APOPTOTIC CELL-DEATH ANALYZED AT THE MOLECULAR-LEVEL BY 2-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 eels pulse-labeled with [S-35]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 relabeled with (32)pi.