Correlation of ouabain-sensitive ion movements with cell-cycle activation

The role of tumor-promoter-induced Na+, K+-ATPase activity in cell proliferation and the extent of coupling of Na+ and K+ movements to cell-cycle control under differing physiological states was examined. Earlier studies indicated that staging of cells in G1 by serum deprivation in the presence of phorbol esters such as phorbol 12-myristate 13-acetate (PMA) induced a state(s) in which postconfluent C3H 10T1/2 fibroblasts were refractory to ouabain inhibition of DNA synthesis, and the present study further examines this property. Previous findings suggested that the promoter can act in either of two ways: (i) it can act by inducing an alternative pathway to S-phase independent of Na+,K+-ATPase activity, or (ii) the promoter can advance the cells in G1 to a point beyond which Na+, K+-ATPase activity is no longer required for the induction of DNA synthesis. When ouabain (0.3 mM) was added simultaneously with tumor promoters, such as dihydroteleocidin B, to cells arrested in the G1 phase, [3H]thymidine incorporation was inhibited greater than 90%. These data suggest that an alternative pathway is not likely the explanation but that tumor promoters advance cells through a dynamic state in G1, during which progression toward S-phase entry is independent of a Na+,K+-ATPase dependent regulatory step. Ouabain sensitivity kinetics measured by two independent methods indicated that the development of ouabain insensitivity is found in G1 approximately equal to 2 hr prior to S phase. This study indicates that the measured ion movements are markedly dependent on cell-cycle state and describes the criteria required to obtain reproducible responses.