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.

For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

https://biblio.ugent.be/publication/8555786/file/8555788.pdf

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

/pdf/molecular-definitions-of-cell-death-subroutines-q5g428rlyi.pdf

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

PLoS BIOLOGY Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor Jean-Philippe Coppe 1 , Christopher K. Patil 1[ , Francis Rodier 1,2[ , Yu Sun 3 , Denise P. Mun oz 1,2 , Joshua Goldstein 1¤ , Peter S. Nelson 3 , Pierre-Yves Desprez 1,4 , Judith Campisi 1,2* 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 2 Buck Institute for Age Research, Novato, California, United States of America, 3 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 California Pacific Medical Center Research Institute, San Francisco, California, United States of America Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA- damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment. Citation: Coppe JP, Patil CK, Rodier F, Sun Y, Mun oz DP, et al. (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12): e301. doi:10.1371/journal.pbio.0060301 Introduction Cancer is a multistep disease in which cells acquire increasingly malignant phenotypes. These phenotypes are acquired in part by somatic mutations, which derange normal controls over cell proliferation (growth), survival, invasion, and other processes important for malignant tumorigenesis [1]. In addition, there is increasing evidence that the tissue microenvironment is an important determinant of whether and how malignancies develop [2,3]. Normal tissue environ- ments tend to suppress malignant phenotypes, whereas abnormal tissue environments such at those caused by inﬂammation can promote cancer progression. Cancer development is restrained by a variety of tumor suppressor genes. Some of these genes permanently arrest the growth of cells at risk for neoplastic transformation, a process termed cellular senescence [4–6]. Two tumor suppressor pathways, controlled by the p53 and p16INK4a/pRB proteins, regulate senescence responses. Both pathways integrate multiple aspects of cellular physiology and direct cell fate towards survival, death, proliferation, or growth arrest, depending on the context [7,8]. Several lines of evidence indicate that cellular senescence is a potent tumor-suppressive mechanism [4,9,10]. Many poten- tially oncogenic stimuli (e.g., dysfunctional telomeres, DNA PLoS Biology | www.plosbiology.org damage, and certain oncogenes) induce senescence [6,11]. Moreover, mutations that dampen the p53 or p16INK4a/pRB pathways confer resistance to senescence and greatly increase cancer risk [12,13]. Most cancers harbor mutations in one or both of these pathways [14,15]. Lastly, in mice and humans, a senescence response to strong mitogenic signals, such as those delivered by certain oncogenes, prevents premalignant lesions from progressing to malignant cancers [16–19]. Academic Editor: Julian Downward, Cancer Research UK, United Kingdom Received June 27, 2008; Accepted October 22, 2008; Published December 2, 2008 Copyright: O 2008 Coppe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abbreviations: CM, conditioned medium; DDR, DNA damage response; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial–mesenchymal transition; GSE, genetic suppressor element; IL, interleukin; MIT, mitoxantrone; PRE, presenescent; PrEC, normal human prostate epithelial cell; REP, replicative exhaustion; SASP, senescence-associated secretory phenotype; SEN, senescent; shRNA, short hairpin RNA; XRA, X-irradiation * To whom correspondence should be addressed. E-mail: jcampisi@lbl.gov [ These authors contributed equally to this work. ¤ Current address: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America December 2008 | Volume 6 | Issue 12 | e301

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

For most species, aging promotes a host of degenerative pathologies that are characterized by debilitating losses of tissue or cellular function. However, especially among vertebrates, aging also promotes hyperplastic pathologies, the most deadly of which is cancer. In contrast to the loss of function that characterizes degenerating cells and tissues, malignant (cancerous) cells must acquire new (albeit aberrant) functions that allow them to develop into a lethal tumor. This review discusses the idea that, despite seemingly opposite characteristics, the degenerative and hyperplastic pathologies of aging are at least partly linked by a common biological phenomenon: a cellular stress response known as cellular senescence. The senescence response is widely recognized as a potent tumor suppressive mechanism. However, recent evidence strengthens the idea that it also drives both degenerative and hyperplastic pathologies, most likely by promoting chronic inflammation. Thus, the senescence response may be the result of antagonistically pleiotropic gene action.

