Effect of intracellular acidity and ionomycin on apoptosis in HL-60 cells

The aim was to investigate in detail the influence of intracellular pH (pH(i)) and intracellular Ca2+ concentration ([Ca2+](i)) on apoptosis in HL-60 human promyelocytic leukaemia cells. The pH(i) was controlled by changing the pH of media as well as by interfering with the pH(i) regulatory mechanisms with 3-amino-6-chloro-5-(1-homopiperidyl)-N-(diaminomethylene) pyrazincarboxamide.(HMA; an inhibitor of Na+/H+ antiport), 4-diiosothiocyanatostilbene-2,2'disulfonic acid, (DIDS; an inhibitor of Na+-dependent HCO3-/Cl- exchange) and nigericin (a K+ ionophore). The [Ca2+](i) was increased with ionomycin, a Ca2+ ionophore. The apoptosis of HL-60 cells was measured with conventional agarose gel electrophoresis for DNA fragmentation and also with the release of H-3 from H-3-thymidine-labelled DNA. Based on the magnitude of DNA fragmentation and H-3 release at different pH(i), it was shown that apoptosis occurred in HL-60 cells when the pH(i) was lowered from normal pH, of 7.4 to about 7.2-6.7 with a peak increase at pH(i) 6.8-6.9. Addition of 4 mu M ionomycin to RPMI 1640 medium, which contained 615 mu M Ca2+, elevated the apoptosis in the cells. Such an increase in apoptosis by ionomycin in HL-60 cells appeared to result from both an increase in [Ca2+](i) and from a decline in pH(i). The results indicate that the acidic intratumour environment may greatly affect the response of neoplastic tissues to hyperthermia, radiation and chemotherapeutic drugs which cause apoptosis.