Volume-sensitive KCl cotransport associated with human cervical carcinogenesis

Abstract

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.

Insulin-like Growth Factor 1 Stimulates KCl Cotransport, Which Is Necessary for Invasion and Proliferation of Cervical Cancer and Ovarian Cancer Cells

“…Our previous studies suggested that the swelling-activated KCC works in concert with ion channels to mediate regulatory volume decrease of cervical cancer cells (8,9). Prompted by the observation of substantially increased swelling-activated KCC by IGF-1, we tested the hypothesis that IGF-1 might similarly enhance cell volume regulation.…”

“…Functional K ϩ ( 86 Rb ϩ ) Efflux Assays-Unidirectional K ϩ efflux was carried out at 37°C as described in detail elsewhere (7,8). In brief, cells were preincubated with isotonic culture medium loaded with 2 Ci/ml 86 Rb ϩ for 2 h at 37°C.…”

“…For KCC4, forward (5Ј-GACT-CGTTTCCGCAAAACC-3Ј) and reverse (5Ј-AGAGTGCCGTGATGCTG-TTGG-3Ј) primers were used to amplify a 783-bp fragment from KCC4 (hKCC4: nt 2498 -3280). PCR was carried out as described in detail elsewhere (8). In brief, after heating the reaction contents for 3 min at 94°C, 30 cycles of PCR were performed (each consisting of incubation of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C).…”

“…KCC3, initially cloned from vascular endothelial cells (4), may have physiological significance in cell proliferation (7). Human cervical carcinogenesis is accompanied by increased expression of mRNA transcripts encoding KCC1, KCC3, and KCC4 (8). Furthermore, KCC is an important modulator of cervical cancer cell proliferation and invasiveness (9).…”

Differential dependence of regulatory volume decrease behavior in rabbit corneal epithelial cells on MAPK superfamily activation

“…On the other hand, the fact that the suppressions of RVD were greater in Cl − -free and high-K + solutions (53 and 44%, respectively) than those obtained with individual Cl − and K + channel inhibitors (27% and 27%, respectively), suggests the existence of a heterogeneous family of volume-sensitive ion transients mechanisms, only some of which are sensitive to NA and 4-AP (Nilius et al, 1996). KCl cotransporter (KCC) is another type of contributor to RVD, which has been described in multiple cell lines, including HCEC and RCEC (Capo-Aponte et al, 2005, 2007aErnest et al, 2005;Lauf and Adragna, 2000;Shen et al, 2000;Wehner et al, 2003). The contribution of this alternative mechanism to RVD is variable in different species, but less than that mediated by Cl − and K + channel activity.…”