Apoptosis resistance of MCF-7 breast carcinoma cells to ionizing radiation is independent of p53 and cell cycle control but caused by the lack of caspase-3 and a caffeine-inhibitable event.
We have shown previously that ionizing radiation (IR) induces a persistent G(2)-M arrest but not cell death in MCF-7 breast carcinoma cells that harbor functional p53 but lack caspase-3. In the present study, we investigated the mechanisms of apoptosis resistance and the roles of p53, caspase-3, and cell cycle arrest in IR-induced apoptosis. The methylxanthine caffeine and the staurosporine analog UCN-01, which can inhibit ATM and Chk kinases, efficiently abrogated the IR-induced G(2)-M arrest and induced mitochondrial activation as judged by the loss of the mitochondrial membrane potential and the release of cytochrome c and Smac/Diablo. However, despite these proapoptotic alterations, cell death and activation of the initiator caspase-9 were not induced in MCF-7 cells but were interestingly only observed after reexpression of caspase-3. Sensitization to IR-induced apoptosis by caffeine or UCN-01 was abrogated neither by cycloheximide nor by pifithrin-alpha, an inhibitor of the transcriptional activity of p53. Furthermore, suppression of p53 by RNA interference could not prevent caffeine- and IR-induced mitochondrial alterations and apoptosis but resulted in an even more pronounced G(2)-M arrest. Collectively, our results clearly show that the resistance of MCF-7 cells to IR-induced apoptosis is caused by two independent events; one of them is a caffeine- or UCN-01-inhibitable event that does not depend on p53 or a release of the G(2)-M arrest. The second event is the loss of caspase-3 that surprisingly seems essential for a fully functional caspase-9 pathway, even despite the previous release of mitochondrial proapoptotic proteins.
Essmann
Frank
F
University of Düsseldorf, Institute of Molecular Medicine, Düsseldorf, Germany.
Engels
Ingo H
IH
Totzke
Gudrun
G
Schulze-Osthoff
Klaus
K
Jänicke
Reiner U
RU
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology* / Q000532:radiotherapy; D002110:Caffeine / Q000494:pharmacology*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000172:deficiency* / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D016195:G2 Phase / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000528:radiation effects; D008938:Mitosis / Q000187:drug effects / Q000528:radiation effects; D011836:Radiation Tolerance / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects / Q000528:radiation effects; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*
D016159:Tumor Suppressor Protein p53; D002110:Caffeine; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases