Adi Kimchi

Self-eating and self-killing: crosstalk between autophagy and apoptosis

Apoptotic cell death is mediated by molecular pathways that culminate in the activation of a family of cysteine proteases, known as the caspases, which orchestrate the dismantling and clearance of the dying cell. However, mounting evidence indicates that a cell that has been treated with an apoptotic inducer can also initiate a suicide programme that does not rely on caspase activation. Here, we present recent findings and discuss the physiological relevance of caspase-independent cell death.

Autophagy as a cell death and tumor suppressor mechanism.

Autophagy is characterized by sequestration of bulk cytoplasm and organelles in double or multimembrane autophagic vesicles, and their delivery to and subsequent degradation by the cells own lysosomal system. Autophagy has multiple physiological functions in multicellular organisms, including protein degradation and organelle turnover. Genes and proteins that constitute the basic machinery of the autophagic process were first identified in the yeast system and some of their mammalian orthologues have been characterized as well. Increasing lines of evidence indicate that these molecular mechanisms may be recruited by an alternative, caspase-independent form of programmed cell death, named autophagic type II cell death. In some settings, autophagy and apoptosis seem to be interconnected positively or negatively, introducing the concept of ‘molecular switches’ between them. Additionally, mitochondria may be central organelles integrating the two types of cell death. Malignant transformation is frequently associated with suppression of autophagy. The recent implication of tumor suppressors like Beclin 1, DAP-kinase and PTEN in autophagic pathways indicates a causative role for autophagy deficiencies in cancer formation. Autophagic cell death induction by some anticancer agents underlines the potential utility of its induction as a new cancer treatment modality.

Life and death partners: apoptosis, autophagy and the cross-talk between them

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 cells 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 cross-talk is a key factor in the outcome of death-related pathologies such as cancer, its development and treatment.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha.

A functional approach of gene cloning was applied to HeLa cells in an attempt to isolate positive mediators of programmed cell death. The approach was based on random inactivation of genes by transfections with antisense cDNA expression libraries, followed by the selection of cells that survived in the presence of the external apoptotic stimulus. An antisense cDNA fragment identical to human cathepsin D aspartic protease was rescued by this positive selection. The high cathepsin D antisense RNA levels protected the HeLa cells from interferon‐gamma‐ and Fas/APO‐1‐induced death. Pepstatin A, an inhibitor of cathepsin D, suppressed cell death in these systems and interfered with the TNF‐alpha‐induced programmed cell death of U937 cells as well. During cell death, expression of cathepsin D was elevated and processing of the protein was affected, which resulted in high steady‐state levels of an intermediate, proteolytically active, single chain form of this protease. Overexpression of cathepsin D by ectopic expression induced cell death in the absence of any external stimulus. Altogether, these results suggest that this well‐known endoprotease plays an active role in cytokine‐induced programmed cell death, thus adding cathepsin D to the growing list of proteases that function as positive mediators of apoptosis.

DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy.

Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3‐domain‐only protein that binds to Bcl‐2 anti‐apoptotic family members. The dissociation of beclin 1 from its Bcl‐2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death‐associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin‐1‐dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl‐XL and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation‐based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.

DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death

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.

DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity

DAP‐kinase was initially identified as a gene whose anti‐sense‐mediated reduced expression protected HeLa cells from interferon‐γ‐induced programmed cell death. It was cloned in our laboratory by a functional gene selection approach. According to its amino acid sequence, this 160 kDa protein was predicted to be a novel type of calmodulin‐regulated serine/threonine kinase which carries ankyrin repeats and the death domain. In this work we have shown that the kinase was autophosphorylated and capable of phosphorylating an exogenous substrate in a Ca2+/calmodulin‐dependent manner. We proved that calmodulin binds directly to the recombinant kinase, and generated a constitutively active kinase mutant by the deletion of the calmodulin‐regulatory domain. By immunostaining and biochemical fractionations we demonstrated that the kinase is localized to the cytoskeleton, in association with the microfilament system, and mapped a region within the protein which is responsible for binding to the cytoskeleton. Several assays attributed a cell death function to the gene. Ectopic expression of wild‐type DAP‐kinase induced the death of target cells, and the killing property depended strictly on the status of the intrinsic kinase activity. Conversely, a catalytically inactive mutant that carried a lysine to alanine substitution within the kinase domain, displayed dominant‐negative features and protected cells from interferon‐γ‐induced cell death. DAP‐kinase is therefore a novel cytoskeletal‐associated cell death serine/threonine kinase whose activation by Ca2+/calmodulin may be linked to the biochemical mechanism underlying the cytoskeletal alterations that occur during cell death.

Autophagy and Cell Death

Autophagy is a physiological and evolutionarily conserved phenomenon maintaining homeostatic functions like protein degradation and organelle turnover. It is rapidly upregulated under conditions leading to cellular stress, such as nutrient or growth factor deprivation, providing an alternative source of intracellular building blocks and substrates for energy generation to enable continuous cell survival. Yet accumulating data provide evidence that the autophagic machinery can be also recruited to kill cells under certain conditions generating a caspase-independent form of programed cell death (PCD), named autophagic cell death. Due to increasing interest in nonapoptotic PCD forms and the development of mammalian genetic tools to study autophagy, autophagic cell death has achieved major prominence, and is recognized now as a legitimate alternative death pathway to apoptosis. This chapter aims at summarizing the recent data in the field of autophagy signaling and autophagic cell death.

DAP kinase links the control of apoptosis to metastasis

DAP kinase is a new type of calcium/calmodulin-dependent enzyme that phosphorylates serine/threonine residues on proteins. Its structure contains ankyrin repeats and the ‘death’ domain, and it is associated with the cell cytoskeleton. The gene encoding DAP kinase was initially isolated as a positive mediator of apoptosis induced by interferon-γ, by using a strategy of functional cloning. We have now tested whether this gene has tumour-suppressive activity. We found that lung carcinoma clones, characterized by their highly aggressive metastatic behaviour and originating from two independent murine lung tumours, did not express DAP kinase, in contrast to their low-metastatic counterparts. Restoration of DAP kinase to physiological levels in high-metastatic Lewis carcinoma cells suppressed their ability to form lung metastases after intravenous injection into syngeneic mice, and delayed local tumour growth in a foreign ‘microenvironment’. Conversely, in vivo selection of rare lung lesions following injection into syngeneic mice of low-metastatic Lewis carcinoma cells or of DAP kinase transfectants, was associated with loss of DAP kinase expression. In situ TUNEL staining of tumour sections revealed that DAP kinase expression from the transgene raised the incidence of apoptosis in vivo. DAP-kinase transfectants also showed increased sensitivity in vitro to apoptotic stimuli, of the sort encountered by metastasizing cells at different stages of malignancy. We propose that loss of DAP kinase expression provides a unique mechanism that links suppression of apoptosis to metastasis.