Apoptotic cell death analyzed at the molecular level by two-dimensional gel electrophoresis.
The pattern of protein expression and phosphorylation after an apoptotic stimulus has been studied in two systems. Bovine aortic endothelial cells were induced to undergo apoptotic cell death by a combination of a cytokine (tumor necrosis factor, TNF) and inhibitors of protein synthesis, like cycloheximide. Two-dimensional (2-DE) electrophoresis of proteins from such cells revealed specific proteolysis of distinct proteins, some at an early stage of apoptosis and some at a later stage. These proteins may have antiapoptotic properties. In rat IPC-81 promyelocytic leukemia cells, cAMP induced apoptosis. 2-DE of such cells pulse-labeled with [35S]methionine revealed two "novel" protein spots (of 30 kDa and 46 kDa, respectively), induced very rapidly by a posttranscriptional mechanism. It is proposed that "dysphosphorylation" may accompany apoptosis in general, since both endothelial cells treated with TNF/cycloheximide and IPC-81 cells treated with cAMP analog or the apoptosis-inducing phosphatase inhibitors okadaic acid or calyculin A all showed altered protein phosphorylation patterns, as revealed by 2-DE electrophoresis of proteins from cells prelabeled with 32Pi.
Robaye
B
B
Institute of Interdisciplinary Research, School of Medicine, Free University of Brussels, Belgium.
Døskeland
A P
AP
Suarez-Huerta
N
N
Døskeland
S O
SO
Dumont
J E
JE
D000818:Animals; D001011:Aorta; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001345:Autoradiography / Q000379:methods; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005453:Fluorescence; D007930:Leucine / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; D013873:Thionucleotides; D014409:Tumor Necrosis Factor-alpha; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D010450:Endopeptidases; D007930:Leucine