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

Topoisomerase I (TOP1) relaxes superhelical DNA through a breakage/rejoining reaction in which the active site tyrosine links covalently to a 3 phosphate at the break site as a transient intermediate. The antitumor drug camptothecin (CPT) and its analogs inhibit the rejoining step of the breakage/rejoining reaction, which traps the enzyme in covalent linkage with DNA (the cleavable complex). Little is known about the fate of cellular TOP1 trapped in the cleavable complex. We have analyzed TOP1 in mammalian cell lines treated with CPT. When CPT-treated cells were lysed with either SDS or alkali and analyzed by Western blotting, greater than 90% of the TOP1 was linked to DNA. Nuclease treatment of the cell lysate to remove the covalently linked DNA from TOP1 revealed a distinct ladder of higher molecular weight bands having properties indicative of multi-ubiquitin (Ub) conjugates of TOP1. Approximately 5-10% of TOP1 was present as these conjugates within minutes of CPT treatment. Consistent with ubiquitination, TOP1 was not modified in ts85 cells at the restrictive temperature for its thermolabile ubiquitin-activating enzyme (E1). Because conjugation with ubiquitin can mark proteins for destruction by the 26S proteasome, we analyzed TOP1 protein levels during prolonged CPT treatment. TOP1 protein levels were reduced to about 25% during CPT treatments of 2-4 h resulting from increased destruction, with the half-life dropping from 10 -16 h down to 1-2 h. The destruction of TOP1, like the formation of Ub-TOP1 conjugates, was not observed in ts85 cells at the restrictive temperature. The destruction of TOP1 was also prevented in cells treated with MG-132 and lactacystin, specific inhibitors of the 26S proteasome. Finally, the multi-Ub conjugates of TOP1 were observed whether or not aphidicolin was included in cotreatment with CPT, indicating that replication fork activity was not involved in making TOP1 a substrate for ubiquitination. These results demonstrate that independent of DNA replication, the TOP1 cleavable complex is ubiquitinated and destroyed in cells treated with antitumor drugs that block the religation step of the TOP1 reaction. DNA topoisomerase I (TOP1)1 is a vital DNA metabolic enzyme as well as a molecular target of antitumor drugs. TOP1 relaxes DNA supercoils by cleaving a single strand of duplex DNA and passing the complimentary DNA strand though the cleaved strand before religation (1). In a transient reaction intermediate, termed the cleavable complex, the enzyme links covalently to DNA through tyrosine 723 (human TOP1) leaving a DNA break having a free 5Ј hydroxyl end. Stopping the TOP1 reaction with strong protein denaturants produces a very low yield of TOP1 linked covalently to DNA. The rarity of this product is a reflection of the transient nature of the "cleaved" reaction intermediate. Antitumor drugs that act on TOP1 inhibit the DNA religation step through a reversible mechanism. The inhibition of religation can be observed as an increase in the yield of the enzyme-DNA covalent complex pro...

IFN-stimulatory gene factor 15 (ISG15) is a ubiquitin-like protein, which is conjugated to many cellular proteins. However, its role in protein degradation is unclear. Here, we show that ISG15 is highly elevated and extensively conjugated to cellular proteins in many tumors and tumor cell lines. The increased levels of ISG15 in tumor cells were found to be associated with decreased levels of polyubiquitinated proteins. Specific knockdown of ISG15 expression using ISG15-specific small interfering RNA (siRNA) was shown to increase the levels of polyubiquitinated proteins, suggesting an antagonistic role of ISG15 in regulating ubiquitin-mediated protein turnover. Moreover, siRNA-mediated down-regulation of the major E2 for ISG15 (UbcH8), which blocked the formation of ISG15 protein conjugates, also increased the levels of polyubiquitinated proteins. Together, our results suggest that the ISG15 pathway, which is deregulated during tumorigenesis, negatively regulates the ubiquitin/proteasome pathway by interfering with protein polyubiquitination/ degradation. (Cancer Res 2006; 66(2): 921-8)

