APOPTOSIS IN C3H-10T1 2 CELLS - ROLES OF INTRACELLULAR PH, PROTEIN-KINASE-C, AND THE NA+/H+ ANTIPORTER/

Changes in intracellular ion concentrations have been correlated with the activation of an endogenous endonuclease and thus internucleosomal DNA cleavage during apoptosis in many cell types. We investigated whe ther intracellular pH could play a significant role in apoptotic initi ation and progression in C3H-10T1/2 cells, a cell strain that does not exhibit double-stranded DNA cleavage during apoptosis. Protein kinase C and the Na+/H+ antiporter, known regulators of intracellular pH, al so were assessed for their involvement in apoptosis of C3H-10T1/2 cell s. When a H+ ionophore was used to clamp intracellular pH to 6.0 or be low, a significant level of apoptosis was induced in these cells withi n 6 h, whereas clamping at pH 6.75 did not induce significant amounts of apoptosis until 36 h after acidification. The acidified cells exhib ited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA clea vage with electrophoresis of genomic DNA. Our results also suggest tha t the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition o f apoptosis in serum withdrawn C3H-10T1/2 cells functions through a se quential activation of protein kinase C and the Na+/H+ antiporter; thu s, an alkalinization or an inhibition of acidification is involved in this apoptotic block. Serum withdrawal itself does not appear to act t hrough a negative effect on either protein kinase C or the Na+/H+ anti porter. TPA was also capable of inhibiting the apoptosis induced by sp ecific inhibitors of protein kinase C and the Na+/H+ antiporter, but t he inhibition was successful only if the TPA was administered at least 20 min prior to the addition of the enzyme inhibitor. These results i ndicate that apoptosis in C3H-10T1/2 cells follows a pathway that invo lves intracellular acidification, but is independent of detectable end onuclease activity. (C) 1997 Wiley-Liss, Inc.