Commitment and Effector Phases of the Physiological Cell Death Pathway Elucidated with Respect to Bcl-2, Caspase,and Cyclin-Dependent Kinase Activities

Physiological cell deaths occur ubiquitously throughout biology and have common attributes, including apoptotic morphology with mitosis-like chromatin condensation and prelytic genome digestion. The fundamental question is whether a common mechanism of dying underlies these common hallmarks of death. Here we describe evidence of such a conserved mechanism in different cells induced by distinct stimuli to undergo physiological cell death. Our genetic and quantitative biochemical analyses of T- and B-cell deaths reveal a conserved pattern of requisite components. We have dissected the role of cysteine proteases (caspases) in cell death to reflect two obligate classes of cytoplasmic activities functioning in an amplifying cascade, with upstream interleukin-1β-converting enzyme-like proteases activating downstream caspase 3-like caspases. Bcl-2 spares cells from death by punctuating this cascade, preventing the activation of downstream caspases while leaving upstream activity undisturbed. This observation permits an operational definition of the stages of the cell death process. Upstream steps, which are necessary but not themselves lethal, are modulators of the death process. Downstream steps are effectors of, and not dissociable from, actual death; the irreversible commitment to cell death reflects the initiation of this downstream phase. In addition to caspase 3-like proteases, the effector phase of death involves the activation in the nucleus of cell cycle kinases of the cyclin-dependent kinase (Cdk) family. Nuclear recruitment and activation of Cdk components is dependent on the caspase cascade, suggesting that catastrophic Cdk activity may be the actual effector of cell death. The conservation of the cell death mechanism is not reflected in the molecular identity of its individual components, however. For example, we have detected different cyclin-Cdk pairs in different instances of cell death. The ordered course of events that we have observed in distinct cases reflects essential thematic elements of a conserved sequence of modulatory and effector activities comprising a common pathway of physiological cell death.Although interest in the process of physiological cell death has grown enormously in recent years, the mechanism of death has remained enigmatic. While the induction of physiological death in diverse cell types is effected by a wide variety of stimuli, a common morphology, described as apoptosis, ensues in all cases. The commonality of morphology has led to the belief that disparate inducers trigger distinct signaling events which ultimately converge in a common biochemical pathway of death. This hypothesis suggests a division of the biochemical process into upstream events that are specific for individual inducers and downstream steps, comprising the common pathway, which bring about the actual demise of the cell.Since most cell deaths in the nematode Caenorhabditis elegans are induced in a lineage-determined program, the simple pathway of death elucidated in that species (17) is likely to be revealing of downstream steps. Cell death in C. elegans is dependent on the activation of Ced3, a cysteine protease (77, 79), and is inhibited by Ced9 (27). In mammalian cells, a group of Ced3 homologs, termed caspases (1), appears to play a role in virtually all of the physiological cell deaths studied to date. These enzymes cleave on the carboxyl-terminal side of aspartate residues within distinct recognition motifs. Each caspase is synthesized as a proenzyme and activated by cleavage at internal sites, potentially by the same or another caspase class (66, 77). This leads to the notion that caspases function in an ordered cascade, with members of one family activating members of the next. Data consistent with this pattern have been obtained from studies in vitro (41, 60, 65).Of the large family of mammalian caspases, caspase 3 is closely homologous to Ced3 and appears to be involved widely in cell deaths (50, 65). Nonetheless, specific caspases seem not to be associated uniquely with distinct cases of death, and gene-targeting experiments reveal that the absence of a single caspase has extremely limited consequences for cell death responsiveness (38, 39).Similarly, a family of ced9-related death response modulatory genes exists in mammals; the most closely related homolog, bcl-2, is functionally interchangeable with ced9 in the worm (28, 73). These gene products do not function in all mammalian cell deaths (61, 72). Moreover, while the products of some bcl-2 gene family members have death-sparing activity (6, 7), others exert the opposite effect (52, 78).Several cellular proteins, among them poly(ADP-ribose) polymerase (PARP), nuclear lamins, fodrin, and DNA-dependent protein kinase (10, 16, 34), are targets for cleavage by various caspases. In cells spared from death, for example by Bcl-2, these proteolytic events do not occur (9, 13, 18). Still, the cleavage of none of these proteins has been shown to be essential for the cell death response (42, 54, 74). The specific consequences of caspase activation which are lethal are unknown.It may be that the consequence of protease activity is the specific activation of distinct death effectors. We have proposed that essential genes involved in cell division may be critically involved in cell death as well and that the difficulty in identifying distal effector steps genetically reflects the indispensable function of those gene products in cell life (67). Data from several groups have shown that cell cycle catastrophes, the precocious expression of mitosis-like cyclin-dependent histone kinases (Cdks), are associated with a variety of physiological cell deaths and that the inhibition of death by Bcl-2 is associated with alterations in the expression and localization of these Cdk proteins (22, 23, 29, 36, 40, 46, 47, 58, 59, 70).We have taken advantage of the death-sparing activities of Bcl-2 and two viral caspase inhibitors, CrmA and p35 (64, 77), to dissect the mechanism of cell death in two separate cellular paradigms. These studies allow us to draw a generalized skeletal pathway of the death-associated biochemical activities discussed above and demonstrate the requisite involvement of these different classes of activities in a conserved and ordered pathway by which cells die physiologically.