It is not surprising that the demise of a cell is a complex well-controlled process. Apoptosis, the first genetically programmed death process identified, has been extensively studied and its contribution to the pathogenesis of disease well documented. Yet, apoptosis does not function alone to determine a cell's fate. More recently, autophagy, a process in which de novo-formed membrane-enclosed vesicles engulf and consume cellular components, has been shown to engage in a complex interplay with apoptosis. In some cellular settings, it can serve as a cell survival pathway, suppressing apoptosis, and in others, it can lead to death itself, either in collaboration with apoptosis or as a back-up mechanism when the former is defective. The molecular regulators of both pathways are inter-connected; numerous death stimuli are capable of activating either pathway, and both pathways share several genes that are critical for their respective execution. The cross-talk between apoptosis and autophagy is therefore quite complex, and sometimes contradictory, but surely critical to the overall fate of the cell. Furthermore, the crosstalk is a key factor in the outcome of death-related pathologies such as cancer, its development and treatment. Autophagy, a process long known to provide a survival advantage to cells undergoing nutrient deprivation or other stresses, has also been more recently linked to the actual death process itself. Thus apoptosis is not the sole means by which the cell can undergo a genetically programmed regulated process by which it undergoes self-elimination. Cell death can occur by several mechanisms and the phenotypic changes that accompany cell death can vary depending on the stimulus and cell setting. In any given death scenario, the cell decides which pathway to use, depending on the nature of the stimulus and the particulars of the cell environment. Furthermore, apoptosis and autophagy are not mutually exclusive pathways. They have been shown to act in synergy and also to counter each other. They share many of the same molecular regulators. In a clinical setting, one cannot predict the outcome of inhibition or activation of one death program without considering the effect on the other. This review will focus on the cross-talk between the autophagic and apoptotic pathways, with an analysis of how this may affect the clinical applications of death suppression/activation to cancer. The process of necrosis, the third means by which the cell can undergo a genetically programmed self-elimination, will not be discussed in detail. Although not intended to provide an exhaustive summary of the recent literature, the discussion will include salient experimental results as examples of the different facets of the apoptosis/autophagy interplay.An issue that has been raised and discussed in the literature is that in many cell settings, autophagy accompanies, rather than causes, cell death. 1 This argument is based on the fact that many studies that claim autophagic cell death prove that autophagy occurs, and that ...

Death-associated protein kinase (DAPk) is the founding member of a newly classified family of Ser/Thr kinases, whose members not only possess significant homology in their catalytic domains, but also share cell death-associated functions. The realization that DAPk is a tumor suppressor gene, whose expression is lost in multiple tumor types, has spurred a flurry of interest in the kinase family and produced an impressive body of literature concerning its function, regulation, and connection to disease. The DAPk family has been linked to several cell death-related signaling pathways, and functions other than cell death have also been proposed. This review presents a thorough structural analysis of the kinases, discusses methods of regulation, clarifies their cellular targets and functions, and shows how these functions are integrated. Although many gaps in our knowledge still remain, the data generated to date can be combined to delineate a place for the DAPk family within the general cell death-signaling network.

Death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1 proteins are Ca+2/calmodulin–regulated Ser/Thr death kinases whose precise roles in programmed cell death are still mostly unknown. In this study, we dissected the subcellular events in which these kinases are involved during cell death. Expression of each of these DAPk subfamily members in their activated forms triggered two major cytoplasmic events: membrane blebbing, characteristic of several types of cell death, and extensive autophagy, which is typical of autophagic (type II) programmed cell death. These two different cellular outcomes were totally independent of caspase activity. It was also found that dominant negative mutants of DAPk or DRP-1 reduced membrane blebbing during the p55/tumor necrosis factor receptor 1–induced type I apoptosis but did not prevent nuclear fragmentation. In addition, expression of the dominant negative mutant of DRP-1 or of DAPk antisense mRNA reduced autophagy induced by antiestrogens, amino acid starvation, or administration of interferon-γ. Thus, both endogenous DAPk and DRP-1 possess rate-limiting functions in these two distinct cytoplasmic events. Finally, immunogold staining showed that DRP-1 is localized inside the autophagic vesicles, suggesting a direct involvement of this kinase in the process of autophagy.

