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

The cyclin-dependent kinase inhibitor p21 WAF1/CIP1 is a major player in cell cycle control and it is mainly regulated at the transcriptional level. Whereas induction of p21 predominantly leads to cell cycle arrest, repression of p21 may have a variety of outcomes depending on the context. In this review, we concentrate on transcriptional repression of p21 by cellular and viral factors, and delve in detail into its possible biological implications and its role in cancer. It seems that the major mode of p21 transcriptional repression by negative regulators is the interference with positive transcription factors without direct binding to the p21 promoter.

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Progression through the cell cycle is regulated by cyclins and cyclin-dependent kinases (Cdks). The cyclin kinase inhibitor p21 (also known as WAF1, CIP1, SDI1, and MDA-6) can induce G1 arrest and block entry into S phase by inactivating Cdks or by inhibiting activity of proliferating cell nuclear antigen (PCNA). In normal cells, p21 exists in quaternary complexes with cyclin, Cdk, and PCNA. Transcription of the p21 gene is activated by p53-dependent and -independent mechanisms. Mice deficient in p21 exhibit no apparent phenotype, although p21 function has been demonstrated to be necessary for p53-mediated G1 arrest following irradiation of p21-deficient mouse embryonic fibroblasts. Thus, the function of p21 under normal circumstances appears to be redundant. p21 is expressed in terminally differentiating cells of a variety of tissues in a p53-independent manner. Overexpression of p21 results in G1 arrest and has been shown to suppress effectively tumor growth in vitro and in vivo. We review the recent literature describing the functional characterization of p21. This protein plays a key role in regulating the cell cycle and may have potential gene therapy applications.

The oncogenic transcription factor forkhead box M1 (FoxM1) is overexpressed in a number of different carcinomas, whereas its expression is turned off in terminally differentiated cells. For this reason, FoxM1 is an attractive target for therapeutic intervention in cancer treatment. As a first step toward realizing this goal, in this study, using a high-throughput, cellbased assay system, we screened for and isolated the antibiotic thiazole compound Siomycin A as an inhibitor of FoxM1. Interestingly, we observed that Siomycin A was able to downregulate the transcriptional activity as well as the protein and mRNA abundance of FoxM1. Consequently, we found that the downstream target genes of FoxM1, such as Cdc25B, Survivin, and CENPB, were repressed. Also, we observed that consistent with earlier reports of FoxM1 inhibition, Siomycin A was able to reduce anchorage-independent growth of cells in soft agar. Furthermore, we found that Siomycin A was able to induce apoptosis selectively in transformed but not normal cells of the same origin. Taken together, our data suggest that FoxM1 inhibitor Siomycin A could represent a useful starting point for the development of anticancer therapeutics.

Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G 2 /M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G 2 /M to the G 1 phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G 2 arrest that is alleviated by activated Akt. Furthermore, the transition from the G 2 /M to the G 1 phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G 2 /M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G 2 /M cell cycle checkpoint and apoptosis in MMRdeficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.Akt, or protein kinase B (PKB), is a serine/threonine kinase that has been implicated in the control of major cellular functions such as transcription, protein synthesis, and carbohydrate and lipid metabolism, and it is a downstream effector of growth factor-mediated cell survival. Normally, Akt is activated by growth factors that activate phosphoinositol-3-kinase (PI3K). Upon activation, PI3K phosphorylates the inositol ring at the D3 position, which in turn serves to anchor Akt to the plasma membrane, where it is phosphorylated and fully activated by the 3-phosphoinositide-dependent kinases PDK1 and PDK2. Phospholipid phosphatases such as PTEN and SHIP decrease the pool of available phospholipids and therefore are negative regulators of Akt. Activated Ras, at least in certain circumstances, can up-regulate PI3K and therefore is a potential activator of Akt as well (13,22). Overall, positive regulators of Akt are commonly up-regulated in human cancers, while PTEN is frequently lost or inactivated by mutations (7, 36). Furthermore, heterozygous deletion of PTEN in mice elicits a wide range of spontaneous tumors; this has been attributed mainly to activation of Akt (12,34,38). Finally, activated forms of Akt induce cellular transformation (4). ...

Proteasome inhibitors are currently in the clinic or in clinical trials, but the mechanism of their anticancer activity is not completely understood. The oncogenic transcription factor FoxM1 is one of the most overexpressed genes in human tumors, while its expression is usually halted in normal non-proliferating cells. Previously, we established that thiazole antibiotics Siomycin A and thiostrepton inhibit FoxM1 and induce apoptosis in human cancer cells. Here, we report that Siomycin A and thiostrepton stabilize the expression of a variety of proteins, such as p21, Mcl-1, p53 and hdm-2 and also act as proteasome inhibitors in vitro. More importantly, we also found that well-known proteasome inhibitors such as MG115, MG132 and bortezomib inhibit FoxM1 transcriptional activity and FoxM1 expression. In addition, overexpression of FoxM1 specifically protects against bortezomib-, but not doxorubicin-induced apoptosis. These data suggest that negative regulation of FoxM1 by proteasome inhibitors is a general feature of these drugs and it may contribute to their anticancer properties.