INVOLVEMENT OF ICE FAMILY PROTEASES IN APOPTOSIS INDUCED BY REOXYGENATION OF HYPOXIC HEPATOCYTES

Cell death due to reoxygenation after hypoxia was characterized in pri mary cultured hepatocytes. Fluorescence and electron microscopic analy ses of reoxygenated hepatocytes revealed morphological characteristics of apoptosis, including chromatin condensation, nuclear fragmentation , and formation of apoptotic bodies. Few necrotic hepatocytes, defined by loss of plasma membrane integrity, mitochondrial swelling, and for mation of large vacuoles, were observed. Activation of interleukin-1 b eta-converting enzyme (ICE)-like and CPP32/Yama-like proteases, which are known to drive apoptosis, was observed during reoxygenation, and a ddition of their respective inhibitors inhibited the induction of apop tosis, indicating the involvement of ICE family proteases in apoptosis by reoxygenation. Production of oxygen radicals was enhanced by reoxy genation of hypoxic cells, and reoxygenation-induced apoptosis was inh ibited by oxygen radical scavengers, suggesting a role for reactive ox ygen species as a triggering factor in cell death. Electrophoretic ana lysis revealed the presence of 50-kb DNA fragments but not oligonucleo somal DNA fragments in reoxygenation-induced apoptotic hepatocytes.