Overexpression of the Na+/K+/Cl? cotransporter gene induces cell proliferation and phenotypic transformation in mouse fibroblasts

Panet, Rivka; Marcus, Miriam; Atlan, Henri

doi:10.1002/(sici)1097-4652(200001)182:1<109::aid-jcp12>3.0.co;2-a

Cited by 72 publications

(41 citation statements)

References 38 publications

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“…Likewise, an overexpression of the NKCC in mouse fibroblasts resulted in increased cell proliferation and a transformed phenotype [7]. Thus these data provide additional corroborative evidence that the NKCC plays an important role in the regulation of cell growth and suggest that the NKCC would be a molecular target for cancer therapy.…”

Section: Discussionmentioning

confidence: 54%

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Furosemide, a Blocker of Na+/K+/2Cl− Cotransporter, Diminishes Proliferation of Poorly Differentiated Human Gastric Cancer Cells by Affecting G0/G1 State

et al. 2006

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Furosemide, a blocker of Na + /K + /2Cl -cotransporter (NKCC), is often used as a diuretic to improve edema, ascites, and pleural effusion of patients with cancers. The aim of the present study was to investigate whether an NKCC blocker affects cancer cell growth. If so, we would clarify the mechanism of this action. We found that poorly differentiated gastric adenocarcinoma cells (MKN45) expressed the mRNA of NKCC1 three times higher than moderately differentiated ones (MKN28) and that the NKCC in MKN45 showed higher activity than that in MKN28. A cell proliferation assay indicates that furosemide significantly inhibited cell growth in MKN45 cells, but not in MKN28 cells. Using flow cytometrical analysis, we found that the exposure to furosemide brought MKN45 cells to spend more time at the G 0 /G 1 phase, but not MKN28 cells. Based on these observations, we indicate that furosemide diminishes cell growth by delaying the G 1 -S phase progression in poorly differentiated gastric adenocarcinoma cells, which show high expression and activity of NKCC, but not in moderately differentiated gastric adenocarcinoma cells with low expression and NKCC activity.Key words: gastric cancer cell, Na + /K + /2Cl -cotransporter (NKCC), furosemide, cell cycle, cell proliferation.In the past two decades, many researchers have reported the characteristics of ion channels/transporters in various types of cells, including cancer cells. Several types of K + , Ca 2+ , and Cl -channels have been identified in colorectal, breast, lung, and prostate cancer cells [1,2]. Some studies indicate that the transepithelial ion transport plays an important role in cell apoptosis [3,4]. Particularly, the modulation of Cl -transport via Cl -channel, K + /Cl -cotransporter (KCC), and Na + /K + /2Cl -cotransporter (NKCC) occurs in cell proliferation [5][6][7]. However, it is unknown whether NKCC affects the proliferation of cancer cells. In the treatment of patients with terminal stage cancers, however, furosemide (an NKCC blocker, a loop diuretic) is often used as a diuretic to keep urine output and to improve edema, ascites, or pleural effusion. Therefore we performed the present study to clarify the effect of furosemide on the proliferation of cancer cells.In the present study, we used two human gastric cancer cell lines, MKN28 and MKN45, which were established from moderately and poorly differentiated adenocarcinoma, respectively [8]. We determined the mRNA and functional expression levels of the NKCC and investigated the inhibitory effect of an NKCC blocker, furosemide, on the proliferation in these two cancer cell lines. We found that mRNA and the functional expression levels of NKCC were both higher in MKN45 than in MKN28 cells. Furthermore, furosemide diminished the cell proliferation, delaying the G 1 -S phase progression in MKN45 cells, but not in MKN28 cells. MATERIALS AND METHODSCell culture. The moderately differentiated human gastric adenocarcinoma cell line, MKN28, and the poorly differentiated human gastric adenocarcinoma cel...

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Section: Discussionmentioning

confidence: 54%

“…Some studies indicate that the transepithelial ion transport plays an important role in cell apoptosis [3,4]. Particularly, the modulation of Cl -transport via Cl -channel, K + /Cl -cotransporter (KCC), and Na + /K + /2Cl -cotransporter (NKCC) occurs in cell proliferation [5][6][7]. However, it is unknown whether NKCC affects the proliferation of cancer cells.…”

mentioning

confidence: 99%

Furosemide, a Blocker of Na+/K+/2Cl− Cotransporter, Diminishes Proliferation of Poorly Differentiated Human Gastric Cancer Cells by Affecting G0/G1 State

et al. 2006

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“…Similar results for both cell types were obtained by using ␣-wNT and TEF-S2 antibodies (data not shown). Scale bar, 20 M. (6,24,48,72, and 96 h) after viral exposure (H) were applied to the SDS-PAGE. The biotinylated subfractions from each treatment regimen were extracted by using the defined volume protocol (see EXPERIMENTAL PROCEDURES for details).…”

Section: Nkcc Is Predominantly An Intracellular Protein In Uninfectedmentioning

confidence: 99%

“…There are reports that mitogens stimulate functional NKCC activity (e.g., Refs. 8,47) and that overexpression of the NKCC protein will stimulate cell proliferation (48). In light of these findings, it is very interesting to note that HCMV infection stimulates the host cells to enter the cell cycle, but causes the cycle to halt at the G 1 /S interface (e.g., Refs.…”

