Apoptosis in C3H-10T1/2 cells: Roles of intracellular pH, protein kinase C, and the Na+/H+ antiporter

Abstract

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 whether intracellular pH could play a significant role in apoptotic initiation 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, also were assessed for their involvement in apoptosis of C3H-10T1/2 cells. When a H+ ionophore was used to clamp intracellular pH to 6.0 or below, a significant level of apoptosis was induced in these cells within 6 h, whereas clamping at pH 6.75 did not induce significant amounts of apoptosis until 36 h after acidification. The acidified cells exhibited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA cleavage with electrophoresis of genomic DNA. Our results also suggest that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition of apoptosis in serum withdrawn C3H-10T1/2 cells functions through a sequential activation of protein kinase C and the Na+/H+ antiporter; thus, an alkalinization or an inhibition of acidification is involved in this apoptotic block. Serum withdrawal itself does not appear to act through a negative effect on either protein kinase C or the Na+/H+ antiporter. TPA was also capable of inhibiting the apoptosis induced by specific inhibitors of protein kinase C and the Na+/H+ antiporter, but the inhibition was successful only if the TPA was administered at least 20 min prior to the addition of the enzyme inhibitor. These results indicate that apoptosis in C3H-10T1/2 cells follows a pathway that involves intracellular acidification, but is independent of detectable endonuclease activity.

Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide

“…These mechanisms are likely to be significant in vivo as well because intracellular acidification is known to occur during apoptosis (19)(20)(21)(22). Significant levels of apoptosis also have been observed by using a proton ionophore under conditions in which intracellular pH is clamped to 6.0 or below, but not to 6.75 (23), consistent with the pH ranges necessary for procaspase-3 autoactivation. Hyperacidification of the mitochondrial intermembrane space, where a substantial portion of the procaspase-3 pool is contained in some cell types (24), also has been demonstrated during apoptosis (25).…”

Apoptosis induced by Na+/H+ antiport inhibition activates the LEI/L-DNase II pathway

“…43,44 To confirm such induction in BHK cells, we exposed these cells to 40 mM HMA for different periods of time 45 and estimated, 24 h after the start of treatment, the cell survival ratio by using a metabolic index provided by the MTT test. In these conditions, the survival rate significantly decreased with increases in exposure time and reached a mean of 6.872% at 24 h of HMA treatment (Figure 2a).…”

Tenidap, an Anti-Inflammatory Agent, Inhibits DNA and Collagen Syntheses, Depresses Cell Proliferation, and Lowers Intracellular pH in Cultured Human Gingival Fibroblasts

“…We have also demonstrated here that 20 M tenidap decreased pH i by 0.12 pH unit. Boyle et al (1997) reported the clamping intracellular pH 6.75 did not induce a significant level of apoptosis of C3H-10T1/2 cells, which pH 6.0 did induce in these cells. Thus, the continuous retention of tenidap in local areas, such as in periodontal pockets, might affect gingival fibroblasts to reduce its growth through apoptosis.…”