ETOPOSIDE-INDUCED APOPTOSIS IN HUMAN HL-60 CELLS IS ASSOCIATED WITH INTRACELLULAR ACIDIFICATION

Apoptosis is a pathway of cell death characterized by internucleosomal digestion of genomic DNA. Such DNA digestion can be induced by both p hysiological stimuli and cytotoxic treatment with many anticancer agen ts. This digestion has generally been considered to be mediated by a C a2+/Mg2+-dependent endonuclease that is activated by increases in intr acellular Ca2+. However. we suggest that an alternate endonuclease, DN ase II, may be a more likely candidate. In these studies, apoptosis wa s induced in human HL-60 cells by a 30-min incubation with the topoiso merase II inhibitor etoposide. DNA digestion characteristic of apoptos is began within 3 h of removal of etoposide. Morphological indication of apoptosis was observed concurrently. Only about 20% of the cells un derwent apoptosis at this time; these appeared to be cells in S phase at the time of etoposide treatment. The remainder of the cells progres sed to the G2 phase and arrested there for at least 48 h. Intracellula r Ca2+ and pH were measured in individual cells by flow cytometry. No changes in intracellular Ca2+ were observed, but an acidification of u p to 1 pH unit occurred in about 15% of the cells and correlated with the time course of appearance of DNA digestion. Cells were sorted on t he basis of intracellular pH and only the acidic cells showed the morp hology and DNA digestion characteristic of apoptosis. These results de monstrate the involvement of DNase II in apoptotic DNA digestion and s uggest mechanisms of pH homeostasis as regulators of apoptosis.