Antimycin A-induced apoptosis of HL-60 cells.
BACKGROUND
Previous experiments in our laboratory investigating apoptosis induced in HL-60 cells by camptothecin (CAM) have revealed that the sequence and rapidity of the apoptotic phenomena in an individual cell depend on the proliferative state of that cell when it encounters CAM. The role of mitochondria in HL-60 apoptosis was explored using an inhibitor of oxidative phosphorylation, antimycin A (AMA).


METHODS
Changes in cell light scatter, binding of annexin V-fluorescein isothiocyanate (FITC), uptake of propidium iodide (PI), and DNA content after membrane fixation/permeabilization were monitored by flow cytometry. Z-VAD-FMK was used to inhibit caspases. Fluorescence microscopy was used to examine cell morphology.


RESULTS
Cells in the G1 phase of the cell cycle were the first to exhibit signs of apoptosis in response to 100 microM AMA and some of these cells disintegrated without exposing to phosphatidylserine (PS). Caspase inhibition prevented fragmentation of DNA, the nucleus, and the cell, but only delayed PS exposure and loss of plasma membrane integrity.


CONCLUSIONS
The highly active mitochondria of G1-phase HL-60 cells make them particularly sensitive to AMA. PS exposure and plasma membrane damage are mediated by noncaspase molecules released from mitochondria. We hypothesize that if mitochondria are subjected to a sufficiently severe insult, whether indirectly as a result of extensive CAM-induced DNA damage or directly by the effect of AMA on electron transport, the nature and quantities of the proapoptotic molecules released are such that apoptosis proceeds to the point of cell disintegration before the PS exposure pathway is complete.
King
Malcolm A
MA
Department of Clinical Immunology, Pacific Laboratory Medicine Services, Royal North Shore Hospital, St. Leonards, Australia. making@doh.health.nsw.gov.au
Radicchi-Mastroianni
Monica A
MA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017304:Annexin A5 / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000968:Antimycin A / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002166:Camptothecin / Q000494:pharmacology; D061945:Caspase Inhibitors; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects; D018922:HL-60 Cells / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D010718:Phosphatidylserines / Q000494:pharmacology; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D017304:Annexin A5; D000900:Anti-Bacterial Agents; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004273:DNA, Neoplasm; D010718:Phosphatidylserines; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D000968:Antimycin A; D002166:Camptothecin