Apoptosis of Ewing's sarcoma cells is accompanied by accumulation of ubiquitinated proteins.
Induction of apoptosis in Ewing's sarcoma cells by ionizing radiation is accompanied by accumulation of ubiquitinated proteins preferentially in the form of conjugates with M(r) greater than 75,000. Furthermore, enhanced antiubiquitin immunofluorescence was detected only in cells that underwent radiation-induced apoptosis, suggesting that the observed alterations in protein ubiquitination are specific to the apoptotic process. To determine the role of the proteasome in apoptosis-associated accumulation of ubiquitin-protein conjugates, we used lactacystin, a highly selective inhibitor of proteasome proteolytic activity. Exposure of Ewing's sarcoma cells to lactacystin resulted in accumulation of ubiquitinated proteins and activation of a Bcl-2-sensitive apoptotic pathways. The latter led to proteolytic cleavage of poly(ADP-ribose) polymerase and fragmentation of nuclear DNA. These findings suggest that proteasome function is required for apoptosis-specific accumulation of ubiquitinated proteins and indicate that functional disorder of the ubiquitin-proteasome system may play an important role in the apoptotic cell death pathway.
Soldatenkov
V A
VA
Department of Radiation Medicine, Georgetown University Medical Center, Washington, D.C. 20007, USA.
Dritschilo
A
A
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017209:Apoptosis / Q000528:radiation effects; D003546:Cysteine Endopeptidases / Q000502:physiology*; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D009097:Multienzyme Complexes / Q000502:physiology*; D009363:Neoplasm Proteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012512:Sarcoma, Ewing / Q000378:metabolism / Q000473:pathology*; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014452:Ubiquitins; C067713:lactacystin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine