Insulin withdrawal-induced cell death in adult hippocampal neural stem cells as a model of autophagic cell death

The term "autophagic cell death" was coined to describe a form of cell death associated with the massive formation of autophagic vacuoles without signs of apoptosis. However, questions about the actual role of autophagy and its molecular basis in cell death remain to be elucidated. We recently reported that adult hippocampal neural stem (HCN) cells undergo autophagic cell death following insulin withdrawal. Insulin-deprived HCN cells exhibit morphological and biochemical markers of autophagy, including accumulation of Beclin 1 and the type II form of microtubule-associated protein 1 light chain 3 (LC3) without evidence of apoptosis. Suppression of autophagy by knockdown of Atg7 reduces cell death, whereas promotion of autophagy with rapamycin augments cell death in insulin-deficient HCN cells. These data reveal a causative role of autophagy in insulin withdrawal-induced HCN cell death. HCN cells have intact apoptotic capability despite the lack of apoptosis following insulin withdrawal. Our study demonstrates that autophagy is the default cell death mechanism in insulin-deficient HCN cells, and provides a genuine model of autophagic cell death in apoptosis-intact cells. Novel insight into molecular mechanisms of this underappreciated form of programmed cell death should facilitate the development of therapeutic methods to cope with human diseases caused by dysregulated cell death.     

Nutrient deprivation induces autophagy as well as apoptosis in Chinese hamster ovary cell culture

During Chinese hamster ovary (CHO) cell culture for foreign protein production, cells are subjected to programmed cell death (PCD). A rapid death at the end of batch culture is accelerated by nutrient starvation. In this study, type II PCD, autophagy, as well as type I PCD, apoptosis, was found to take place in two antibody-producing CHO cell lines, Ab1 and Ab2, toward the end of batch culture when glucose and glutamine were limiting. The evidence of autophagy was observed from the accumulation of a common autophagic marker, a 16 kDa form of LC3-II during batch culture. Moreover, a significant percentage of the total cells (80% of Ab1 cells and 86% of Ab2 cells) showed autophagic vacuoles containing cytoplasmic material by transmission electron microscopy. An increased level of PARP cleavage and chromosomal DNA fragmentation supported that starvation-induced apoptosis also occurred simultaneously with autophagy.     

The plant cell death suppressor Adi3 interacts with the autophagic protein Atg8h

The tomato AGC protein kinase Adi3 is known to function as a suppressor of PCD and silencing of Adi3 leads to spontaneous cell death on leaves and stems. In an effort to isolate Adi3 interacting proteins, a yeast two-hybrid screen was carried out and identified the autophagy protein Atg8h as an Adi3 interactor. This interaction occurred independent of the kinase activity status of Adi3. Silencing of genes involved in autophagy is known to eliminate the restriction of pathogen-induced PCD to a few cells and leads to run away PCD. Cosilencing Adi3 with several autophagy genes lead to the same run away cell death suggesting Adi3 may be involved in autophagic regulation of PCD.     

Autophagy can be a killer even in apoptosis-competent cells

Despite abundant evidence for autophagic cell death as a morphological type, the notion that autophagy can actually contribute mechanistically to the cell's death is controversial. In cells capable of apoptosis, autophagic cell death has been dismissed by some authors as a morphologically unusual form of apoptosis. But strong recent evidence for autophagy-mediated death of cells rendered incapable of apoptosis has been criticized on the grounds that this cell death is too artificial to be relevant to normal cells. We here argue from our own and other recent evidence that autophagy can mediate the death even of apoptosis-competent cells.     