Section: Hcmv Infection Of Mrc-5 Cells Leads To a Perinuclear Accumulmentioning

confidence: 99%

Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection

Maglova¹,

Crowe²,

Russell³

2004

American Journal of Physiology-Cell Physiology

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previously reported that Na-K-Cl-cotransporter (NKCC) function and microsomal protein expression are both dramatically reduced late in human cytomegalovirus (HCMV) infection of a human fibroblast cell line (MRC-5). We now report DNA microarray data showing that no significant HCMV-dependent NKCC gene repression can be detected 30 h postexposure (PE) to the virus. Consequently, we used plasma membrane biotinylation and subsequent subcellular fractionation in combination with semiquantitative immunoblotting and confocal microscopy to investigate the possibility that intracellular redistribution of the NKCC protein after HCMV infection could be a cause of the HCMV-induced loss of NKCC ion transport function. Our results show that the lifetime of plasmalemmal NKCC protein in quiescent, uninfected MRC-5 cells is ϳ48 h, and Ͻ20% of the total expressed NKCC protein are in the plasma membrane. The remainder (ϳ80%) was detected as diffusely distributed, small punctate structures in the cytoplasm. Following HCMV infection: 1) NKCC protein expression in the plasmalemma was sharply reduced (ϳ75%) within 24 h PE and thereafter continued to slowly decrease; 2) total cellular NKCC protein content remained unchanged or slightly increased during the course of the viral infection; and 3) HCMV infection caused NKCC protein to accumulate in the perinuclear region late in the HCMV infection (72 h PE). Thus our results imply that, in the process of productive HCMV infection, NKCC protein continues to be synthesized, but, instead of being delivered to the plasma membrane, it is clustered in a large, detergentsoluble perinuclear structure.sodium-potassium-chloride-cotransporter; human fibroblast cell line; perinuclear accumulation

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“…This association was described in human skin fibroblasts, in which stimulation of bumetanide-sensitive NKCC by different mitogens was shown to be essential for cell proliferation (Panet and Atlan, 1991), and the NKCC inhibitors-bumetanide and furosemide-were shown to inhibit cell proliferation (Panet et al, 1994). Additional evidence for the dependence of proliferation on amount of NKCC1 comes from the finding that gene overexpression augments cell proliferation in human skin fibroblasts (Panet et al, 2000). In the current study, however, the association between tonicity-induced increases in NKCC1 activity and proliferation is species-specific.…”

Section: Discussionmentioning

confidence: 85%

Fate of hypertonicity-stressed corneal epithelial cells depends on differential MAPK activation and p38MAPK/Na–K–2Cl cotransporter1 interaction

Capó-Aponte

Wang

Bildin

et al. 2007

Experimental Eye Research

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The capacity of the corneal epithelium to adapt to hypertonic challenge is dependent on the ability of the cells to upregulate the expression and activity of cell membrane-associated Na-K-2Cl cotransporter1 (NKCC1). Yet, the signaling pathways that control this response during hypertonic stress are still unclear. We studied stress-induced changes in proliferation and survival capacity of SV40-immortalized human (HCEC) and rabbit (RCEC) corneal epithelial cells as a function of (i) the magnitude of the hypertonic challenge, (ii) differential changes in activation of mitogen-activated protein kinase (MAPK), and (iii) the extent of p38MAPK interaction with NKCC1. Cells were incubated in hypertonic (up to 600 mOsm) media for varying time periods up to 24 h. Phosphorylated forms of p44/42, p38, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) MAPK were immunoprecipitated from cell lysates, and the amount of each activated NKCC1-associated MAPK was evaluated by Western blot/ECL assay. DNA integrity was assessed by electrophoresis in a 2% agarose gel. Cell survival and proliferation were evaluated based on three criteria: protein content, cell count, and the MTT assay. Exposure to media of 325-350 mOsm increased proliferation of HCEC up to 75%, whereas this response was limited to <16% in RCEC. At higher osmolarities, cell proliferation decreased in both species. SAPK/JNK activity increased 150-fold in HCEC and <10-fold in RCEC, while DNA fragmentation occurred only in HCEC. Compared to HCEC, the better RCEC survival rate was associated with higher p38MAPK activity and near complete restoration of p44/42MAPK activity after the first 30 min. In both cell lines, the amount of phospho-NKCC1 that coimmunoprecipitated with phospho-p38MAPK was proportional to the magnitudes of their respective activation levels. However, no such associations occurred between amounts of phosphorylated p44/42MAPK or SAPK/JNK and phospho-NKCC1. Under isotonic conditions, with bumetanide-induced inhibition of RCEC and HCEC NKCC1 activities, p44/42MAPK activity declined by 40 and 60%, respectively. Such declines led to proportional decreases in cell proliferation. Survival of hypertonicity-stressed corneal epithelial cells depends both on p38MAPK activation capacity and the ability of p38MAPK to stimulate NKCC1 activity through protein-protein interaction. The level of NKCC1 activation affects the extent of cell volume recovery and, in turn, epithelial survival capacity.

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Overexpression of the Na+/K+/Cl? cotransporter gene induces cell proliferation and phenotypic transformation in mouse fibroblasts

Cited by 72 publications

References 38 publications

Furosemide, a Blocker of Na+/K+/2Cl− Cotransporter, Diminishes Proliferation of Poorly Differentiated Human Gastric Cancer Cells by Affecting G0/G1 State

Furosemide, a Blocker of Na+/K+/2Cl− Cotransporter, Diminishes Proliferation of Poorly Differentiated Human Gastric Cancer Cells by Affecting G0/G1 State

Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection

Fate of hypertonicity-stressed corneal epithelial cells depends on differential MAPK activation and p38MAPK/Na–K–2Cl cotransporter1 interaction

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