APOPTOTIC CELL-DEATH ANALYZED AT THE MOLECULAR-LEVEL BY 2-DIMENSIONALGEL-ELECTROPHORESIS

The pattern of protein expression and phosphorylation after an apoptot ic stimulus has been studied in two systems. Bovine aortic endothelial cells were induced to undergo apoptotic cell death by a combination o f a cytokine (tumor necrosis factor, TNF) and inhibitors of protein sy nthesis, like cycloheximide. Two-dimensional (2-DE) electrophoresis of proteins from such cells revealed specific proteolysis of distinct pr oteins, some at an early stage of apoptosis and some at a later stage. These proteins may have antiapoptotic properties. In rat IPC-81 promy elocytic leukemia cells, cAMP induced apoptosis. 2-DE of such eels pul se-labeled with [S-35]methionine revealed two ''novel'' protein spots (of 30 kDa and 46 kDa, respectively), induced very rapidly by a posttr anscriptional mechanism. It is proposed that '''dysphosphorylation'' m ay accompany apoptosis in general, since both endothelial cells treate d with TNF/cycloheximide and IPC-81 cells treated with cAMP analog or the apoptosis-inducing phosphatase inhibitors okadaic acid or calyculi n A all showed altered protein phosphorylation patterns, as revealed b y 2-DE electrophoresis of proteins from cells relabeled with (32)pi.