Interplay between autophagy and programmed cell death in mammalian neural stem cells

Mammalian neural stem cells (NSCs) are of particular interest because of their role in brain development and function. Recent findings suggest the intimate involvement of programmed cell death (PCD) in the turnover of NSCs. However, the underlying mechanisms of PCD are largely unknown. Although apoptosis is the best-defined form of PCD, accumulating evidence has revealed a wide spectrum of PCD encompassing apoptosis, autophagic cell death (ACD) and necrosis. This mini-review aims to illustrate a unique regulation of PCD in NSCs. The results of our recent studies on autophagic death of adult hippocampal neural stem (HCN) cells are also discussed. HCN cell death following insulin withdrawal clearly provides a reliable model that can be used to analyze the molecular mechanisms of ACD in the larger context of PCD. More research efforts are needed to increase our understanding of the molecular basis of NSC turnover under degenerating conditions, such as aging, stress and neurological diseases. Efforts aimed at protecting and harnessing endogenous NSCs will offer novel opportunities for the development of new therapeutic strategies for neuropathologies. [BMB Reports 2013; 46(8): 383-390]     

A Calpain-Like Protease Inhibits Autophagic Cell Death

Programmed cell death (PCD) plays critical roles during development and in disease states. One form of programmed cell death utilizes autophagy - a cellular mechanism of degrading bulk cytosolic components - to destroy cells. Previously, the broad-spectrum caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) was shown to induce autophagic cell death. The mechanism of zVAD-induced cell death was proposed to require caspase-8 inhibition. In our report, we extend these findings to show that - as is the case for apoptosis - induction of autophagic cell death in response to zVAD results in phosphatidylserine exposure prior to loss of membrane integrity. Additionally, we show that caspase-8 inhibition is insufficient to cause autophagic cell death. Rather, the activity of a calpain-like protease must also be blocked. These results reveal the existence of an autophagic PCD-inhibiting calpain-like cysteine protease.     

Lepidopteran larval midgut during prepupal instar: digestion or self-digestion?

Programmed cell death (PCD) is crucial in body restructuring during metamorphosis of holometabolous insects (those that have a pupal stage between the final larval and adult stages). Besides apoptosis, an increasing body of evidence indicates that in several insect species programmed autophagy also plays a key role in these developmental processes. We have recently characterized the midgut replacement process in Heliothis virescens larva, during the prepupal phase, responsible for the formation of a new pupal midgut. We found that the elimination of the old larval midgut epithelium is obtained by a combination of apoptotic and autophagic events. In particular, autophagic PCD completely digests decaying tissues, and provides nutrients that are rapidly absorbed by the newly formed epithelium, which is apparently functional at this early stage. The presence of both apoptosis and autophagy in the replacement of midgut cells in Lepidoptera offers the opportunity to investigate the functional peculiarities of these PCD modalities and if they share any molecular mechanism, which may account for possible cross-talk between them.     

Mitochondrial ROS generation for regulation of autophagic pathways in cancer

Mitochondria, the main source of reactive oxygen species (ROS), are required for cell survival; yet also orchestrate programmed cell death (PCD), referring to apoptosis and autophagy. Autophagy is an evolutionarily conserved lysosomal degradation process implicated in a wide range of pathological processes, most notably cancer. Accumulating evidence has recently revealed that mitochondria may generate massive ROS that play the essential role for autophagy regulation, and thus sealing the fate of cancer cell. In this review, we summarize mitochondrial function and ROS generation, and also highlight ROS-modulated core autophagic pathways involved in ATG4–ATG8/LC3, Beclin-1, p53, PTEN, PI3K–Akt–mTOR and MAPK signaling in cancer. Therefore, a better understanding of the intricate relationships between mitochondrial ROS and autophagy may ultimately allow cancer biologists to harness mitochondrial ROS-mediated autophagic pathways for cancer drug discovery.     

Does Autophagy Contribute To Cell Death?

Autophagy (specifically macroautophagy) is an evolutionarily conserved catabolic process where the cytoplasmic contents of a cell are sequestered within double membrane vacuoles, called autophagosomes, and subsequently delivered to the lysosome for degradation. Autophagy can function as a survival mechanism in starving cells. At the same time, extensive autophagy is commonly observed in dying cells, leading to its classification as an alternative form of programmed cell death. The functional contribution of autophagy to cell death has been a subject of great controversy. However, several recent loss-of-function studies of autophagy (Atg) genes have begun to address the roles of autophagy in both cell death and survival. Here, we review the emerging evidence in favor of and against autophagic cell death, discuss the possible roles that autophagic degradation might play in dying cells, and identify salient issues for future investigation.     

Does autophagy contribute to cell death?

Autophagy (specifically macroautophagy) is an evolutionarily conserved catabolic process where the cytoplasmic contents of a cell are sequestered within double membrane vacuoles, called autophagosomes, and subsequently delivered to the lysosome for degradation. Autophagy can function as a survival mechanism in starving cells. At the same time, extensive autophagy is commonly observed in dying cells, leading to its classification as an alternative form of programmed cell death. The functional contribution of autophagy to cell death has been a subject of great controversy. However, several recent loss-of-function studies of autophagy (atg) genes have begun to address the roles of autophagy in both cell death and survival. Here, we review the emerging evidence in favor of and against autophagic cell death, discuss the possible roles that autophagic degradation might play in dying cells, and identify salient issues for future investigation.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.     

Role of Arabidopsis RabG3b and autophagy in tracheary element differentiation

The vascular system of plants consists of two conducting tissues, xylem and phloem, which differentiate from procambium cells. Xylem serves as a transporting system for water and signaling molecules and is formed by sequential developmental processes, including cell division/expansion, secondary cell wall deposition, vacuole collapse and programmed cell death (PCD). PCD during xylem differentiation is accomplished by degradation of cytoplasmic constituents, and it is required for the formation of hollow vessels, known as tracheary elements (TEs). Our recent study revealed that the small GTPase RabG3b acts as a regulator of TE differentiation through its autophagic activation. By using an Arabidopsis in vitro cell culture system, we showed that autophagy is activated during TE differentiation. Overexpression of a constitutively active RabG3b (RabG3bCA) significantly enhances both autophagy and TE differentiation, which are consistently suppressed in transgenic plants overexpressing a dominant negative form (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid-insensitive mutant bri1-301 and an autophagy mutant atg5-1. On the basis of our results, we propose that RabG3b functions as a component of autophagy and regulates TE differentiation by activating the process of PCD.     

Autophagy and apoptosis in Chinese hamster ovary cell culture

Upon nutrient deprivation during Chinese hamster ovary (CHO) cell culture for foreign protein production, cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. However, only apoptosis has drawn attention in the field of CHO cell culture. Numerous studies on engineering genes or supplementing essential nutrients or chemical additives to culture media to overcome cell death induced by various stimuli have been limited to apoptosis. Recently, autophagic morphologies were demonstrated by the processing of LC3 into the 16 kDa LC3-II form, and the accumulation of multiple autophagosomes in CHO cell culture. Therefore, it seems worthwhile to revisit the issue of cell death in CHO cell culture with the concept of autophagy in mind, in order to achieve a maximum production of foreign proteins by protecting cells from both types of PCD.     

Real-time observation of autophagic programmed cell death of<Emphasis Type="Italic"> Drosophila</Emphasis> salivary glands in vitro

Autophagy, a form of programmed cell death (PCD) that is morphologically distinguished from apoptosis, is thought to be as prevalent as apoptosis, at least during development. In insect metamorphosis, the steroid hormone 20-hydroxyecdysone (ecdysone) activates autophagic PCD to eliminate larval structures that are no longer needed. However, in comparison with apoptosis, there are not many studies on the regulation mechanisms of autophagy. To provide a useful model for studying autophagic PCD, I established an in vitro culture system that enables real-time observation of the autophagic cell destruction of Drosophila salivary glands. The new system revealed that de novo gene expression was still required for the destruction of salivary glands dissected from phanerocephalic pupae. This indicates the usefulness of the system for exploring genes that participate in the last processes of autophagic PCD.Edited by N. Satoh     

Regulation of autophagy by polyphenolic compounds as a potential therapeutic strategy for cancer

Autophagy, a lysosomal degradation pathway for cellular constituents and organelles, is an adaptive and essential process required for cellular homeostasis. Although autophagy functions as a survival mechanism in response to cellular stressors such as nutrient or growth factor deprivation, it can also lead to a non-apoptotic form of programmed cell death (PCD) called autophagy-induced cell death or autophagy-associated cell death (type II PCD). Current evidence suggests that cell death through autophagy can be induced as an alternative to apoptosis (type I PCD), with therapeutic purpose in cancer cells that are resistant to apoptosis. Thus, modulating autophagy is of great interest in cancer research and therapy. Natural polyphenolic compounds that are present in our diet, such as rottlerin, genistein, quercetin, curcumin, and resveratrol, can trigger type II PCD via various mechanisms through the canonical (Beclin-1 dependent) and non-canonical (Beclin-1 independent) routes of autophagy. The capacity of these compounds to provide a means of cancer cell death that enhances the effects of standard therapies should be taken into consideration for designing novel therapeutic strategies. This review focuses on the autophagy- and cell death-inducing effects of these polyphenolic compounds in cancer.     

Cell death and tissue reorganization in Rhynchosciara americana (Sciaridae: Diptera) metamorphosis and their relation to molting hormone titers

Programmed cell death (PCD) is a focal topic for understanding processes underlying metamorphosis in insects, especially so in holometabolous orders. During adult morphogenesis it allows for the elimination of larva-specific tissues and the reorganization of others for their functionalities in adult life. In Rhynchosciara, this PCD process could be classified as autophagic cell death, yet the expression of apoptosis-related genes and certain morphological aspects suggest that processes, autophagy and apoptosis may be involved. Aiming to reveal the morphological changes that salivary gland and fat body cells undergo during metamorphosis we conducted microscopy analyses to detect chromatin condensation and fragmentation, as well as alterations in the cytoplasm of late pupal tissues of Rhynchosciara americana. Transmission electron microscopy and confocal microscopy revealed cells in variable stages of death. By analyzing the morphological structure of the salivary gland we observed the presence of cells with autophagic vacuoles and apoptotic bodies and DNA fragmentation was confirmed with the TUNEL assay in salivary gland. The reorganization of fat body occurs with discrete detection of cell death by TUNEL assay. However, both salivary gland histolysis and fat body reorganization occur under control of the hormone ecdysone.     

程序性细胞死亡与肿瘤

程序性细胞死亡（programmed cell death,PCD）是指由基因控制的细胞自主的有序性死亡方式,涉及一系列基因的激活、表达以及调控等。目前,经典细胞凋亡被称为Ⅰ型PCD,而自噬性细胞死亡称为Ⅱ型PCD,坏死样程序性细胞死亡则被称为Ⅲ型PCD,它们在肿瘤的发生、发展及治疗过程中起非常重要的作用。该文结合国内外最新研究进展主要针对不同细胞死亡模式及其相互作用、关键作用蛋白,细胞自噬与肿瘤发生,细胞自噬、凋亡与肿瘤治疗作一简要综述,并展望发展前景,提出在肿瘤治疗中如何利用不同死亡模式的协同作用最大限度地发挥其临床应用价值。     

Autophagy promotes caspase-dependent cell death during Drosophila development

The relationship between autophagic cell death and apoptosis is a poorly understood aspect of programmed cell death (PCD). We have examined this relationship by studying the elimination of an extra-embryonic tissue, known as the amnioserosa (AS), during Drosophila development. The AS becomes autophagic during the final stages of embryogenesis; ultimately, however, the elimination of the AS involves caspase-dependent nuclear fragmentation, tissue dissociation, and engulfment by phagocytic macrophages. Mutants that are defective in the activation or execution of caspase-dependent PCD fail to degrade and eliminate the AS but show no abatement in AS autophagy. Sustained autophagy does not, therefore, necessarily result in cell death. Surprisingly, the down-regulation of autophagy also results in a persistent AS phenotype and reduced cell death. Conversely, up-regulation of autophagy results in caspase-dependent premature AS dissociation. These observations are consistent with the interpretation that autophagy is a prerequisite for caspase-dependent cell death in the AS.     

Vacuolar cell death in plants

Vacuolar programmed cell death (PCD) is indispensable for plant development and is accompanied by a dramatic growth of lytic vacuoles, which gradually digest cytoplasmic content leading to self-clearance of dying cells. Our recent data demonstrate that vacuolar PCD critically requires autophagy and its upstream regulator, a caspase-fold protease metacaspase. Furthermore, both components lie downstream of the point of no return in the cell-death pathway. Here we consider the possibilities that i) autophagy could have both cytotoxic and cytoprotective roles in the vacuolar PCD, and ii) metacaspase could augment autophagic flux through targeting an as yet unknown autophagy repressor.