stabilizing compound, did not alter the basal Cl- conductance, but inhibited the activation of volume-sensitive Cl- channels as well as the process of RVD in a dose-dependent manner. These data support the notion that functional integrity of actin filaments and microtubules plays critical roles in maintaining the RVD responses and activation of Cl- channels in human cervical cancer HT-3 cells.
The present study was carried out to identify the specific protein kinase C (PKC) isoform involved in regulatory volume decrease (RVD) responses, and to investigate the signal transduction pathways underlying the activation of volume‐sensitive chloride channels in human cervical cancer HT‐3 cells. The role of Ca ²⁺ in RVD and in the activation of chloride currents was also studied.
The time course of RVDs was prolonged by microinjection of PKC‐α antibody but not by PKC‐β or PKC‐γ antibody, and also by exposure to Ca ²⁺ ‐free medium, in particular when combined with microinjection of EDTA. Immunofluorescence staining showed that hypotonic superfusion evoked the translocation of PKC‐α to the cell membrane, whereas PKC‐β or PKC‐γ remained unaffected. The translocation of PKC‐α was observed a few minutes after hypotonic stress, reaching peak intensity at 30 min, and returned to the cytoplasm 60 min after hypotonic exposure. Western blot analyses showed an increased PKC‐α level in terms of intensity and phosphorylation in the cell membrane, while neither PKC‐β nor PKC‐γ was activated upon hyposmotic challenge.
Whole‐cell patch‐clamp studies demonstrated that neomycin and PKC blockers such as staurosporine and H7 inhibited volume‐sensitive chloride currents. The inhibitory effect of neomycin on chloride currents can be reversed by the PKC activator phorbol 12‐myristate, 13‐acetate (PMA). Moreover, the PKC inhibitor and PKC‐α antibody, but not PKC‐β or PKC‐γ antibody, significantly attenuated the chloride currents. The activation of volume‐sensitive chloride currents were insensitive to the changes of intracellular Ca ²⁺ but required the presence of extracellular Ca ²⁺ .
Our results suggest the involvement of PKC‐α and extracellular Ca ²⁺ in RVD responses and the activation of volume‐sensitive chloride channels in HT‐3 cells.
According to previous studies hyposmotic swelling of Madin Darby Canine Kidney (MDCK) cells leads to a marked decrease of cell membrane resistance. The present study has been performed to identify the underlying ion channels using the patch-clamp technique: reduction of extracellular osmolarity to 230 mmol/liter leads to a transient activation of K+ channels and a sustained activation of anion channels. The K+ channels are inwardly rectifying with a single-channel slope conductance of 56 +/- 3 pS at -50 mV (cell negative) and of 29 +/- 2 pS at 0 mV PD across the patch (150 mmol/liter K+ in pipette). The same channels are activated by an increase of intracellular calcium activity, as shown previously. The anion channels display a single-channel slope conductance of 41 +/- 4 pS at -50 mV (cell negative) and of 25 +/- 3 pS at 0 mV PD across the patch (150 mmol/liter Cl- in pipette). The channel is anion selective and conducts both bicarbonate and chloride with a preference for bicarbonate. Its open probability is not affected by changing intracellular calcium from 0.1-10 mumol/liter. The channels observed explain the effects of cell swelling on PD, ion selectivity and resistance of the cell membrane in MDCK cells.
1. Ion channels and their possible relation to cell proliferation have been studied in a human melanoma cell line (IGR 1). Membrane currents were recorded by the patch-clamp technique using the cell-attached, cell-free and whole-cell mode. Cell growth was monitored by counting the number of cells at different days after seeding and [3H]thymidine incorporation. 2. A voltage-dependent 10 pS non-inactivating potassium channel (delayed rectifier) is the most commonly observed ion channel in this type of human cell. The channel is active at the normal resting potential and can be blocked by tetraethylammonium chloride (TEA) and also by a membrane-permeable cyclic adenosine monophosphate (8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, cyclic AMP). A second type of potassium channel shows properties similar to voltage-dependent A-type potassium channels with complete inactivation. 3. A voltage-independent, non-selective cation channel with a single-channel conductance of approximately 20 pS could be seen in only 8% of the patches. Its properties of modulation are still unknown. 4. The incidence of the 10 pS, non-inactivated potassium channel was maximal at the fourth day after seeding (in 89% of the patches) and was significantly reduced at the seventh day (in 35% of the patches). 5. [3H]thymidine incorporation is maximal at the third day after seeding and is reduced when cells are grown in the presence of TEA or cyclic AMP. This peak of maximal [3H]thymidine incorporation correlated with the incidence of non-inactivated potassium channels. 6. In the presence of TEA or cyclic AMP, growth of the cells is inhibited. We suppose that due to block of potassium channels, most of the melanoma cells are not able to enter the S-phase in the cell division cycle. 7. It is concluded that delayed rectifier potassium channels are involved in the control of melanoma cell proliferation. A similar finding has been reported for K+ channels in T-lymphocytes and human breast carcinoma cells. It is suggested that potassium channels may be involved in controlling the driving force for a calcium influx thereby interacting with Ca(2+)-dependent cell cycle control proteins.
Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba2+ and hence presumably to Ca2+. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca2+ permeability, as part of the volume-activated signal system. In isolated inside-out patches a Ca(2+)-dependent, inwardly rectifying K+ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mM K+ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K+ channel takes place after an increase in Ca2+ from 10(-7) to 10(-6) M which is in the physiological range. Patch-clamp studies in cell-attached mode show K+ channels with spontaneous activity and with characteristics similar to those of the K+ channel seen in excised patches. The single-channel conductance for outward current at 5 mM external K+ is estimated at about 7 pS. A K+ channel with similar properties can be activated in the cell-attached mode by addition of Ca2+ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K+ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K+ channels following addition of Ca2+ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K+ channels in the presence of 3 mM Ca2+ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca2+ and could not be activated by membrane stretch (suction). In cell-attached mode single-channel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca2+ plus ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)
Substantial experimental evidence accumulated over the past 8 years has indicated an etiological role for specific human papillomavirus (HPV) types in anogenital cancer and its premalignant precursors. Virus infection and viral gene expression emerge as necessary but obviously not sufficient factors for cancer induction. Additional modifications of host cell genes appear to be required for malignant progression of infected cells. The expression of viral oncoproteins in cells infected by "high-risk" types (e.g., HPV 16, HPV 18), in contrast to "low-risk" types (e.g., HPV 6, HPV 11), results in chromosomal instability and apparently in accumulation of mutational events. These "endogenous" modifications seem to be most important in the pathogenesis of premalignant lesions and tumor progression. Exogenous mutagens should act as additional cofactors.
Ion channels, and ion fluxes in general, appear to regulate a wide variety of processes important to lymphocyte function in normal and disease states. These include resting ionic homeostasis and the more complex signaling events involved in activation, proliferation, cytotoxic function, and volume regulation. The wider application of patch-clamp and microfluorimetry techniques to lymphocytes has helped to clarify some issues and raised many more. It seems likely that rapid progress will be made in our understanding of these areas through a combination of immunological, biochemical, and electrophysiological approaches.
The finding that astrocytes possess glutamate-sensitive ion channels hinted at a previously unrecognized signaling role for
these cells. Now it is reported that cultured hippocampal astrocytes can respond to glutamate with a prompt and oscillatory
elevation of cytoplasmic free calcium, visible through use of the fluorescent calcium indicator fluo-3. Two types of glutamate
receptor--one preferring quisqualate and releasing calcium from intracellular stores and the other preferring kainate and
promoting surface-membrane calcium influx--appear to be involved. Moreover, glutamate-induced increases in cytoplasmic free
calcium frequently propagate as waves within the cytoplasm of individual astrocytes and between adjacent astrocytes in confluent
cultures. These propagating waves of calcium suggest that networks of astrocytes may constitute a long-range signaling system
within the brain.
Cystic fibrosis (CF) is a genetic disease characterized by abnormal regulation of epithelial cell chloride channels. Nonepithelial cells, including lymphocytes and fibroblasts, may exhibit a similar defect. Two independent techniques were used to assess the macroscopic chloride permeability (PCl) of freshly isolated B lymphocytes and of B and T lymphocyte cell lines. Values for PCl increased specifically during the G1 phase of the cell cycle and could be further enhanced by increasing intracellular adenosine 3',5'-monophosphate (cAMP) or calcium. In lymphocytes from CF patients, regulation of PCl during the cell cycle and by second messengers was absent. Characterization of the cell cycle-dependent expression of the chloride permeability defect in lymphocytes from CF patients increases the utility of these cells in the analysis of the functional consequences of mutations in the CF gene.
