Publications (29)238.27 Total impact
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Article: Guidelines for the use and interpretation of assays for monitoring autophagy.
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ABSTRACT: 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.Autophagy 04/2012; 8(4):445-544. · 7.45 Impact Factor -
Article: Mitochondrial complex III: Tuner of autophagy.
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ABSTRACT: Using chemical approaches, Ma et al. in this issue of Chemistry & Biology identify mitochondrial complex III as a specific positive regulator of autophagy. This study brings us a step closer to understanding the mechanism by which basal autophagy is coupled to cellular energy flux.Chemistry & biology 11/2011; 18(11):1348-9. · 6.52 Impact Factor -
Article: Use of the ODD-luciferase transgene for the non-invasive imaging of spontaneous tumors in mice.
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ABSTRACT: In humans, imaging of tumors provides rapid, accurate assessment of tumor growth and location. In laboratory animals, however, the imaging of spontaneously occurring tumors continues to pose many technical and logistical problems. Recently a mouse model was generated in which a chimeric protein consisting of HIF-1α oxygen-dependent degradation domain (ODD) fused to luciferase was ubiquitously expressed in all tissues. Hypoxic stress leads to the accumulation of ODD-luciferase in the tissues of this mouse model which can be identified by non-invasive bioluminescence measurement. Since solid tumors often contain hypoxic regions, we performed proof-of-principle experiments testing whether this transgenic mouse model may be used as a universal platform for non-invasive imaging analysis of spontaneous solid tumors. ODD-luciferase transgenic mice were bred with MMTV-neu/beclin1+/- mice. Upon injection of luciferin, bioluminescent background of normal tissues in the transgenic mice and bioluminescent signals from spontaneously mammary carcinomas were measured non-invasively with an IVIS Spectrum imaging station. Tumor volumes were measured manually and the histology of tumor tissues was analyzed. Our results show that spontaneous mammary tumors in ODD-luciferase transgenic mice generate substantial bioluminescent signals, which are clearly discernable from background tissue luminescence. Moreover, we demonstrate a strong quantitative correlation between the bioluminescent tumor contour and the volume of palpable tumors. We further demonstrate that shrinkage of the volume of spontaneous tumors in response to chemotherapeutic treatment can be determined quantitatively using this system. Finally, we show that the growth and development of spontaneous tumors can be monitored longitudinally over several weeks. Thus, our results suggest that this model could potentially provide a practical, reliable, and cost-effective non-invasive quantitative method for imaging spontaneous solid tumors in mice.PLoS ONE 01/2011; 6(3):e18269. · 4.09 Impact Factor -
Article: Autophagic degradation of mitochondria in white adipose tissue differentiation.
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ABSTRACT: Recent work has revealed that autophagy plays a significant role in the process of white adipocyte differentiation. In both in vitro and in vivo model systems, autophagy inactivation by targeted deletion of essential autophagy genes results in alterations in white adipocyte structure. In both models, postdifferentiation cells exhibit atypical morphology, with many small lipid droplets and large numbers of mitochondria, rather than the single large lipid droplet and relatively few mitochondria observed in normal white adipocytes. The role of autophagy as the primary means of the degradation of mitochondria has long been studied, and it is likely that a deficiency in the degradation of mitochondria contributes to the unusual phenotypes observed in mice with autophagy-deficient adipose tissue, including reduced adiposity, resistance to diet-induced obesity, and increased insulin sensitivity. What is not yet known is whether the process of mitochondria-specific autophagy, often referred to as "mitophagy," is specifically induced during adipogenesis or if a general increase in the nonspecific autophagic degradation of mitochondria plays a role in normal adipose differentiation. Despite remaining questions, these findings not only establish the critical role of autophagy in white adipose tissue development, but also suggest that the manipulation of autophagy in adipose tissue may provide novel therapeutic opportunities for metabolic diseases.Antioxidants & Redox Signaling 12/2010; 14(10):1971-8. · 8.20 Impact Factor -
Article: Autophagy and the degradation of mitochondria.