DNA topoisomerase II (TOP2) cleavable complexes represent an unusual type of DNA damage characterized by reversible TOP2-DNA cross-links and DNA double strand breaks. Many antitumor drugs and physiological stresses are known to induce TOP2 cleavable complexes leading to apoptotic cell death and genomic instability. However, the molecular mechanism(s) for repair of TOP2 cleavable complexes remains unclear. In the current studies, we show that TOP2 cleavable complexes induced by the prototypic TOP2 poison VM-26 are proteolytically degraded by the ubiquitin/26 S proteasome pathway. Surprisingly the TOP2␤ isozyme is preferentially degraded over TOP2␣ isozyme. In addition, transcription inhibitors such as 5,6-dichlorobenzimidazole riboside and camptothecin can substantially block VM-26-induced TOP2␤ degradation. These results are consistent with a model in which the repair of TOP2␤ cleavable complexes may involve transcription-dependent proteolysis of TOP2␤ to reveal the protein-concealed double strand breaks.DNA topoisomerases are double-edged swords. They are essential for many important processes of DNA such as DNA replication, RNA transcription, chromosome condensation/decondensation, and chromosome segregation (1). However, due to their delicate act on DNA, they are also highly vulnerable to xenobiotics and physiological stresses to produce topoisomerase-mediated DNA damage, mostly in the form of topoisomerase cleavable complexes (2-5). So far, five human DNA topoisomerases, topoisomerase I (TOP1), 1 TOP2␣, TOP2␤, TOP3␣, and TOP3␤, have been identified and characterized, and the first three have been demonstrated to be important molecular targets for antitumor drugs (1, 6 -10).Both hTOP2 isozymes have been demonstrated to be the cellular targets for many clinically useful anticancer drugs such as VP-16 (etoposide) and doxorubicin (11-13). In the presence of these TOP2-directed drugs (TOP2 poisons), TOP2 isozymes are trapped as their covalent reaction intermediates, the reversible TOP2 cleavable complexes in which each TOP2 subunit is covalently linked to the 5Ј-phosphoryl ends of the four-base staggered double strand breaks (14,15). While the double strand breaks within the TOP2 cleavable complexes are normally concealed by TOP2, many of the cellular effects of TOP2 cleavable complexes are clearly indicative of DNA damage. For example, TOP2 cleavable complexes induced by TOP2 poisons are known to induce DNA damage responses (e.g. G 2 arrest, elevation of sister-chromatid exchanges, NFB activation, and p53 stabilization) (16 -19). DNA repair mutant cells (e.g. ataxia telangiectasia, progeroid Werner's syndrome, and Rad52) are also known to be hypersensitive to TOP2 poisons (20 -22). However, how TOP2-concealed DNA strand breaks are converted to DNA damage signals is still unknown. Inhibitor studies have suggested that both DNA replication and RNA transcription may be important for processing TOP2 cleavable complexes into DNA damage signals (23-25).Repair of topoisomerase cleavable complexes is conceptually challe...

Camptothecin (CPT) class of compounds has been demonstrated to be effective against a broad spectrum of tumors. Their molecular target has been firmly established to be human DNA topoisomerase I (topo I). CPT inhibits topo I by blocking the rejoining step of the cleavage/religation reaction of topo‐I, resulting in accumulation of a covalent reaction intermediate, the cleavable complex. The primary mechanism of cell killing by CPT is S‐phase‐specific killing through potentially lethal collisions between advancing replication forks and topo‐I cleavable complexes. Collisions with the transcription machinery have also been shown to trigger the formation of long‐lived covalent topo‐I DNA complexes, which contribute to CPT cytotoxicity. Two novel repair responses to topo‐I‐mediated DNA damage involving covalent modifications of topo‐I have been discovered. The first involves activation of the ubiquitin/26S proteasome pathway, leading to degradation of topo‐I (CPT‐induced topo‐I downregulation). The second involves SUMO conjugation to topo‐I. The potential roles of these new mechanisms for repair of topo‐I‐mediated DNA damage in determining CPT sensitivity/resistance in tumor cells are discussed.

Topoisomerase I (Top I)-DNA covalent complexes represent a unique type of DNA lesion whose repair and processing remain unclear. In this study, we show that Top I-DNA covalent complexes transiently arrest RNA transcription in normal nontransformed cells. Arrest of RNA transcription is coupled to activation of proteasomal degradation of Top I and the large subunit of RNA polymerase II. Recovery of transcription occurs gradually and depends on both proteasomal degradation of Top I and functional transcription-coupled repair (TCR). These results suggest that arrest of the RNA polymerase elongation complex by the Top I-DNA covalent complex triggers a 26S proteasome-mediated signaling pathway(s) leading to degradation of both Top I and the large subunit of RNA polymerase II. We propose that proteasomal degradation of Top I and RNA polymerase II precedes repair of the exposed single-strand breaks by TCR.

