Km. Boyle et al., APOPTOSIS IN C3H-10T1 2 CELLS - ROLES OF INTRACELLULAR PH, PROTEIN-KINASE-C, AND THE NA+/H+ ANTIPORTER/, Journal of cellular biochemistry, 67(2), 1997, pp. 231-240
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to serum withdrawn cells, but failed to show internucleosomal DNA clea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f apoptosis in serum withdrawn C3H-10T1/2 cells functions through a se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.