The tumor suppressor functions of p19(ARF) have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. Here we describe another cellular function of p19(ARF), involving a short isoform (smARF, short mitochondrial ARF) that localizes to a Proteinase K-resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, which lacks the nucleolar functional domains. The human p14(ARF) mRNA likewise produces a shorter isoform. smARF is maintained at low levels via proteasome-mediated degradation, but it increases in response to viral and cellular oncogenes. Ectopic expression of smARF reduces mitochondrial membrane potential (DeltaPsim) without causing cytochrome c release or caspase activation. The dissipation of DeltaPsim does not depend on p53 or Bcl-2 family members. smARF induces massive autophagy and caspase-independent cell death that can be partially rescued by knocking down ATG5 or Beclin-1, suggesting a different prodeath function for this short isoform.

Toxins convert the hepatocellular response to tumor necrosis factor-α (TNF-α) stimulation from proliferation to cell death, suggesting that hepatotoxins somehow sensitize hepatocytes to TNF-α toxicity. Because nuclear factor-κB (NF-κB) activation confers resistance to TNF-α cytotoxicity in nonhepatic cells, the possibility that toxin-induced sensitization to TNF-α killing results from inhibition of NF-κB-dependent gene expression was examined in the RALA rat hepatocyte cell line sensitized to TNF-α cytotoxicity by actinomycin D (ActD). ActD did not affect TNF-α-induced hepatocyte NF-κB activation but decreased NF-κB-dependent gene expression. Expression of an IκB superrepressor rendered RALA hepatocytes sensitive to TNF-α-induced apoptosis in the absence of ActD. Apoptosis was blocked by caspase inhibitors, and TNF-α treatment led to activation of caspase-2, caspase-3, and caspase-8 only when NF-κB activation was blocked. Although apoptosis was blocked by the NF-κB-dependent factor nitric oxide (NO), inhibition of endogenous NO production did not sensitize cells to TNF-α-induced cytotoxicity. Thus NF-κB activation is the critical intracellular signal that determines whether TNF-α stimulates hepatocyte proliferation or apoptosis. Although exogenous NO blocks RALA hepatocyte TNF-α cytotoxicity, endogenous production of NO is not the mechanism by which NF-κB activation inhibits this death pathway.

Autophagy and apoptosis constitute important determinants of cell fate and engage in a complex interplay in both physiological and pathological settings. The molecular basis of this crosstalk is poorly understood and relies, in part, on "dual-function" proteins that operate in both processes. Here, we identify the essential autophagy protein Atg12 as a positive mediator of mitochondrial apoptosis and show that Atg12 directly regulates the apoptotic pathway by binding and inactivating prosurvival Bcl-2 family members, including Bcl-2 and Mcl-1. The binding occurs independently of Atg5 or Atg3 and requires a unique BH3-like motif in Atg12, characterized by interaction studies and computational docking. In apoptotic cells, knockdown of Atg12 inhibited Bax activation and cytochrome c release, while ectopic expression of Atg12 antagonized the antiapoptotic activity of Mcl-1. The interaction between Atg12 and Bcl-2 family members may thus constitute an important point of convergence between autophagy and apoptosis in response to specific signals.

Significant numbers of myocytes die by apoptosis during myocardial infarction. The molecular mechanism of this process, however, remains largely unexplored. To facilitate a molecular genetic analysis, we have developed a model of ischemia-induced cardiac myocyte apoptosis in the mouse. Surgical occlusion of the left coronary artery results in apoptosis, as indicated by the presence of nucleosome ladders and in situ DNA strand breaks. Apoptosis occurs mainly in cardiac myocytes, and is shown for the first time to be limited to hypoxic regions during acute infarction. Since hypoxia-induced apoptosis in other cell types is dependent on p53, and p53 is induced by hypoxia in cardiac myocytes, we investigated the necessity of p53 for myocyte apoptosis during myocardial infarction. Myocyte apoptosis occurs as readily, however, in the hearts of mice nullizygous for p53 as in wild-type littermates. These data demonstrate the existence of a p53-independent pathway that mediates myocyte apoptosis during myocardial infarction. ( J.

Damage to endoplasmic reticulum (ER) homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase (DAPk) as an important component in the ER stress-induced cell death pathway. DAPkÀ/À mice are protected from kidney damage caused by injection of the ER stress-inducer tunicamycin. Likewise, the cell death response to ER stress-inducers is reduced in DAPkÀ/À primary fibroblasts. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion. We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser 308 by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.