Radiosensitive cell lines derived from X‐ray cross complementing group 5 (XRCC5), SCID mice and a human glioma cell line lack components of the DNA‐dependent protein kinase, DNA‐PK, suggesting that DNA‐PK plays an important role in DNA double‐strand break repair. Another enzyme implicated in DNA repair, poly(ADP‐ribose) polymerase, is cleaved and inactivated during apoptosis, suggesting that some DNA repair proteins may be selectively targeted for destruction during apoptosis. Here we demonstrate that DNA‐PKcs, the catalytic subunit of DNA‐PK, is preferentially degraded after the exposure of different cell types to a variety of agents known to cause apoptosis. However, Ku, the DNA‐binding component of the enzyme, remains intact. Degradation of DNA‐PKcs was accompanied by loss of DNA‐PK activity. One cell line resistant to etoposide‐induced apoptosis failed to show degradation of DNA‐PKcs. Protease inhibitor data implicated an ICE‐like protease in the cleavage of DNA‐PKcs, and it was subsequently shown that the cysteine protease CPP32, but not Mch2alpha, ICE or TX, cleaved purified DNA‐PKcs into three fragments of comparable size with those observed in cells undergoing apoptosis. Cleavage sites in DNA‐PKcs, determined by antibody mapping and microsequencing, were shown to be the same for CPP32 cleavage and for cleavage catalyzed by extracts from cells undergoing apoptosis. These observations suggest that DNA‐PKcs is a critical target for proteolysis by an ICE‐like protease during apoptosis.

In this report, we describe the cloning and characterization of Boo, a novel anti-apoptotic member of the Bcl-2 family. The expression of Boo was highly restricted to the ovary and epididymis implicating it in the control of ovarian atresia and sperm maturation. Boo contains the conserved BH1 and BH2 domains, but lacks the BH3 motif. Like Bcl-2, Boo possesses a hydrophobic C-terminus and localizes to intracellular membranes. Boo also has an N-terminal region with strong homology to the BH4 domain found to be important for the function of some anti-apoptotic Bcl-2 homologues. Chromosomal localization analysis assigned Boo to murine chromosome 9 at band d9. Boo inhibits apoptosis, homodimerizes or heterodimerizes with some death-promoting and -suppressing Bcl-2 family members. More importantly, Boo interacts with Apaf-1 and forms a multimeric protein complex with Apaf-1 and caspase-9. Bak and Bik, two pro-apoptotic homologues disrupt the association of Boo and Apaf-1. Furthermore, Boo binds to three distinct regions of Apaf-1. These results demonstrate the evolutionarily conserved nature of the mechanisms of apoptosis. Like Ced-9, the mammalian homologues Boo and Bcl-x L interact with the human counterpart of Ced-4, Apaf-1, and thereby regulate apoptosis.

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A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1␤-converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.Apoptosis or programmed cell death is a physiological mechanism responsible for the elimination of unwanted cells during the process of development and the removal of self-reactive lymphocytes (1, 2). The biochemical mechanisms underlying apoptosis remain unclear, but several genes implicated in the process have been identified (reviewed in refs. 3 and 4). It is evident from recent results that a growing family of cysteine proteases which share similarity with the interleukin-1␤-converting enzyme (ICE) play a central role in the execution phase of apoptosis (reviewed in refs. 5 and 6). Identification of this family of ICE-like proteases is largely due to investigations of the regulation of cell death in Caenorhabditis elegans (7). In these studies several genes controlling cell death, including ced3, ced4, and ced9, have been identified. Yuan et al. (8) demonstrated significant sequence homology between Ced3 and ICE, which cleaves inactive pro-interleukin-1␤ to an active form (9), and overexpression of this protein in fibroblasts resulted in apoptosis (10). To date, eight homologs of ICE have been