Increased basolateral membrane K conductance accompanies stimulation of Cl secretion across canine trachea. To assess the K conductance properties, we permeabilized the apical membranes with amphotericin B and monitored the current and conductance caused by K flow across the basolateral membranes. Under basal unstimulated conditions, two K conductances could be distinguished by blockers. One was inhibited only by barium; the other was sensitive also to quinidine and lidocaine. The permeabilities of the basal conductance pathways to K and Rb were similar (PK/PRb approximately equal to 1.5). The secretory agonist, epinephrine, selectively increased the quinidine-insensitive conductance, implicating it in the Cl secretory response. Cell swelling induced a third conductance with a low permeability to Rb (PK/PRb approximately equal to 10) that was quinidine sensitive. In tissues not treated with amphotericin, neither quinidine nor Rb-for-K replacement inhibited transepithelial Cl secretion. Thus neither of the quinidine-sensitive K conductances (basal or swelling induced) contribute to the increase in basolateral K conductance during Cl secretion. Cell shrinkage inhibited all three conductances and secretion, suggesting that the initial priority of the cell is volume regulation.
Recent work on selected aspects of the cellular and molecular physiology of cell volume regulation is reviewed. First, the physiological significance of the regulation of cell volume is discussed. Membrane transporters involved in cell volume regulation are reviewed, including volume-sensitive K+ and Cl- channels, K+, Cl- and Na+, K+, 2Cl- cotransporters, and the Na+, H+, Cl-, HCO3-, and K+, H+ exchangers. The role of amino acids, particularly taurine, as cellular osmolytes is discussed. Possible mechanisms by which cells sense their volumes, along with the sensors of these signals, are discussed. The signals are mechanical changes in the membrane and changes in macromolecular crowding. Sensors of these signals include stretch-activated channels, the cytoskeleton, and specific membrane or cytoplasmic enzymes. Mechanisms for transduction of the signal from sensors to transporters are reviewed. These include the Ca(2+)-calmodulin system, phospholipases, polyphosphoinositide metabolism, eicosanoid metabolism, and protein kinases and phosphatases. A detailed model is presented for the swelling-initiated signal transduction pathway in Ehrlich ascites tumor cells. Finally, the coordinated control of volume-regulatory transport processes and changes in the expression of organic osmolyte transporters with long-term adaptation to osmotic stress are reviewed briefly.
Astrocyte proliferation was studied in primary cultures of rat spinal cord by [3H]thymidine uptake and was significantly reduced by culturing cells for 24 h in the presence of agents known to block astrocytic K+ channels: Cs+, Ba2+, 4-AP and tetraethylammonium (TEA). To determine whether effects were mediated by changes in Vm or pHi, these parameters were studied electrophysiologically or ratiometrically, using BCECF. Of the four K+ channel blockers, only Ba2+ depolarized astrocytes significantly. However, all four K+ channel blockers resulted in an alkaline shift in pHi. Under culture conditions that altered pHi in a defined way, proliferation strongly depended on pHi, with highest rates at pH approximately 6.7 and growth inhibition at more acidic or alkaline conditions. These observations suggest that astrocyte proliferation is sensitive to changes in pHi and that K+ channel blockers may exhibit their antiproliferative effects through changes in pHi.
Swelling of bovine aortic endothelial cells activates Ca(2+)-dependent K+ channels. To determine the role of Ca2+ in this response, we examined the effect of cell swelling on intracellular Ca2+ concentration ([Ca2+]i), and the role of [Ca2+]i in swelling-activated K+ efflux. Basal [Ca2+]i, measured by fura 2 fluorescence, was 62 nM and increased by 36 nM in hypotonic medium (220 mosmol/l) compared with a 277 nM increase in response to extracellular ATP. In cells loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA), the increases induced by swelling and by ATP were reduced to 13 and 20 nM, respectively. Exposure to hypotonic medium (220 mosmol/kg) or to the Ca2+ ionophore A-23187 stimulated a furosemide-insensitive 86Rb efflux consistent with activation of K+ channels. The swelling-activated efflux was inhibited 16% by 5 mM tetraethylammonium and 24% by 23 mM tetrabutylammonium, but not by 100 microM quinidine, a pattern similar to that previously observed for swelling-activated K+ channels in cell-attached patches. The effects of A-23187 and hypotonic swelling on 86Rb efflux were completely additive, suggesting Ca(2+)-independent activation by cell swelling. Removal of Ca2+ from the external medium or loading of cells with BAPTA to buffer intracellular Ca2+ blocked the activation of 86Rb efflux by A-23187, but not by hypotonic swelling. Hypertonic medium (440 mosmol/kg by the addition of sucrose) attenuated the increased 86Rb efflux in response to A-23187. We conclude that the activation of K+ efflux in swollen endothelial cells occurs independently of changes in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)