Ubiquitin͞26S proteasome-dependent degradation of topoisomerase I (TOP1) has been suggested to be a unique repair response to TOP1-mediated DNA damage. In the current study, we show that treatment of mammalian cells or yeast cells expressing human DNA TOP1 with camptothecin (CPT) induces covalent modification of the TOP1 by SUMO-1͞Smt3p, a ubiquitin-like protein. This conclusion is based on the following observations: (i) Mammalian DNA TOP1 conjugates induced by CPT were cross-reactive with SUMO-1͞Smt3p-specific antibodies both in yeast expressing human DNA TOP1 as well as mammalian cells. (ii) The formation of TOP1 conjugates was shown to be dependent on UBC9, the E2 enzyme for SUMO-1͞Smt3p. (iii) TOP1 physically interacts with UBC9. (iv) Ubc9 mutant yeast cells expressing human DNA TOP1 was hypersensitive to CPT, suggesting that UBC9͞SUMO-1 may be involved in the repair of TOP1-mediated DNA damage.UBC9 ͉ Smt3p ͉ ubiquitin T opoisomerase-mediated DNA damage represents an unique type of DNA damage whose importance has become increasingly appreciated. Many antibiotics, anticancer drugs, toxins, carcinogens, and physiological stresses are known to abort the catalytic cycles of topoisomerases resulting in the formation of topoisomerase-mediated DNA damage (1-7). Despite its importance, little is known about the molecular basis for the repair of this unique type of DNA damage.Recent studies have demonstrated that topoisomerase I-(TOP1) mediated DNA damage activates a ubiquitin͞proteasome pathway, resulting in degradation of TOP1 (down-regulation of TOP1) (8). It has been speculated that this pathway may represent a unique repair mechanism for TOP1-mediated DNA damage because down-regulation of human TOP1 (hTOP1) is expected to result in resistance͞tolerance to TOP1 poisons (8).Human SUMO-1 (small ubiquitin-like modifier) is an 11-kDa protein that shares 18% sequence homology with ubiquitin (9-11). Smt3p, which is 48% identical to human SUMO-1, has been identified in yeast (12,13). SUMO-1͞Smt3p is activated by a heterodimeric E1 enzyme in both the yeast and mammalian systems (13)(14)(15). In addition, UBC9, an E2 enzyme, has been shown to specifically conjugate SUMO-1͞Smt3p to the target proteins (11,(16)(17)(18). Recently, an enzyme Ulp1p, which specifically cleaves proteins from SUMO-1͞Smt3p conjugates and is distinct from isopeptidases for ubiquitin conjugates, also has been identified in yeast (19). These results suggest that the SUMO-1͞Smt3p pathway is very similar to the ubiquitin pathway but distinct E1 and E2 enzymes as well as proteases are involved in these pathways. The biological function of the SUMO-1͞Smt3p pathway appears diverse. UBC9 was originally identified to play an essential function in G 2 -M cell cycle progression in yeast (20). However, UBC9 is also known to be important for DNA repair (21). It has been shown that SUMO-1 can be conjugated to a number of proteins such as PML (22), RanGAP1 (23, 24), I B␣ (25), FAS͞apolipoprotein-1 (26), p53 (27, 28), RAD51, and RAD52 (29). A growing number of pr...

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 downregulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process.Eukaryotic DNA topoisomerase I (Top1) 3 catalyzes the breakage/reunion of DNA by transiently nicking one strand of the DNA duplex, through the formation of a reversible Top1-DNA covalent complex (reviewed in Refs. 1-4). It has been well established that anticancer camptothecins (e.g. irinotecan and topotecan) as well as various structurally perturbed DNAs (e.g. UV adducts, abasic sites, base mismatches, uracil incorporation, nicks and gaps, and oxidized DNA lesions) stabilize reversible Top1-DNA covalent complexes, often referred to as Top1 cleavable or cleavage complexes (reviewed in Refs. 3 and 5).Studies using camptothecins (CPTs) have generated a wealth of information on the structure and biology of Top1 cleavage complexes (reviewed in Refs. 3 and 5). It is well established that CPTs exert their antitumor activity through their specific stabilization of Top1 cleavage complexes (6). Top1 cleavage complexes are unique DNA lesions, characterized by their reversibility and Top1-concealed single-strand breaks (SSBs) (6). It has been hypothesized that reversible Top1 cleavage complexes are processed into DNA damage through their interactions with cellular machineries associated with active DNA replication and transcription (7-11).The role of active DNA replication in the processing of Top1 cleavage complexes into DNA damage was initially sugges...