Monitoring of premitotic and postmitotic apoptosis in gamma-irradiated HL-60 cells by the mitochondrial membrane protein-specific monoclonal antibody APO2.7.
BACKGROUND
Majority of hematopoietic cells die by apoptosis after irradiation with ionizing radiation. In present study it is shown that human promyelocytic leukemia HL-60 cells can undergo two different types of apoptosis, premitotic and postmitotic.


METHODS
HL-60 cells were irradiated with doses 8 and 20 Gy. For apoptosis detection APO2.7 antigen (mitochondrial membrane specific protein) expression without and with permeabilization by digitonin was used. This method was compared with flow-cytometric analysis of cell light scattering properties and determination of subG1 DNA.


RESULTS
Cells irradiated with high dose (20 Gy) died rapidly by premitotic apoptosis (interphase death) from all phases of cell cycle. 2 hours after irradiation cells with subdiploid DNA content and cells stained by APO2.7 after digitonin permeabilization appeared. After 6 hours 40% of cells were apoptotic, nonapoptotic cells were mainly in G1-phase. Lower dose (8 Gy) after 6 hours of irradiation caused accumulation of cells in S-phase. After 24 hours majority of cells was in G2-phase and apoptotic cells appeared (subG1 peak, APO2.7 with permeabilization).


CONCLUSIONS
Data presented herein indicate that mitochondrial membrane protein-specific antibody APO2.7 after permeabilization is a useful marker for detection of early apoptotic cells dying by premitotic and postmitotic apoptosis.
Mareková
M
M
Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic. marekova@lfhk.cuni.cz
Vávrová
J
J
Vokurková
D
D
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects*; D015415:Biomarkers / Q000032:analysis; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry / Q000379:methods*; D005720:Gamma Rays; D018922:HL-60 Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D033681:Mitochondrial Membrane Transport Proteins / Q000276:immunology / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology* / Q000528:radiation effects*
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D033681:Mitochondrial Membrane Transport Proteins