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ABSTRACT: The cellular process of macromolecular degradation known as macroautophagy has long been known to play a role in the elimination of mitochondria. Over the past decade, much progress has been made in the development of systems by which the nature and mechanism of mitochondria degradation may be studied. Recent findings imply that the degradation of mitochondria via autophagy may be more specific and more tightly regulated than originally thought, and have led to designation of this specific type of autophagy as "mitophagy". In this review we provide a brief history of the development of mitophagy models and their associated discoveries.Mitochondrion 06/2010; 10(4):309-15. · 3.62 Impact Factor -
Article: Autophagy and adipogenesis: implications in obesity and type II diabetes.
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ABSTRACT: Obesity is a direct result of the accumulation of white adipose tissue (WAT). In this study, the role of autophagy in the differentiation of white adipose tissue was studied by deleting the autophagy-related 7 (atg7) gene from adipose tissue in mice. This deletion results in a striking phenotype at the cellular, tissue and whole-organism levels. Adipose tissue deposits in the mutant mice are much smaller in mass than those observed in their wild-type counterparts, and mutant adipocytes exhibit unusual morphological characteristics including multilocular lipid droplets and greatly increased numbers of mitochondria. The knockout mice are noticeably slimmer than their wild-type littermates, despite parity in food and water consumption. The mutant mice also exhibit higher basal physical activity levels and an array of metabolic changes revealed through blood tests. Most importantly, these mice show resistance to high-fat diet-induced obesity and markedly increased sensitivity to insulin. These findings establish a new function for autophagy and provide a new model system for use in the search for treatments for obesity and type II diabetes.Autophagy 01/2010; 6(1):179-81. · 7.45 Impact Factor -
Article: Targeted deletion of autophagy-related 5 (atg5) impairs adipogenesis in a cellular model and in mice.
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ABSTRACT: Mammalian white adipocytes have a unique structure in which nearly the entire cell volume is occupied by a single large lipid droplet, while the surrounding cytoplasm occupies minimal space. The massive cytoplasmic remodeling processes involved in the formation of this unique cellular structure are poorly defined. Autophagy is a membrane trafficking process leading to lysosomal degradation of cytoplasmic components. Here, we investigated the functional role of atg5, a gene encoding an essential protein required for autophagy, in adipocyte differentiation in a cellular model and in mice. Massive autophagy was activated when wild-type primary mouse embryonic fibroblasts (MEFs) were induced for adipocyte differentiation. Importantly, the autophagy deficient primary atg5(-/-) MEFs exhibited dramatically reduced efficiency in adipogenesis. Time-lapse microscopy revealed that atg5(-/-) MEFs initially appeared to differentiate normally; however, a majority of the differentiating atg5(-/-) cells ultimately failed to undergo further morphological transformation and eventually died, likely through apoptosis. Consistent with these in vitro results, histological analysis revealed that the atg5(-/-) late-stage embryos and neonatal pups had much less subcutaneous perilipin A-positive adipocytes. Consistently, when treated with chloroquine, a functional inhibitor of autophagy, wild-type MEFs exhibited drastically reduced efficiency of adipocyte differentiation. Taken together, these findings demonstrated that Atg5 is involved in normal adipocyte differentiation, suggesting an important role of autophagy in adipogenesis.Autophagy 11/2009; 5(8):1118-30. · 7.45 Impact Factor -
Article: Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis.
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ABSTRACT: White adipocytes have a unique structure in which nearly the entire cell volume is occupied by one large lipid droplet. However, the molecular and cellular processes involved in the cytoplasmic remodeling necessary to create this structure are poorly defined. Autophagy is a membrane trafficking process leading to lysosomal degradation. Here, we investigated the effect of the deletion of an essential autophagy gene, autophagy-related gene 7 (atg7), on adipogenesis. A mouse model with a targeted deletion of atg7 in adipose tissue was generated. The mutant mice were slim and contained only 20% of the mass of white adipose tissue (WAT) found in wild-type mice. Interestingly, approximately 50% of the mutant white adipocytes were multilocular. The mutant white adipocytes were smaller with a larger volume of cytosol and contained more mitochondria. These cells exhibited altered fatty acid metabolism with increased rates of beta-oxidation and reduced rates of hormone-induced lipolysis. Consistently, the mutant mice had lower fed plasma concentrations of fatty acids and the levels decreased at faster rates upon insulin stimuli. These mutant mice exhibited increased insulin sensitivity. The mutant mice also exhibited markedly decreased plasma concentrations of leptin but not adiponectin, lower plasma concentrations of triglyceride and cholesterol, and they had higher levels of basal physical activity. Strikingly, these mutant mice were resistant to high-fat-diet-induced obesity. Taken together, our results indicate that atg7, and by inference autophagy, plays an important role in normal adipogenesis and that inhibition of autophagy by disrupting the atg7 gene has a unique anti-obesity and insulin sensitization effect.Proceedings of the National Academy of Sciences 11/2009; 106(47):19860-5. · 9.68 Impact Factor -
Article: Productive Chlamydia trachomatis lymphogranuloma venereum 434 infection in cells with augmented or inactivated autophagic activities.
