Gamma irradiation of human leukaemic cells HL-60 and MOLT-4 induces decrease in Mcl-1 and Bid, release of cytochrome c, and activation of caspase-8 and caspase-9.
OBJECTIVE
Apoptosis is significantly controlled by proteins of Bcl-2 (B-cell lymphoma 2) family promoting cell death or maintaining cell survival. We selected two representatives of Bcl-2 family (anti-apoptotic Mcl-1 - myeloid cell line-1 and pro-apoptotic Bid - Bcl-2 homology domain 3 interacting death agonist), cytochrome c (cyt-c), and two initial caspases (-8 and -9) to evaluate their function in ionizing radiation (IR)-induced apoptosis in human leukaemic cell lines diverging in p53 (TP53 tumor suppressor gene) status.


METHODS
A total of 30 microg of proteins of whole-cell lysates or 10 microg of mitochondrial protein fractions were electrophoretically separated and analyzed by Western-blotting.


RESULTS
Here we show that in both HL-60 (p53 null) and MOLT-4 (p53 wild type) leukaemic cells the amount of Mcl-1 initially increased after irradiation by sublethal but not by lethal dose and later (when apoptosis occurred) it decreased in a dose-dependent manner. Caspase-8 was cleaved and afterwards the amount of Bid decreased as it was truncated. We also found cyt-c release from the inner mitochondrial membrane space into cytoplasm to be dose-dependent and it was followed by induction of apoptosis. In the p53-null cells caspase-8 was activated prior caspase-9, whereas the cells harboring p53 exhibited a simultaneous activation of both initial caspases.


CONCLUSIONS
IR induced a decrease in Mcl-1, activation of Bid, caspase-8, and -9, and release of cyt-c. Presented data indicate that both extrinsic and intrinsic apoptosis signalling pathways were activated in HL-60 and MOLT-4 cells upon exposure to IR regardless to the p53 status.
Tichý
Ales
A
Department of Radiobiology, Faculty of Health Sciences in Hradec Králové, University of Defence, Brno. tichy@pmfhk.cz
Záskodová
Darina
D
Pejchal
Jaroslav
J
Rezácová
Martina
M
Osterreicher
Jan
J
Vávrová
Jirina
J
Cerman
Jaroslav
J
D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism*; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism*; D004789:Enzyme Activation / Q000528:radiation effects; D005720:Gamma Rays / Q000009:adverse effects*; D006801:Humans; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D045304:Cytochromes c; D053181:Caspase 8; D053453:Caspase 9