Eag1 and Bestrophin 1 Are Up-regulated in Fast-growing Colonic Cancer Cells

Abstract

An increasing number of studies demonstrate proliferative effects of membrane ion channels (1). Voltage-gated K ϩ channels and other types of K ϩ channels are expressed in numerous types of tumors, where they may serve as diagnostic and prognostic markers and potential drug targets (2-4). Eag1 channels are probably necessary for progression through the G 1 phase and G 0 /G 1 transition of the cell cycle (5). A recent study demonstrates the hyperpolarizing effects of Eag1 and other Kv channels on the membrane voltage of T 84 cells, which supports intracellular pH regulation and Ca 2ϩ increase necessary for proliferation (6).Much less is known about the role of Ca 2ϩ -activated Cl Ϫ channels in cell proliferation (7). This may be due to the ongoing controversy regarding the molecular nature of Ca 2ϩ -activated Cl Ϫ channels (8). A family of putative Ca 2ϩ -activated Cl Ϫ channels has been identified that also controls cell-cell adhesion, apoptosis, and the cell cycle. However, the structure and biophysical properties of these channels are poorly understood (9,10 In the present report we demonstrate that both voltage-gated Eag1 K ϩ channels and bestrophin 1 (Best1) Cl Ϫ channels support proliferation of fast-growing T 84 colonic carcinoma cells. The fast-growing T 84 cell clone was obtained through spontaneous transformation of slow-growing T 84 cells. In contrast to slow-growing T 84 cells, transformed cells do not form polarized monolayers and show a remarkable up-regulation of Eag1 and Best1 expression. We demonstrate that both currents are in charge of enhanced cell proliferation. MATERIALS AND METHODSCell Culture and Proliferation Studies-Human colorectal carcinoma epithelial T 84 cells (ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen, Karlsruhe, Germany) at 5% CO 2 and 37°C. Cells were seeded on fibronectin (Invitrogen)/collagen (Cellon)-coated glass coverslips or permeable supports (Snapwell, Costar). Typically these cells grow slowly as polarized monolayers (T 84 -slow). Because of spontaneous transformation, a T 84 cell line was selected that grew remarkably faster (T 84 -fast). For proliferation assays, cells were plated at a density of 2000 cells/ 0.35 cm 2 and incubated 2 days later with either niflumic acid (0.01-100 mM) or astemizole (0.5-5000 nM). Cell proliferation was assessed by 5-bromo-2Ј-deoxyuridine (BrdUrd) 3 incorporation using an enzyme-linked immunosorbent assay kit (Roche Diagnostics, Penzberg, Germany) and cell counting. The cell number was assessed after fixation in 3.7% formaldehyde and 0.5% Triton X-100 for 30 min at room temperature and after staining with Mayer's hemalaun (Merck, Darmstadt, Germany) for 5 min. Digitized microscopic images were taken (Fluovert FS, Leitz), and nuclei were counted using imaging software (TINA version 2.09g). Toxicity of the blockers was * This work was supported by Deutsche Forschungsgemei...

Role of anoctamins in cancer and apoptosis

“…These results suggest that very effective suppression of Ano1 expression is necessary to eliminate the pro-proliferative effects of Ano1, suggesting that low expression of Ano1 is sufficient to induce this effect. While siRNA had no effect in HT 29 cells, 1 mM staurosporine (figure 1a black arrow, dashed lines) inhibited proliferation and induced cell apoptosis in colonic carcinoma cells, as demonstrated in an earlier publication [28]. Thus, enhanced expression of Ano1 does not seem to enhance proliferation in every cell type [27,29].…”

“…This inverse correlation between low Ano1 levels and upregulation of mTOR [51] suggests that Ano1 may be inhibitory on proliferation of mouse intestinal epithelial cells, similar to HT 29 cells. Interestingly, a fast growing subclone of T 84 colonic epithelial cells (T 84 fast) is lacking expression of Ano1, when compared with the slowly growing parental cells (T 84 slow) [28] ( figure 5a,b). Notably, treatment of fast growing T 84 cells with the mTOR-inhibitor rapamycin reduced proliferation and induced expression of Ano1 ( figure 5c,d).…”

Rapamycin inhibits oncogenic intestinal ion channels and neoplasia in APCMin/+ mice

“…From another mechanistic viewpoint, we have previously shown that the development of intestinal cancer is characterized by the expression of oncogenic K þ ion channels, such as voltage-gated K þ channels of the ether-a-go-go (Eag) family and large conductance Ca 2 þ -activated K þ channels Spitzner et al, 2007Spitzner et al, , 2008. Ion channels have been found to support the proliferation and development of cancer cells (Kunzelmann, 2005).…”

“…It has been reported that APC Min/ þ mice show upregulated phosphoinositide-3 kinase-Akt signaling in intestinal tumors (Moran et al, 2004;Spitzner et al, 2008), which is a main route of triggering mTOR. Besides this quite direct involvement of mTOR in tumor formation in the gut, APC mutations have also been related to alterations in Wnt-Akt-b-catenin signaling, which is commonly dysregulated in colorectal cancers (Fodde et al, 2001;Dihlmann et al, 2005).…”

Proteomic Analysis of Potential Breast Cancer Biomarkers

“…Nevertheless, the related study in bestrophin-1 shows the protein improves intracellular Ca 2+ signaling and increases cell growth rate in colonic carcinoma cells. The proliferation of the cells is significantly suppressed by bestrophin-1 RNA interference treatment (Spitzner et al, 2008). This indicates bestrophin-3 may be a potential target for breast cancer therapy.…”