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

Abstract

The aim was to investigate in detail the influence of intracellular pH (pH i) and intracellular Ca 2+ concentration ([Ca 2+] 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 HCO 3−/C1 − exchange) and nigericin (a K + ionophore). The [Ca 2+] i was increased with ionomycin, a Ca 2+ ionophore. The apoptosis of HL-60 cells was measured with conventional agarose gel electrophoresis for DNA fragmentation and also with the release of 3H from 3H-thymidine-labelled DNA. Based on the magnitude of DNA fragmentation and 3H release at different pH i it was shown that apoptosis occurred in HL-60 cells when the pH i was lowered from normal pH i of 7.4 to about 7.2-6.7 with a peak increase at pH i 6.8-6.9. Addition of 4 μM ionomycin to RPMI 1640 medium, which contained 615 μM Ca 2+, 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 [Ca 2+] 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.