Activation of a low pH-dependent nuclease by apoptotic agents

Abstract

Intracellular acidification caused by agents such as UV(C), etoposide or ceramide accompanies the progression of apoptosis. It is suggested that cellular acidosis may set favorable conditions for a dormant, low pH-dependent (acidic) nuclease, which could be involved in intranucleosomal genome degradation, a hallmark of programmed cell death. Here we show that exposure of HL-60 cells to acidotic/ apoptotic agents results in the several-fold activation of a novel low pH-dependent (acidic) nuclease activity, as revealed by zymography. Its activity, which resides in nuclei, is associated with four polypeptides with apparent M r of 56, 48, 45 and 40 kDa. Treatment of HeLa cells with UV(C) or ceramide causes also the up-regulation of an acidic nuclease activity which is represented by 70 and 62 kDa polypeptides. These observations suggest that acidic nuclease activation can be induced by the same apoptotic agents in different cell types. In HL-60 cells, acidic nuclease up-regulation triggered by acidotic agents follows the induction of AP-1 transcription factor active complexes and accompanies the progression of apoptosis. Inhibition of AP-1 factor activity caused by either anti-caspase/anti-acidotic agent Zn 2+ or curcumin, an inhibitor of AP-1 binding to DNA and c-jun synthesis, protects cells from genome destruction. Acidic nuclease activation, however, is only partially inhibited by these factors. We propose that (i) the up-regulation of an acidic nuclease activity is governed by a regulatory pathway different from that responsible for AP-1 factor induction, caspases activation and intracellular acidification, and (ii) activation of an acidic nuclease does not cause any deleterious effects when AP-1 transcription factor induction, caspases activation and intracellular acidification are down-regulated. Thus, the acidic nuclease up-regulation alone is not a sufficient prerequisite for apoptosis.

Nuclear Translocation of DNase II and Acid Phosphatase during Radiation-induced Apoptosis in HL60 Cells

“…Zymography assays demonstrated that the 32-kDa protein is likely to be the nuclease responsible for generation of TUNEL-positive cells after g-irradiation. These results resemble those in UV(C)-irradiation of HL60 cells (17). However, when HL60 cells were irradiated with a high dose of g-rays, and/or incubated after irradiation, there was a tendency for DNase II and acid phosphatase in the nuclear extract to decrease.…”

“…Zymography assays were done for endonuclease activity (17). Twelve percent of the polyacrylamide gels were copolymerized with calf thymus DNA (0.2 mg/ml) and stored overnight at 48C and 20 ml nuclear extracts were mixed with equal volumes of the sample buffer and separated at 100 mV for 90 min.…”

“…Recently, it has been shown that acidic nuclease, detected in HL60 cells upon activation by UV irradiation, is represented by four polypeptides of apparent molecular weights 56-, 48-, 45-, and 40-kDa (17). TUNEL assay was sensitive in detecting very early signs of apoptosis in HL60 cells at the various UV irradiation time periods tested (18).…”

Alterations of intracellular pH homeostasis in apoptosis: origins and roles

“…40 Regarding the nuclear amount of acidic endonucleases, it is important to stress that the activity of DNase II-like 29-31 kDa polypeptides identified in fibroblasts and also that of low-pHdependent nucleases unrelated to DNase II in HL60 and HeLa cells have been shown to be upregulated upon exposure to diverse acidotic/apoptotic stimuli such as anoxia, UV or etoposide. 37,38 Nonetheless, since this upregulation was not sufficient to cause any deleterious effects when caspase activation and intracellular acidification were downregulated, Famulski et al 38 postulated that the acidic nuclease upregulation alone was not a sufficient prerequisite for the execution of apoptosis. In this context, other cellular targets for the apoptosis-related cytosolic acidification should be sought ( Figure 5).…”

Acidification induces Bax translocation to the mitochondria and promotes ultraviolet light-induced apoptosis

“…Although the importance of cytosol acidification is still controversial, mounting evidence suggests that cytosol acidification at least contributes to cytochrome c release, caspase and endonuclease activation, and apoptosis in some cell systems. Reduction of the cytosolic pH (intracellular acidification) due to proton channel inhibition can induce apoptosis, and conversely, prevention of cytosol acidification can block apoptosis [12,[17][18][19][20][21]. However, it remains unclear how cytosol acidification facilitates apoptosis.…”