Overexpression of the Na(+)/K(+)/Cl(-) cotransporter gene induces cell proliferation and phenotypic transformation in mouse fibroblasts.

Na(+)/K(+)/Cl(-) cotransporter activity is stimulated in early G(1) phase of the cell cycle and this stimulation was shown to be an essential event in fibroblast cell proliferation. In order to elucidate further the role of the Na(+)/K(+)/Cl(-) cotransporter in cell proliferation, we overexpressed the gene encoding the Na(+)/K(+)/Cl(-) cotransporter in mouse fibroblasts, and analyzed cellular phenotypic changes. Mouse Balb/c 3T3 cells were stably transfected with the cDNA of the shark rectal gland Na(+)/K(+)/Cl(-) cotransporter gene (NKCC1), and expressed in a mammalian vector under the cytomegalovirus promoter (Balb/c-NKCC1 cells). The transfected cells exhibited up to 10-fold greater bumetanide-sensitive Rb(+) influx compared to the control cells. The Balb/c-NKCC1 cells have acquired a typical transformation phenotype indicated by: (1) Loss of contact inhibition exhibited by growth to a higher cell density in confluent cultures, and formation of cell foci; (2) proliferation in low serum concentrations; and (3) formation of cell colonies in soft agar. The control cells transfected with the NKCC1 gene inserted in the opposite orientation in the vector retained their normal phenotype. Furthermore, the two specific inhibitors of the Na(+)/K(+)/Cl(-) cotransporter activity; bumetanide and furosemide inhibited the clonogenic efficiency in the NKCC1 transfected cells. These control experiments indicate that the apparent transformation phenotype acquired by the Balb/c-NKCC1 cells was not merely associated with the process of transfection and selecting for the neomycin-resistant clones, but rather with the overexpression of the Na(+)/K(+)/Cl(-) cotransporter gene. In order to ascertain that the regulated and normal expression of the Na(+)/K(+)/Cl(-) cotransporter control cell proliferation, the effect of bumetanide a specific inhibitor of the cotransporter, was tested on Balb/c 3T3 cell proliferation, induced by fibroblasts growth factor (FGF) and fetal calf serum (FCS). Bumetanide inhibited synchronized Balb/c 3T3 cell exit from the G(0)/G(1) arrest and entering S-phase. The inhibition was reversible, as removal of bumetanide completely released cell proliferation. Taken together, these results propose that the NKCC1 gene is involved in the control of normal cell proliferation, while its overexpression results in apparent cell transformation, in a manner similar to some protooncogenes. Copyright 2000 Wiley-Liss, Inc.