The purpose of this study was to determine if potassium channel activity is required for the proliferation of MCF-7 human mammary carcinoma cells. We examined the sensitivities of proliferation and progress through the cell cycle to each of nine potassium channel antagonists. Five of the potassium channel antagonists produced a concentration-dependent inhibition of cell proliferation with no evidence of cytotoxicity following a 3-day or 5-day exposure to drug. The IC50 values for these five drugs, quinidine (25 microM), glibenclamide (50 microM), linogliride (770 microM), 4-aminopyridine (1.6 mM), and tetraethylammonium (5.8 mM) were estimated from their respective concentration-response curves. Four other potassium channel blockers were tested at supra-maximal channel blocking concentrations, including charybdotoxin (200 nM), iberiotoxin (100 nM), margatoxin (10 nM), and apamin (500 nM), and they had no effect on MCF-7 cell proliferation, viability, or cell cycle distribution. Of the five drugs that inhibited proliferation, only quinidine, glibenclamide, and linogliride also affected the cell cycle distribution. Cell populations exposed to each of these drugs for 3 days showed a statistically significant accumulation in G0/G1 phase and a significant proportional reduction in S phase and G2/M phase cells. The inhibition of cell proliferation correlated significantly with the extent of cell accumulation in G0/G1 phase and the threshold concentrations for inhibition of growth and G0/G1 arrest were similar. The G0/G1 arrest produced by quinidine and glibenclamide were reversed by removing the drug, and cells released from arrest entered S phase synchronously with a lag period of approximately 24 hours. Based on the differential sensitivity of cell proliferation and cell cycle progression to the nine potassium channel antagonists, we conclude that inhibition of ATP-sensitive potassium channels in these human mammary carcinoma cells, reversibly arrests the cells in the G0/G1 phase of the cell cycle, resulting in an inhibition of cell proliferation.
There have been few investigations into the role of ion channels in mammalian early embryonic development, despite studies showing that changes in ion channel activity accompany the early embryonic development of non-mammalian species and the proliferation of mammalian cells. Here we report that a large-conductance, voltage-activated K+ channel is active in unfertilized mouse oocytes but is rarely observed in later embryos. The channel activity is linked to the cell cycle, being active throughout M and G1 phases, and switching off during the G1-to-S transition. These changes in channel activity are accompanied by corresponding shifts in membrane potential. Inactivation of the channel during S/G2 can be prevented by exposing the oocytes to dibutyryl cyclic AMP or forskolin, an activator of adenylyl cyclase. Inhibition of protein synthesis with puromycin did not prevent inactivation of the channel at the end of G1 or its subsequent reactivation at the end of G2, indicating that the channel activity is not regulated by mitosis-promoting factor or cyclins.
The annual incidence rates (crude and age-standardized) and numbers of new cases of 18 different cancers have been estimated for the year 1985 in 24 areas of the world. The total number of new cancer cases (excluding non-melanoma skin cancer) was 7.6 million, 52% of which occur in developing countries. The most common cancer in the world today is lung cancer, accounting for 17.6% of cancers of men worldwide, and 22% of cancers in men in the developed countries. Stomach cancer is now second in frequency (it was slightly more common than lung cancer in 1980) and breast cancer--by far the most important cancer of women (19.1% of the total)--is third. There are very large differences in the relative importance of the different cancers by world area. The major cancers of developed countries (other than the 3 already named) are cancers of the colon-rectum and prostate, and, in developing countries, cancers of the cervix uteri, mouth and pharynx, liver and oesophagus. The implications of these patterns for cancer control, and specifically prevention, are discussed. Tobacco smoking and chewing are almost certainly the major preventable causes of cancer today.
1. We have measured changes in cell volume, membrane potential and ionic currents in distal nephron A6 cells following a challenge with hypotonic solutions (HTS). 2. The volume increase induced by HTS is compensated by a regulatory volume decrease (RVD), which is inhibited by both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and quinine. Quinine (500 microM) completely blocked RVD, whereas 100 microM NPPB delayed and attenuated RVD. 3. The resting potential in A6 cells was -52.3 +/- 4.8 mV (n = 53), and shifted to -35.1 +/- 2.2 mV (n = 33) during HTS. 4. Resting membrane current in A6 cells was 0.35 +/- 0.12 pA pF-1 at -80 mV and 0.51 +/- 0.16 pA pF-1 at +80 mV (n = 5). During cell swelling these values increased to 11.5 +/- 1.1 and 29.3 +/- 2.8 pA pF-1 (n = 29), respectively. 5. Quinine (500 microM) completely blocked the HTS-activated current at -15 mV, the reversal potential for Cl- currents, but exerted only a small block at -100 mV (K+ equilibrium potential). NPPB (100 microM) inhibited the current at both potentials almost to the same extent. The HTS-induced net current reversed at -41 +/- 2.5 mV (n = 15), which is close to the measured resting potential during HTS. 6. The quinine-insensitive current reversed near the Cl- equilibrium potential. The quinine-sensitive current reversed near the K+ equilibrium potential. The respective conductances activated by HTS at the zero-current potential were 2.1 +/- 0.7 nS for K+ and 5.2 +/- 1.3 nS for Cl- (n = 15). 7. Single channel analysis unveiled activation of at least two different channels during HTS. A 36 pS channel reversing at the Cl- equilibrium potential showed increased open probability at depolarized potentials. HTS also activated a K+ channel with a 29 pS conductance in high-K+ extracellular solutions (130 mM) or 12 pS in 2.5 mM K+. 8. This coactivation of K+ and Cl- channels shifts the membrane potential towards a value between EK and ECl (the reversal potentials for K+ and Cl-), where a net efflux of Cl- (Cl- inward current) and K+ (K+ outward current) under zero-current conditions occurs. Block of either the K+ or the Cl- conductance will shift the zero-current potential towards the equilibrium potential of the unblocked channel, preventing net efflux of osmolytes and RVD. This coactivation of K+ and Cl- currents causes a shift of osmolytes out of the cells, which almost completely accounts for the observed RVD.