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ABSTRACT: Autophagy, a eukaryotic cellular activity leading to the degradation of cellular components, serves as a defense mechanism against facultative intracellular bacteria as well as a growth niche for the obligate intracellular bacterium Coxiella burnetii. We here demonstrate that the obligate intracellular bacterial pathogen Chlamydia trachomatis lymphogranuloma venereum strongly induced autophagy in the middle of the chlamydial developmental cycle (24 h after infection), a time point with maximal level of chlamydial replication, but not during the early stages with low overall chlamydial metabolism (before 8 h). No autophagy induction was evident in cells exposed to heat- and UV-inactivated elementary bodies (EBs, the infectious form of Chlamydia) or to inocula from which EBs had been removed before inoculation. Blocking chlamydial development with chloramphenicol also prevented autophagy induction in cells infected with infectious EBs. It appears that autophagy is activated primarily in response to the metabolic stress consequent to chlamydial replication. However, autophagy-defective ATG5(-/-) cells supported chlamydial development as efficiently as autophagy-proficient ATG5(+/+) cells.FEMS Microbiology Letters 02/2009; 292(2):240-9. · 2.04 Impact Factor -
Article: Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.
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ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.Autophagy 02/2008; 4(2):151-75. · 7.45 Impact Factor -
Article: Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis.
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ABSTRACT: Autophagy is a catabolic process involving self-digestion of cellular organelles during starvation as a means of cell survival; however, if it proceeds to completion, autophagy can lead to cell death. Autophagy is also a haploinsufficient tumor suppressor mechanism for mammary tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in breast carcinomas. However, the mechanism by which autophagy suppresses breast cancer remains elusive. Here we show that allelic loss of beclin1 and defective autophagy sensitized mammary epithelial cells to metabolic stress and accelerated lumen formation in mammary acini. Autophagy defects also activated the DNA damage response in vitro and in mammary tumors in vivo, promoted gene amplification, and synergized with defective apoptosis to promote mammary tumorigenesis. Therefore, we propose that autophagy limits metabolic stress to protect the genome, and that defective autophagy increases DNA damage and genomic instability that ultimately facilitate breast cancer progression.Genes & Development 08/2007; 21(13):1621-35. · 11.66 Impact Factor -
Article: Autophagy suppresses tumor progression by limiting chromosomal instability.
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ABSTRACT: Autophagy is a bulk degradation process that promotes survival under metabolic stress, but it can also be a means of cell death if executed to completion. Monoallelic loss of the essential autophagy gene beclin1 causes susceptibility to metabolic stress, but also promotes tumorigenesis. This raises the paradox that the loss of a survival pathway enhances tumor growth, where the exact mechanism is not known. Here, we show that compromised autophagy promoted chromosome instability. Failure to sustain metabolism through autophagy was associated with increased DNA damage, gene amplification, and aneuploidy, and this genomic instability may promote tumorigenesis. Thus, autophagy maintains metabolism and survival during metabolic stress that serves to protect the genome, providing an explanation for how the loss of a survival pathway leads to tumor progression. Identification of this novel role of autophagy may be important for rational chemotherapy and therapeutic exploitation of autophagy inducers as potential chemopreventive agents.Genes & Development 07/2007; 21(11):1367-81. · 11.66 Impact Factor -
Article: The regulation of AMPK beta1, TSC2, and PTEN expression by p53: stress, cell and tissue specificity, and the role of these gene products in modulating the IGF-1-AKT-mTOR pathways.
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ABSTRACT: The insulin-like growth factor 1 (IGF-1)-AKT-mTOR pathways sense the availability of nutrients and mitogens and respond by signaling for cell growth and division. The p53 pathway senses a variety of stress signals which will reduce the fidelity of cell growth and division, and responds by initiating cell cycle arrest, senescence, or apoptosis. This study explores four p53-regulated gene products, the beta1 and beta2 subunits of the AMPK, which are shown for the first time to be regulated by the p53 protein, TSC2, PTEN, and IGF-BP3, each of which negatively regulates the IGF-1-AKT-mTOR pathways after stress. These gene products are shown to be expressed under p53 control in a cell type and tissue-specific fashion with the TSC2 and PTEN proteins being coordinately regulated in those tissues that use insulin-dependent energy metabolism (skeletal muscle, heart, white fat, liver, and kidney). In addition, these genes are regulated by p53 in a stress signal-specific fashion. The mTOR pathway also communicates with the p53 pathway. After glucose starvation of mouse embryo fibroblasts, AMPK phosphorylates the p53 protein but does not activate any of the p53 responses. Upon glucose starvation of E1A-transformed mouse embryo fibroblasts, a p53-mediated apoptosis ensues. Thus, there is a great deal of communication between the p53 pathway and the IGF-1-AKT and mTOR pathways.Cancer Research 05/2007; 67(7):3043-53. · 7.86 Impact Factor -
Chapter: The p53 Network
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ABSTRACT: Cancer arises through a series of mutations in selected oncogenes, tumor suppressor genes, or genes involved in DNA repair or replication. The tumor suppressor gene products frequently monitor the efficiency of cellular duplication by populating checkpoints in the process of cell division. When defective, the tumor suppressor genes can lead to inherited predispositions in the development of cancers. Almost every human cancer contains mutations in the tumor suppressor pathways of p53, retinoblastoma (Rb), or both. Each of these pathways receives a complex set of signals and reports from the extracellular and intracellular environments of a cell and in response regulate “go-no go” decisions in the cell cycle. This chapter will review some of the origins of research into the p53 gene and its protein. This will form a basis for understanding the other chapters of this book and provide a foundation upon which new facts are built. It also points to important future directions for this field.03/2007: pages 1-23; -
Article: Metabolic catastrophe as a means to cancer cell death.
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ABSTRACT: During tumorigenesis, normal growth mechanisms are deregulated and safeguards that eliminate abnormal cells by apoptosis are disabled. Tumor cells must also increase nutrient uptake and angiogenesis to support the upregulation of metabolism necessary for unrestricted growth. In addition, they have to rely on inefficient energy production by glycolysis. This glycolytic state can result from mutations that promote cell proliferation, the hypoxic tumor microenvironment and perhaps mitochondrial malfunction. Moreover, the very signals that enable unrestricted cell proliferation inhibit autophagy, which normally sustains cells during nutrient limitation. In tumors, inactivation of the autophagy pathway may enhance necrosis and inflammation and promote genomic instability, which can further enhance tumor growth. Thus, tumor cells cannot adapt efficiently to metabolic stress and could be induced to die by metabolic catastrophe, in which high energy demand is contrasted by insufficient energy production. Efforts to exploit this unique metabolic state clinically previously focused mainly on detecting tissue displaying increased glycolytic metabolism. The challenge now is to induce metabolic catastrophe therapeutically as an approach to killing the unkillable cells.Journal of Cell Science 03/2007; 120(Pt 3):379-83. · 6.11 Impact Factor -
Article: Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis.
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ABSTRACT: Defective apoptosis renders immortalized epithelial cells highly tumorigenic, but how this is impacted by other common tumor mutations is not known. In apoptosis-defective cells, inhibition of autophagy by AKT activation or by allelic disruption of beclin1 confers sensitivity to metabolic stress by inhibiting an autophagy-dependent survival pathway. While autophagy acts to buffer metabolic stress, the combined impairment of apoptosis and autophagy promotes necrotic cell death in vitro and in vivo. Thus, inhibiting autophagy under conditions of nutrient limitation can restore cell death to apoptosis-refractory tumors, but this necrosis is associated with inflammation and accelerated tumor growth. Thus, autophagy may function in tumor suppression by mitigating metabolic stress and, in concert with apoptosis, by preventing death by necrosis.Cancer Cell 08/2006; 10(1):51-64. · 26.57 Impact Factor -
Article: A matter of life or death (or both): understanding autophagy in cancer.
Clinical Cancer Research 05/2006; 12(7 Pt 1):1961-5. · 7.74 Impact Factor -
Article: Elongation factor-2 kinase regulates autophagy in human glioblastoma cells.
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ABSTRACT: Elongation factor-2 kinase (eEF-2 kinase), also known as Ca(2+)/calmodulin-dependent kinase III, regulates protein synthesis by controlling the rate of peptide chain elongation. The activity of eEF-2 kinase is increased in glioblastoma and other malignancies, yet its role in neoplasia is uncertain. Recent evidence suggests that autophagy plays an important role in oncogenesis and that this can be regulated by mammalian target of rapamycin (mTOR). Because eEF-2 kinase lies downstream of mTOR, we studied the role of eEF-2 kinase in autophagy using human glioblastoma cell lines. Knockdown of eEF-2 kinase by RNA interference inhibited autophagy in glioblastoma cell lines, as measured by light chain 3 (LC3)-II formation, acidic vesicular organelle staining, and electron microscopy. In contrast, overexpression of eEF-2 kinase increased autophagy. Furthermore, inhibition of autophagy markedly decreased the viability of glioblastoma cells grown under conditions of nutrient depletion. Nutrient deprivation increased eEF-2 kinase activity and decreased the activity of S6 kinase, suggesting an involvement of mTOR pathway in the eEF-2 kinase regulation of autophagy. These results suggest that eEF-2 kinase plays a regulatory role in the autophagic process in tumor cells; and eEF-2 kinase is a downstream member of the mTOR signaling; eEF-2 kinase may promote cancer cell survival under conditions of nutrient deprivation through regulating autophagy. Therefore, eEF-2 kinase may be a part of a survival mechanism in glioblastoma and targeting this kinase may represent a novel approach to cancer treatment.Cancer Research 04/2006; 66(6):3015-23. · 7.86 Impact Factor -
Article: Coordination and communication between the p53 and IGF-1-AKT-TOR signal transduction pathways.
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ABSTRACT: Over the past 10 years the signal transduction networks for p53, IGF-1-AKT, and TOR pathways have been assembled in worms, flies, and mammals, and their functions elucidated. In the past 1-2 years a number of genes and their proteins have been identified that permit extensive communication and coordination between these pathways. These three pathways are involved in sensing and integrating signals arising from nutrient and growth factor availability, signals from sensory and sexual organs, and intrinsic and extrinsic stress signals. In turn these pathways regulate cell growth, proliferation, and death. These networks are central to our understanding of a variety of physiological and pathological conditions, including cancer, diabetes, and longevity.Genes & Development 03/2006; 20(3):267-75. · 11.66 Impact Factor -
Article: The coordinate regulation of the p53 and mTOR pathways in cells.
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ABSTRACT: Cell growth and proliferation requires an intricate coordination between the stimulatory signals arising from nutrients and growth factors and the inhibitory signals arising from intracellular and extracellular stresses. Alteration of the coordination often causes cancer. In mammals, the mTOR (mammalian target of rapamycin) protein kinase is the central node in nutrient and growth factor signaling, and p53 plays a critical role in sensing genotoxic and other stresses. The results presented here demonstrate that activation of p53 inhibits mTOR activity and regulates its downstream targets, including autophagy, a tumor suppression process. Moreover, the mechanisms by which p53 regulates mTOR involves AMP kinase activation and requires the tuberous sclerosis (TSC) 1/TSC2 complex, both of which respond to energy deprivation in cells. In addition, glucose starvation not only signals to shut down mTOR, but also results in the transient phosphorylation of the p53 protein. Thus, p53 and mTOR signaling machineries can cross-talk and coordinately regulate cell growth, proliferation, and death.Proceedings of the National Academy of Sciences 07/2005; 102(23):8204-9. · 9.68 Impact Factor
Top co-authors
Top Journals
Institutions
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2004–2011
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Robert Wood Johnson University Hospital
New Brunswick, NJ, USA
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2006
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Institute for Advanced Study
Jersey City, NJ, USA
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2003
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The Rockefeller University
New York City, NY, USA
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