Boyle K M, Irwin J P, Humes B R, Runge S W
Department of Biology, University of Central Arkansas, Conway 72035, USA.
J Cell Biochem. 1997 Nov 1;67(2):231-40.
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 The acidified cells exhibited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA cleavage with electrophoresis of genomic DNA. Our results also suggest that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition of apoptosis in serum withdrawn C3H-10T1/2 cells functions through a sequential activation of protein kinase C and the Na+/H+ antiporter; thus, an alkalinization or an inhibition of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.
在许多细胞类型的凋亡过程中，细胞内离子浓度的变化与内源性核酸内切酶的激活相关，进而与核小体间DNA裂解相关。我们研究了细胞内pH值是否在C3H-10T1/2细胞（一种在凋亡过程中不表现出双链DNA裂解的细胞系）的凋亡起始和进展中发挥重要作用。还评估了已知的细胞内pH调节剂蛋白激酶C和Na+/H+反向转运蛋白在C3H-10T1/2细胞凋亡中的作用。当使用H+离子载体将细胞内pH值钳制在6.0或更低时，这些细胞在6小时内诱导出显著水平的凋亡，而将pH值钳制在6.75时，直到酸化后36小时才诱导出大量凋亡。酸化的细胞呈现出典型的凋亡形态和染色质浓缩，类似于血清饥饿的细胞，但在基因组DNA电泳中未显示出核小体间DNA裂解。我们的结果还表明，12-氧-十四烷酰佛波醇-13-乙酸酯（TPA）介导的对血清饥饿的C3H-10T1/2细胞凋亡的抑制作用是通过蛋白激酶C和Na+/H+反向转运蛋白的顺序激活发挥作用的；因此，碱化或酸化抑制参与了这种凋亡阻滞。血清饥饿本身似乎不是通过对蛋白激酶C或Na+/H+反向转运蛋白的负面影响起作用。TPA也能够抑制蛋白激酶C和Na+/H+反向转运蛋白的特异性抑制剂诱导的凋亡，但只有在添加酶抑制剂之前至少20分钟给予TPA，抑制才会成功。这些结果表明，C3H-10T1/
