Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease.
Protein isoprenylation is a post-translational modification essential for the biological activity of G-proteins. Inhibition of protein isoprenylation by lovastatin (LOV) induces apoptosis in HL-60 cells, a process of active cell death characterized by the internucleosomal degradation of genomic DNA. In this article we show that LOV-induced apoptosis is associated with intracellular acidification and that activation of the Na+/H+ antiporter induces a raise in pHi which is sufficient to prevent or arrest DNA digestion. First, LOV induced a decrease in pHi which was dose-dependent and correlated with the extent of DNA degradation. Flow cytometry analysis revealed that this acidification was due to the appearance of a subpopulation of cells whose pHi was 0.9 pH units below control values. Cell sorting experiments demonstrated that DNA degradation had occurred only in those cells which had suffered intracellular acidification. LOV-induced apoptosis could be suppressed by mevalonate supplementation, inhibition of protein synthesis, and protein kinase C activation by phorbol myristate acetate. In all three cases, intracellular acidification was abolished. Inhibition of the Na+/H+ antiporter by 5-N-ethyl-N-isopropyl amiloride induced DNA degradation in HL-60 cells per se and suppressed the protective effect of phorbol myristate acetate. LOV-induced intracellular acidification was not due to a complete inhibition of the Na+/H+ antiporter. In fact, LOV-treated cells were able to respond to phorbol myristate acetate stimulation of the Na+/H+ antiporter with a marked increase in pHi. This effect was accompanied by a rapid arrest of DNA digestion. These observations illustrate the strong pH dependence of LOV-induced DNA degradation, thus providing a connection between the activation of the Na+/H+ antiporter and the suppression of apoptosis.
Pérez-Sala
D
D
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientifícas, Madrid, Spain.
Collado-Escobar
D
D
Mollinedo
F
F
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002453:Cell Cycle / Q000187:drug effects*; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008148:Lovastatin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008798:Mevalonic Acid / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007546:Isoquinolines; D010879:Piperazines; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D003513:Cycloheximide; D008148:Lovastatin; D011493:Protein Kinase C; D004706:Endodeoxyribonucleases; D013755:Tetradecanoylphorbol Acetate; D008798:Mevalonic Acid; C039614:ethylisopropylamiloride