Volume-activated Cl- currents (ICl,vol) and cell growth have been measured in cultured endothelial cells from bovine pulmonary artery (CPAE) in the absence and presence of compounds which block these currents. The anti-oestrogen drug tamoxifen, which efficiently arrests the growth of breast cancer cells (1), inhibits both ICl,vol and cell proliferation with IC50 of 3.8 and 4.8 micromol/l respectively.NPPB and quinine, which also block ICl,vol, inhibit the growth of CPAE cells as well. Current and cell growth were closely correlated under all these conditions. We conclude that ICl,vol might be involved in the control of endothelial cell growth and thus might be important for the modulation of vascularisation and vascular remodelling.
Previous study shows volume-sensitive chloride currents are induced by hypotonicity in human cervical cancer cell lines, but not in normal cervical epithelium. To ascertain whether the preferential activation of these channels in cancer cell lines could be similarly and directly detected in cervical cancer tissues, we studied volume-sensitive chloride channels on the primary culture cells of invasive cervical carcinoma using the whole-cell patch-clamp technique. The process of regulatory volume decrease (RVD) was also studied using electronic cell sizing to measure cell volume. Results demonstrate that, in these cultured cells, RVD was mediated in part by chloride loss through the volume-sensitive Cl- channels. A small background current with a slope conductance of 0.32 +/- 0.07 nS/pF at +30 mV (n=60 cells from 10 different samples) was observed. Hypotonicity induced a fast activating and outward rectifying current which was reversed at about 0 mV, and the slope conductance at +30 mV was increased by 10-fold to 3.62 +/- 0.62 nS/pF. These effects were readily reversed by returning the cells to isotonic medium. Moreover, DIDS, NPPB, and 1,9-dideoxyforskolin, reversibly abolished the volume-sensitive Cl- currents. The EC50 required for the inhibitory effect of DIDS, NPPB and 1,9-dideoxyforskolin was 150, 120, and 50 microM, respectively. Volume-sensitive Cl- channels were ubiquitously expressed in cultured cells from 10 samples of different cancer stages, histopathologic types, and state of HPV DNA positivity. Interestingly, similar outward rectifying chloride currents were activated by intracellular 300 microM GTP gamma S. It is proposed that this Cl- conductance may play an important role leading to RVD in human cervical cancer.
The effect of osmotic cell swelling on the permeability of HeLa cells to a range of structurally unrelated solutes including taurine, sorbitol, thymidine, choline, and K+ (96Rb+) was investigated. For each solute tested, reduction in the osmolality of the medium from 300 to 200 mosmol/kgH2O caused a significant increase in the unidirectional influx rate. In each case, the osmotically activated transport component was nonsaturable up to external substrate concentrations of 50 mM. Inhibitors of the swelling-activated anion channel of HeLa cells [quinine, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, niflumate, 1,9-dideoxyforskolin, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and tamoxifen] blocked the osmotically activated influx of each of the different substrates tested, as well as the osmotically activated efflux of taurine and I-. Tamoxifen and NPPB were similarly effective at blocking the osmotically activated efflux of 96Rb+. The simplest of several hypotheses consistent with the data is that the osmotically activated transport of the different solutes tested here is via a swelling-activated anion-selective channel that has a significant cation permeability and a minimum pore diameter of 8-9 A.
