Apoptosis: Orderly Dismantling

For this article, Nadia S. Halim interviewed Shigekazu Nagata, professor of genetics, Osaka University Medical School, Japan, coauthor of this Nature paper. Data from the Web of Science (ISI, Philadelphia) show that this paper has been cited significantly more often than the average paper of the same type and age. M. Enari, H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu, and S. Nagata, "A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD," Nature, 391:43-50, January 1, 1998. (Cited in about 430 papers since publication) Apoptosis isn't like a bomb going off in a cell. On the contrary, it signals an orderly dismantling of the cell, which results in a quiet demise. For years scientists knew degradation of DNA in the nucleus was a hallmark of this process. But the identity of the nuclease involved was a mystery until a couple of years ago. Shigekazu Nagata defined some key signaling steps in apoptosis, which culminated in the identification of a novel DNase, one activated exclusively in apoptosis. The results were published in this 1998 Nature paper. In 1991 Nagata's lab identified a cDNA encoding Fas and characterized Fas as a membrane protein belonging to the tumor necrosis factor (TNF) receptor family.1 Subsequently, they found the Fas ligand (FasL) in a cytotoxic T-cell clone and showed that it induces apoptosis by binding to Fas.2 By identifying both a death factor and its receptor, Nagata had discovered a trigger for cytokine-induced apoptotic cell death. This was only one of many steps involved in the highly ordered process. He went on to show that Fas binding to its ligand activates a cascade of caspase proteases in most apoptotic cases.3 The activated caspases cleave various cellular substrates, which may be responsible for the morphological changes that occur in dying cells. Fas-induced apoptosis also causes DNA degradation. Nagata noted that cytosolic fractions from Fas-engaged apoptotic cells contained a factor that could degrade DNA in isolated nuclei, whereas similar extracts from living cells could not. Interestingly, the extracts from living cells gained the ability to break down DNA when treated with caspase 3.4 Three years later, Nagata's group purified and cloned the protein that is activated by caspase 3. Molecular and enzymatic analysis of the protein, designated CAD (caspase-activated DNase), indicated that it was complexed with an inhibitor, ICAD (inhibitor of CAD), in proliferating cells. When cells undergo apoptosis, caspases cleave ICAD, releasing CAD to enter the nucleus and degrade chromosomal DNA. The Nagata lab also showed that overexpression of ICAD in human Jurkat cells blocks DNA degradation during apoptosis.5 In these studies CAD and ICAD were purified from mouse cells, but the sequence of murine ICAD is closely homologous to a human protein, DFF45. Like ICAD, DFF45 can be cleaved by caspase 3 to activate nuclease activity. Nagata feels this paper is highly cited because the apoptosis field is so hot. "The molecular mechanism for the apoptotic DNA fragmentation was not clear for a long time. Our two [January 1998] Nature papers clarified the mechanism," adds Nagata. Since the publication of this paper, the Nagata lab has done some fundamental work on the CAD/ICAD system. Most recently, he has uncovered the different functions for the two forms of ICAD, ICAD-L, and ICAD-S, which are produced by alternative splicing.6 But there are still many questions that need to be answered. Cells die even when the CAD system is blocked, and DNA is not fragmented during apoptosis. So what is the purpose of having a mechanism for DNA fragmentation during apoptosis? "We want to know the physiological roles of the system," he comments. References 1. N. Itoh et al., "The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis," Cell, 66:233-43, 1991. 2. T. Suda et al., "Molecular cloning and expression of the Fas ligand: a novel member of the tumor necrosis factor family," Cell, 75:1169-78,1993. 3. M. Enari et al., "Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis," Nature, 380:723-6, 1996. 4. M. Enari et al., "Apoptosis by a cytosolic extract from Fas-activated cells," EMBO Journal, 14:5201-8, 1995. 5. H. Sakahira et al., "Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis," Nature, 391:96-9, 1998. 6. H. Sakahira et al., "Functional differences of two forms of the inhibitor of caspase-activated DNase, ICAD-L, and ICAD-S," Journal of Biological Chemistry, 274:15740-4, May 28, 1999.

Apoptosis: Orderly Dismantling

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