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Members of the interleukin-1 beta-converting enzyme (ICE)/CED-3 protease family have been implicated in apoptosis in both vertebrates and invertebrates. Using primary culture methods, we report that neurons and astrocytes require the activity of the ICE/CED-3 family of proteases to undergo apoptosis induced by staurosporine, ceramide, and serum-free media. We show that specific inhibitors of ICE/CED-3 proteases can inhibit apoptosis and that cytosolic fractions from apoptosing neurons, but not healthy cells, induced apoptosis in a cell-free system. Cell extracts from neurons induced to undergo apoptosis contained ICE/ CED-3 protease activity. To determine which member of the ICE/CED-3 family was activated in neurons and astrocytes during apoptosis, we developed a novel affinity-labeling technique that labeled the active site cysteine and identified a 17-kDa subunit of the activated protease. The affinity-labeled 17-kDa protease subunit shares antigenic and molecular mass identity with the processed form of CPP32 on immunoblots, suggesting that CPP32 may be the principal effector in the apoptotic pathway in neurons and astrocytes. In time-course experiments, the activation of CPP32 preceded the detection of PARP cleavage and DNA laddering, suggesting that processing of CPP32 is a very early event in apoptosis of neurons and astrocytes and may be involved in the proteolytic action on specific cellular targets. The affinity-labeling technique developed and used in this report with neural cells allows for the sensitive detection, purification, and identification of ICE/CED-3 proteases that may be activated in other cells types under a variety of conditions, including certain diseased states.

Cell death has been described as either apoptotic, in which the cell actively participates, or necrotic, in which the cell is felt to be passive. The proto-oncogene bcl-2 has been shown to inhibit apoptosis in some hematopoietic and neural cells, by an unknown mechanism. We demonstrate that bcl-2 inhibits the necrosis of neural cells induced by glutathione depletion. This finding demonstrates that bcl-2 does not inhibit the cellular death program directly; rather, bcl-2 modulates a cellular process that leads to apoptosis under some conditions but necrosis under others.