1. An increase in cell volume activates, in most mammalian cells, a Cl- current, ICl,vol. This current is involved in a variety of cellular functions, such as the maintenance of a constant cell volume, pH regulation, and control of membrane potential. It might also play a role in the regulation of cell proliferation and in the processes that control transition from proliferation to differentiation. This review focuses on various aspects of this current, including its biophysical characterisation and its functional role for various cell processes. 2. Volume-activated Cl- channels show all outward rectification. Iodide is more permeable than chloride. In some cell types, ICl,vol inactivates at positive potentials. Single channel conductance can be divided mainly into two groups: small (< 5 pS) and medium conductance channels (around 50 pS). 3. The pharmacology and modulation of these channels are reviewed in detail, and suggest the existence of an heterogeneous family of multiple volume-activated Cl- channels. 4. Molecular candidates for this channel (i.e. ClC-2, a member of the ClC-family of voltage-dependent Cl- channels, the mdr-1 encoded P-glycoprotein, the nucleotide-sensitive pICln protein and phospholemman) will be discussed.
We have used the whole cell configuration of the patch-clamp technique to investigate volume-activated Cl- currents in BC3H1 and C2C12 cells, two mouse muscle cell lines that can be switched from a proliferating to a differentiated musclelike state. Reducing the extracellular osmolality by 40% evoked large Cl- currents in proliferating BC3H1 and C2C12 cells. These currents were outwardly rectifying and had an anion permeability sequence as follows: I- > Br- > Cl- > gluconate. They were inhibited by >50% by flufenamic acid (500 microM), niflumic acid (500 microM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) but were relatively insensitive to tamoxifen (100 microM). A reduction in the serum concentration in the culture medium induced growth arrest in both cell lines, and the cells started to differentiate into spindle-shaped nonfusing muscle cells (BC3H1) or myotubes (C2C12). This differentiation was accompanied by a drastic decrease in the magnitude of the volume-activated Cl- currents. The close correlation between volume-activated Cl- currents and cell proliferation suggests that these currents may be involved in cell proliferation.
We recently demonstrated expression of a novel, glioma-specific Cl- current in glial-derived tumor cells (gliomas), including stable cell lines such as STTG1, derived from a human anaplastic astrocytoma. We used STTG1 cells to study whether glioma Cl- channel (GCC) activity is regulated during cell cycle progression. Cells were arrested in defined stages of cell cycle (G0, G1, G1/S, S, and M phases) using serum starvation, mevastatin, hydroxyurea, demecolcine, and cytosine beta-D-arabinofuranoside. Cell cycle arrest was confirmed by measuring [3H]thymidine incorporation and by DNA flow cytometry. Using whole cell patch-clamp recordings, we demonstrate differential changes in GCC activity after cell proliferation and cell cycle progression was selectively altered; specifically, channel expression was low in serum-starved, G0-arrested cells, increased significantly in early G1, decreased during S phase, and increased after arrest in M phase. Although the link between the cell cycle and GCC activity is not yet clear, we speculate that GCCs are linked to the cytoskeleton and that cytoskeletal rearrangements associated with cell division lead to the observed changes in channel activity. Consistent with this hypothesis, we demonstrate the activation of GCC by disruption of F-actin using cytochalasin D or osmotic cell swelling.
1. Previous studies demonstrate that volume-sensitive chloride currents are distinctly activated in cervical cancer cells, but not in human papillomavirus (HPV)-immortalized and normal cervical cells. In the present study, the Na+-independent volume-activated transport of taurine in three cervical cell types was investigated.
2. Osmotic swelling of cervical cancer HT-3 cells suspended i. Na+-free hypotonic medium led to increased membrane uptake of taurine. This taurine uptake was effectively blocked by various C1- channel blockers with a similar potency in blocking volume-sensitive C1- channels: l,9-dideoxyforskolin > 5-nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB) > 4-acetamido-4′-isothiocyanastilbene-2,2′-disulphonic acid (SITS) > 4,4′-diisothio-cyanatostilbene-2,2-disulphonic acid (DIDS)>furosemide. The taurine influx was also abolished by pertussis toxin. In contrast, Na+-independent volume-activated taurine transport was not significantly activated in HPV-immortalized Z183A cells and in normal cervical cells.
3. Exposure of HT-3 cells to hypotonic medium also resulted in a marked increase in taurine efflux. The volume-activated taurine efflux was osmolality dependent and the pattern of pharmacological inhibition by C1- channel blockers was indistinguishable from that for taurine uptake.
4. These results suggest that volume-sensitiv. C1- channels in HT-3 cells can mediate the transport of amino acids. In addition, the pertussis toxin-sensitive G-protein is linked with the activation of this transport mechanism.