/pdf/aging-cellular-senescence-and-cancer-3wk3hl6x7h.pdf

Aging, Cellular Senescence, and Cancer

We have determined the genomic structure of the candidate tumor suppressor gene DICE1 (DDX26). The DICE1 gene colocalizes with microsatellite marker D13S284 telomeric to the RB1 gene in chromosomal region 13q14.3. The DICE1 gene encodes 18 exons that are preceded by a GC-rich promoter region. CpG sites flanking a predicted TATA box were found to be hypermethylated in tumor cells that exhibited decreased DICE1 expression. This suggests tumor-specific transcriptional silencing of the DICE1 gene may occur. Aberrantly spliced products were detected in two of three DICE1 expressing cell lines. The predicted DICE1 amino acid sequence is evolutionarily conserved in mouse, fruit fly (D. melanogaster), and nematode (C. elegans). A DEAD box characteristic of ATP-dependent helicases is the predominant motif found in DICE1 and its mouse and fruit fly homologues. Motifs other than the DEAD box are reminiscent of members of the helicase superfamily II but there is considerable variation from the typical DEAD box helicases. Expression of DICE1 green fluorescent fusion protein showed a preferential localization of DICE1 in the nucleus. This suggests that DICE1 is involved in nuclear processes such as DNA repair, transcription, or RNA splicing.

Molecular characterization of the DICE1 (DDX26) tumor suppressor gene in lung carcinoma cells

A method of inhibiting the proliferation and causing the differentiation of undifferentiated cells comprising contacting the undifferentiated cells with an effective amount of an antisense oligonucleotide having a sequence which is complementary to a region of the IGF-1 receptor RNA. The sequence of the antisense oligonucleotide is selected from an oligodeoxynucleotide sequence complementary to codons -29 to -24 of the signal sequence of the IGF-1 receptor and an oligoribonucleotide sequence complementary to codons 1 to 309 of the sequence of the IGF-1 receptor. The oligoribonucleotide sequence may be provided by an expression vector.

Method of inhibiting the proliferation and causing the differentiation of cells with IGF-1 receptor antisense oligonucleotides

Stem cells are defined by their unique ability to self-renew and their multipotent differentiation capacity, thus maintaining tissue homeostasis throughout the life of a multicellular organism. Stem cells reside in niches characterized by hypoxia and low reactive oxygen species (ROS), both of which are critical for maintaining the potential for self-renewal and stemness. Until recently, the focus in stem cell biology has been on the adverse effects of ROS, particularly the damaging effects of ROS accumulation on tissue aging and the development of cancer. However, it has become increasingly clear that, in some cases, redox status plays an important role in stem cell maintenance, that is, regulation of the cell cycle. In fact, ROS at low levels function as signaling molecules to mediate cell proliferation, migration, and differentiation and gene expression. ROS levels in stem and progenitor cells have a clear correlation with cellular functions and are regulated by a fine-tuning of the balance between ROS-generating and antioxidant defense systems.

This special issue tries to fully decipher the underlying molecular mechanisms involved in the maintenance of stem cell self-renewal, which is critical to address the important role of redox homeostasis in the regulation of both self-renewal and differentiation of stem cells.

The safe use of stem cells for clinical applications still requires culture improvements to obtain functional cells. The review of S. Sart et al. investigates the roles of ROS in the maintenance of self-renewal, proliferation, and differentiation of mesenchymal stem cells (MSCs) and pluripotent stem cells (PSCs) and highlights that the tight control of stem cell microenvironment, including cell organization, metabolic, and mechanical environments, may be an effective approach to regulate endogenous ROS generation. While high levels of ROS have detrimental effects on stem cells through the induction of oxidative stress, physiological levels of ROS play an important role in the regulation of stem cell fate decision. Mild levels of ROS act as secondary messengers by interfering with various signaling pathways that regulate stem cell proliferation, survival, and differentiation. However, the contribution of the specific site of ROS production and the specific type of ROS to the regulation of stem cell fate requires further delineation. In addition, the exact threshold levels of ROS discerning between the role as damaging molecules or as enhancers of stem cell signaling pathway are not clearly defined. Combined with accurate ROS measurement, regulation of biochemical and biomechanical environment of stem cells to modulate redox status can lead to the controlled proliferation and differentiation of stem cells towards various biomedical applications.

