In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Reactive oxygen species (ROS) have been identified as signaling molecules in various pathways regulating both cell survival and cell death. Autophagy, a self-digestion process that degrades intracellular structures in response to stress, such as nutrient starvation, is also involved in both cell survival and cell death. Alterations in both ROS and autophagy regulation contribute to cancer initiation and progression, and both are targets for developing therapies to induce cell death selectively in cancer cells. Many stimuli that induce ROS generation also induce autophagy, including nutrient starvation, mitochondrial toxins, hypoxia, and oxidative stress. Some of these stimuli are under clinical investigation as cancer treatments, such as 2-methoxyestrodial and arsenic trioxide. Recently, it was demonstrated that ROS can induce autophagy through several distinct mechanisms involving Atg4, catalase, and the mitochondrial electron transport chain (mETC). This leads to both cell-survival and cell-death responses and could be selective toward cancer cells. In this review, we give an overview of the roles ROS and autophagy play in cell survival and cell death, and their importance to cancer. Furthermore, we describe how autophagy is mediated by ROS and the implications of this regulation to cancer treatments.

Autophagy is involved in human diseases and is regulated by reactive oxygen species (ROS) including superoxide (O 2 KÀ ) and hydrogen peroxide (H 2 O 2 ). However, the relative functions of O 2 KÀ and H 2 O 2 in regulating autophagy are unknown. In this study, autophagy was induced by starvation, mitochondrial electron transport inhibitors, and exogenous H 2 O 2 . We found that O 2 KÀ was selectively induced by starvation of glucose, L-glutamine, pyruvate, and serum (GP) whereas starvation of amino acids and serum (AA) induced O 2 KÀ and H 2 O 2 . Both types of starvation induced autophagy and autophagy was inhibited by overexpression of SOD2 (manganese superoxide dismutase, Mn-SOD), which reduced O 2 KÀ levels but increased H 2 O 2 levels. Starvation-induced autophagy was also inhibited by the addition of catalase, which reduced both O 2 KÀ and H 2 O 2 levels. Starvation of GP or AA also induced cell death that was increased following treatment with autophagy inhibitors 3-methyladenine, and wortamannin. Mitochondrial electron transport chain (mETC) inhibitors in combination with the SOD inhibitor 2-methoxyestradiol (2-ME) increased O 2 KÀ levels, lowered H 2 O 2 levels, and increased autophagy. In contrast to starvation, cell death induced by mETC inhibitors was increased by 2-ME. Finally, adding exogenous H 2 O 2 induced autophagy and increased intracellular O 2 KÀ but failed to increase intracellular H 2 O 2 . Taken together, these findings indicate that O 2 KÀ is the major ROS-regulating autophagy.

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H 2 O 2 ) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H 2 O 2 or 2-ME-induced cell death. H 2 O 2 and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H 2 O 2 or 2-ME through overexpression of manganesesuperoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H 2 O 2 -or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H 2 O 2 or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in nontransformed cells.

Autophagy is a self-digestion process important for cell survival during starvation. It has also been described as a form of programmed cell death. Mitochondria are important regulators of autophagy-induced cell death and damaged mitochondria are often degraded by autophagosomes. Inhibition of the mitochondrial electron transport chain (mETC) induces cell death through generating reactive oxygen species (ROS). The role of mETC inhibitors in autophagy-induced cell death is unknown. Herein, we determined that inhibitors of complex I (rotenone) and complex II (TTFA) induce cell death and autophagy in the transformed cell line HEK 293, and in cancer cell lines U87 and HeLa. Blocking the expression of autophagic genes (beclin 1 and ATG5) by siRNAs or using the autophagy inhibitor 3-methyladenine (3-MA) decreased cell death that was induced by rotenone or TTFA. Rotenone and TTFA induce ROS production, and the ROS scavenger tiron decreased autophagy and cell death induced by rotenone and TTFA. Overexpression of manganese-superoxide dismutase (SOD2) in HeLa cells decreased autophagy and cell death induced by rotenone and TTFA. Furthermore, blocking SOD2 expression by siRNA in HeLa cells increased ROS generation, autophagy and cell death induced by rotenone and TTFA. Rotenone- and TTFA-induced ROS generation was not affected by 3-MA, or by beclin 1 and ATG5 siRNAs. By contrast, treatment of non-transformed primary mouse astrocytes with rotenone or TTFA failed to significantly increase levels of ROS or autophagy. These results indicate that targeting mETC complex I and II selectively induces autophagic cell death through a ROS-mediated mechanism.

Caspases are activated during apoptosis and cleave specific proteins, resulting in the irreversible commitment to cell death. The signal transduction proteins MEKK1, p21-activated kinase 2, and focal adhesion kinase are caspase substrates that contribute to the cell death response when cleaved. Thirty additional signaling proteins were screened for their ability to be cleaved during apoptosis. Twenty-two of these proteins were not affected in Jurkat cells stimulated to undergo apoptosis by Fas ligation, exposure to ultraviolet-C or incubation with etoposide. Ras GTPase-activating protein was found to be a caspase substrate whose cleavage followed the same time course as that for activation of caspase activity and the cleavage of MEKK1 and focal adhesion kinase. Four additional proteins, Cbl, Cbl-b, Raf-1, and Akt-1, were cleaved later in the apoptotic response. Apoptosis is regulated by a series of biochemical events that commit a cell to death. A common feature of cells undergoing apoptosis is the activation of caspases, a family of aspartic acid-directed proteases (1). Caspase substrates are rapidly being identified, but the general assumption is that caspases recognize a limited set of cellular proteins (2). Caspase-mediated proteolysis of specific proteins results in an irreversible commitment of cells to undergo apoptosis characterized by cytoplasmic shrinkage, membrane blebbing, nuclear condensation, and DNA fragmentation.Caspase-dependent cleavage can inactivate protein substrates. Examples include poly(ADP-ribose) polymerase, lamin, and focal adhesion kinase. Cleavage of poly(ADP-ribose) polymerase abolishes its DNA repair ability in cells undergoing apoptosis, nuclear lamin degradation contributes to nuclear condensation, and cleavage of focal adhesion kinase impairs the ability of cells to maintain matrix adherence (2). In contrast, there are examples, besides the caspases themselves (3), where cleavage actually activates the substrate; examples of such substrates include MEKK1, 1 p21-activated kinase, protein kinase C␦, and gelsolin. When cleaved, each of these proteins contributes to the apoptotic response (4 -9).Signal transduction pathways involving the mitogen-activated protein kinases (MAPKs) including the ERKs, JNKs, and p38/HOG1 kinase, have been shown to differentially contribute to pro-and anti-apoptotic pathways (10). In addition, the phosphatidylinositol 3-phosphate-regulated protein kinase Akt has been shown to have significant anti-apoptotic signaling properties (11). This is, at least in part, mediated by the ability of Akt to phosphorylate and inactivate BAD, a pro-apoptotic member of the Bcl family (12,13).In this study, we have surveyed a large set of proteins that are involved in pathways regulating cell growth, cell survival, or cell death, including members of the MAPK network. Among 30 signaling proteins tested, we have identified five new protease substrates, RasGAP, Raf1, Akt-1, Cbl, and Cbl-b, that were found to be cleaved in a caspase-dependent manner during the apoptotic resp...

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Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.