To survive, cells have to avoid excessive alterations of cell volume that jeopardize structural integrity and constancy of intracellular milieu. The function of cellular proteins seems specifically sensitive to dilution and concentration, determining the extent of macromolecular crowding. Even at constant extracellular osmolarity, volume constancy of any mammalian cell is permanently challenged by transport of osmotically active substances across the cell membrane and formation or disappearance of cellular osmolarity by metabolism. Thus cell volume constancy requires the continued operation of cell volume regulatory mechanisms, including ion transport across the cell membrane as well as accumulation or disposal of organic osmolytes and metabolites. The various cell volume regulatory mechanisms are triggered by a multitude of intracellular signaling events including alterations of cell membrane potential and of intracellular ion composition, various second messenger cascades, phosphorylation of diverse target proteins, and altered gene expression. Hormones and mediators have been shown to exploit the volume regulatory machinery to exert their effects. Thus cell volume may be considered a second message in the transmission of hormonal signals. Accordingly, alterations of cell volume and volume regulatory mechanisms participate in a wide variety of cellular functions including epithelial transport, metabolism, excitation, hormone release, migration, cell proliferation, and cell death.
Previous studies show that the regulatory volume decrease (RVD) in human cervical cells with different tumour potential may be mediated by different ion channels. The signalling events involved in regulating these channel activities are not clear. To screen the possible mechanisms involved in cell volume regulation in these cells, we examine intracellular mechanisms and second messengers listed as follows: phospholipase C (PLC), phospholipase A2 (PLA2), tyrosine kinase (TK), protein kinase C (PKC), protein kinase A (PKA), and cAMP. The involvement of G-protein was also studied. Our results showed that PLC signalling with downstream activation of PKC was involved in the cell volume regulation of cervical cancer cells. On the other hand, different PKC isoforms that were not related to upstream PLC regulation were involved in the RVD of human papillomavirus (HPV)-immortalised and normal cervical epithelia. Furthermore, GTP-gamma S facilitated the process of RVD in cervical cancer cells, while pertussis toxin retarded this process. In contrast, neither GTP-gamma S nor pertussis toxin showed effect on the RVD responses of HPV-immortalised and normal cervical cells.
1. The present study was carried out to identify the specific protein kinase C (PKC) isoform involved in regulatory volume decrease (RVD) responses, and to investigate the signal transduction pathways underlying the activation of volume-sensitive chloride channels in human cervical cancer HT-3 cells. The role of Ca2+ in RVD and in the activation of chloride currents was also studied. 2. The time course of RVDs was prolonged by microinjection of PKC-alpha antibody but not by PKC-beta or PKC-gamma antibody, and also by exposure to Ca2+-free medium, in particular when combined with microinjection of EDTA. Immunofluorescence staining showed that hypotonic superfusion evoked the translocation of PKC-alpha to the cell membrane, whereas PKC-beta or PKC-gamma remained unaffected. The translocation of PKC-alpha was observed a few minutes after hypotonic stress, reaching peak intensity at 30 min, and returned to the cytoplasm 60 min after hypotonic exposure. Western blot analyses showed an increased PKC-alpha level in terms of intensity and phosphorylation in the cell membrane, while neither PKC-beta nor PKC-gamma was activated upon hyposmotic challenge. 3. Whole-cell patch-clamp studies demonstrated that neomycin and PKC blockers such as staurosporine and H7 inhibited volume-sensitive chloride currents. The inhibitory effect of neomycin on chloride currents can be reversed by the PKC activator phorbol 12-myristate, 13-acetate (PMA). Moreover, the PKC inhibitor and PKC-alpha antibody, but not PKC-beta or PKC-gamma antibody, significantly attenuated the chloride currents. The activation of volume-sensitive chloride currents were insensitive to the changes of intracellular Ca2+ but required the presence of extracellular Ca2+. 4. Our results suggest the involvement of PKC-alpha and extracellular Ca2+ in RVD responses and the activation of volume-sensitive chloride channels in HT-3 cells.