Regarding the site of ROS production and the damaging role of ROS in human stem cells the article of T. Maraldi et al. suggests a particular role of NAD(P)H oxidase enzyme family. In this study they show that Nox4 nuclear expression depends on the donor and it correlates with the expression of redox transcription factors involved in stemness regulation and with the differentiation potential. Taken together, these results suggest that nuclear Nox4 regulation may have important effects on stem cell capability through modulation of transcription factors and DNA damage and the extensive donor-to-donor heterogeneity.

The study of O. G. Lyublinskaya et al. focuses on the involvement of ROS in the process of MSCs “waking up” and entering the cell cycle after the quiescence. Using human endometrial MSCs, they showed that intracellular basal ROS levels are positively correlated with the proliferative status of the cell cultures. In fact, they observed that physiologically relevant levels of ROS are required for the initiation of human MSC proliferation and that low levels of ROS due to the antioxidant treatment can block the stem cell self-renewal.

Despite many attractive advantages of MSCs, replicative senescence of MSCs occurs, eventually decreasing their functional capacities. MSC aging is proposed to potentially contribute to organism aging leading to loss of tissue homeostasis. It is essential to consider the role of ROS in the aging process of MSCs since MSCs and their regenerative potential have revealed a critical therapeutic approach to aging. In lung diseases, the excessive amount of ROS from the ambient air might be associated with respiratory failure through unsuspected mechanisms that regulate declines in the residual stem cell function with age. However, the review of S.-R. Yang concludes that much work remains to be done to understand the ROS-mediated mechanisms that regulate residual stem cells. Elucidating these mechanisms will be critical to understand how stem cell based or antioxidant therapies are effective in certain tissues including lungs.

Regarding the role of ROS in the differentiation process the article of S. Kostyuk et al. describes that GC-rich fragments in the pool of cell-free DNA (cfDNA), circulating throughout the bloodstream of both healthy people and patients with various diseases, can potentially induce oxidative stress and DNA damage response and affect the direction of MSCs differentiation in human adipose-derived MSCs. Such a response may be one of the causes of obesity or osteoporosis.

Similar to normal stem cells, cancer stem cells (CSCs) also show lower intracellular ROS levels, suggesting that maintenance of a reduced intracellular environment is associated with an undifferentiated state. CSCs are responsible for cancer recurrence after chemotherapy or radiotherapy. However, the role of ROS in CSCs remains poorly understood and intensive research is desperately needed.

The review of S. Ding focuses on ROS generation and removal in CSCs and their effects on CSC self-renewal and differentiation through the modulating of ROS-dependent cellular processes. While information on ROS regulation in CSCs are limited, there is fast emerging evidence that ROS may play essential role in the self-renew and differentiation ability of CSCs. The review concludes that targeting CSCs via ROS and antioxidant enzymes regulation holds a great promise in cancer therapy.

Similarly, the review of J. Liu and Z. Wang analyses the particular role of ROS in CSCs. Increasing evidence proposes that a low concentration of ROS can maintain the stemness of CSCs and contribute to tumorigenesis and development. Various systems exist for the regulation of ROS in CSCs. For example, under hypoxic environment, increased levels of ROS induce the expression of HIFs in CSCs, resulting in the upregulation of CSC markers and the reduction of intracellular ROS level and thus facilitating CSCs survival and proliferation. Consequently, the induction of oxidative stress appears to be a promising approach for the preferential killing of cancer cells, including CSCs. In addition, a more detailed investigation of therapies with direct or indirect effects on ROS will help to define a made-to-order therapeutic schedule with a lower tendency toward promoting the development of resistance to treatment.


Tullia Maraldi

Cristina Angeloni

Elisa Giannoni

Christian Sell

/pdf/reactive-oxygen-species-in-stem-cells-151du2xkpm.pdf

Reactive Oxygen Species in Stem Cells

The regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations which induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several ETC protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and VDAC levels. In addition, mTOR activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

/pdf/inhibition-of-mtor-prevents-ros-production-initiated-by-2kizoxeh2m.pdf

Christian Sell

Papers

Molecular characterization of the DICE1 (DDX26) tumor suppressor gene in lung carcinoma cells

Method of inhibiting the proliferation and causing the differentiation of cells with IGF-1 receptor antisense oligonucleotides

Reactive Oxygen Species in Stem Cells

Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts.