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.