1. An outwardly rectifying Cl- (ORCl) current of murine osteoclasts was activated by hypotonic stimulation. The current was characterized by rapid activation, little inactivation, strong outward rectification, blockage by DIDS and permeability to organic acids (pyruvate and glutamate). 2. The hypotonically activated ORCl current was inhibited by intracellular dialysis with an ATP-free pipette solution, but not by replacement of ATP with a poorly hydrolysable ATP analogue adenosine 5'-O-(3-thiotriphosphate). The current amplitude was reduced when intracellular alkalinity increased over the pH range 6.6-8.0. 3. Intracellular application of cytochalasin D occasionally activated the ORCl current without hypotonic stress, but inhibited activation of the ORCl current by hypotonic stimulation. The hypotonically activated ORCl current was unaffected by a non-actin-depolymerizing cytochalasin, chaetoglobosin C, but partially inhibited by deoxyribonuclease I. 4. Removal of extracellular Ca2+ inhibited activation of the ORCl current by hypotonic shock, but did not reduce the current once activated. The hypotonically activated ORCl current was partially decreased by intracellular dialysis with 20 mM EGTA. 5. With 10 mM Ca2+ in the extracellular medium, the ORCl current was activated in response to more minor decreases in osmolarity than with 1 mM Ca2+. The increased sensitivity to hypotonicity was mimicked by increasing the intracellular Ca2+ level (pCa 6.5). 6. These results suggest that hypotonic stimulation and a rise in the extracellular Ca2+ level synergistically activate the ORCl channel of murine osteoclasts, and that the activating process is modified by multiple intracellular factors (pH, ATP and actin cytoskeletal organization).
Whole-cell recordings were used to identify in MCF-7 human breast cancer cells the ion current(s) required for progression through G1 phase of the cell cycle. Macroscopic current-voltage curves were fitted by the sum of three currents, including linear hyperpolarized, linear depolarized and outwardly rectifying currents. Both linear currents, but not the outwardly rectifying current, were increased by 1 microm intracellular Ca(2+) and blocked by 2 mm intracellular ATP. When tested at concentrations previously shown to inhibit proliferation by 50%, linogliride, glibenclamide and quinidine inhibited the linear hyperpolarized current, and quinidine and linogliride inhibited the linear depolarized current; none of these agents affected the outwardly rectifying current. In contrast, tetraethylammonium completely inhibited the outwardly rectifying current, but did not inhibit either linear current. Changing the bath solution to symmetric K(+) shifted the reversal potential of the linear hyperpolarized current from near the K(+) equilibrium potential (-84 mV) to -4 mV. Arrest of the cell cycle in early G1 by quinidine was associated with significantly smaller linear hyperpolarized currents, without a change in the linear depolarized or outwardly rectifying currents, but this reduction was not observed with arrest by lovastatin at a site approximately 6 hr later in G1. The linear hyperpolarized current was significantly larger in ras-transformed than in untransformed cells. We conclude that the linear hyperpolarized current is an ATP-sensitive K(+) current required for progression of MCF-7 cells through G1 phase.
Arrest of spinal cord astrocytes at defined stages of the cell cycle clock causes significant changes in the expression of voltage-activated Na(+) and K(+) currents. Arrest of actively proliferating astrocytes in G1/G0 by all-trans-retinoic acid induces premature expression of inwardly rectifying K(+) currents (IK(IR)) typically expressed only in differentiated astrocytes. By contrast, arrest in S phase by ara-C or Aphidicolin leads to a greater than twofold increase in "delayed" outwardly rectifying currents (IK(D)) and a concomitant decrease in IK(IR). Pharmacological blockade of IK(D) by TEA and 4AP caused proliferating astrocytes to arrest in G0/G1, suggesting that activity of these channels is required for G1/S checkpoint progression. Conversely, in quiescent astrocytes, inhibition of IK(IR) by 30 microM BaCl(2) led to an increase in astrocyte proliferation and to an increase in the number of cells in S phase from 5% to 26%. These data suggest that a downregulation of K(IR) promotes cell cycle progression through the G1/S checkpoint. Blockade of IK(IR) in actively proliferating cells, however, leads to an accumulation in G2/M, suggesting that reappearance of this current may be critical for progression beyond DNA synthesis. Interestingly, Na(+) currents (INa(+)) are increased greater than fourfold in S phase-arrested cells, yet their pharmacological blockade by TTX has no effect on cell cycle progression. However, the resting membrane potential of S phase-arrested cells increases profoundly, and manipulation of membrane potential by the application of low concentrations of ouabain, or reduction of extracellular potassium, induces the accumulation of quiescent astrocytes in S phase of the cell cycle, suggesting that either depolarization or intracellular sodium, or both, play an important role in promoting astrocyte proliferation.
This study investigates the volume-sensitive KCI 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 beta-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 6 nM, respectively. Conversely, protein kinase inhibitors, chelerythrine and staurosporine, increased Cl- dependent 86Rb+ efflux. NEM (1 mM) 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 co-transport systems work synergistically for volume regulation in human cervical cancer cells.
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. 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 [mu]M. 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 [mu]M) arrested 88 +/- 1.4% of cells at the G0/G1 stage. 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. 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. This study provides new information on the functional significance of VRACs in the cell cycle clock of human cervical cancer cells.