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

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We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH3 demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH3 into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH3–mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH3–mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH3 and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts.

Multiple myeloma (MM) is an incurable B-cell malignancy, requiring new therapeutic strategies. We have found that synthetic alkyl-lysophospholipids (ALPs) edelfosine and perifosine induced apoptosis in MM cell lines and patient MM cells, whereas normal B and T lymphocytes were spared. ALPs induced recruitment of Fas/CD95 death receptor, Fas-associated death domain-containing protein, and procaspase-8 into lipid rafts, leading to the formation of the death-inducing signaling complex (DISC) and apoptosis. TNF-related apoptosis-inducing ligand receptor-1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5, as well as Bid, were also recruited into lipid rafts, linking death receptor and mitochondrial signaling pathways. ALPs induced mitochondrial cytochrome c release. Bcl-X(L) overexpression prevented cytochrome c release and apoptosis. A Fas/CD95-deficient MM subline expressing DR4 and DR5 was resistant to edelfosine. Fas/CD95 retrovirus transduction bestowed edelfosine sensitivity in these cells. A Fas/CD95 mutant lacking part of the intracellular domain was ineffective. Lipid raft disruption prevented ALP-induced Fas/CD95 clustering, DISC formation, and apoptosis. ALP-induced apoptosis was Fas/CD95 ligand (FasL/CD95L) independent. ALP-induced recruitment of death receptors in lipid rafts potentiated MM cell killing by FasL/CD95L and TRAIL. These data uncover a novel lipid raft-mediated therapy in MM involving concentration of death receptors in membrane rafts, with Fas/CD95 playing a major role in ALP-mediated apoptosis.

IntroductionThe plasma membrane contains microdomains named membrane rafts, consisting of dynamic assemblies of cholesterol and sphingolipids. [1][2][3] The presence of saturated hydrocarbon chains in sphingolipids allows for cholesterol to be tightly intercalated, leading to the presence of distinct liquid-ordered phases, membrane rafts, dispersed in the liquid-disordered matrix, and thereby more fluid, lipid bilayer. 4 One key property of membrane rafts is that they can include or exclude proteins to varying degrees. Membrane rafts may serve as foci for recruitment and concentration of signaling molecules at the plasma membrane, and thus they have been implicated in signal transduction from cell surface receptors. 3 Antitumor ether phospholipids constitute a novel class of promising cancer chemotherapeutic drugs. [5][6][7] The ether lipid 1-Ooctadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH 3 ; edelfosine) exerts a selective cytotoxic action against transformed cells, 5-7 and has become the effective standard and prototype of the antitumor ether phospholipids. Encouraging clinical research on the use of ET-18-OCH 3 in purging leukemic bone marrow prior to autologous bone marrow transplantation has been reported. 8,9 ET-18-OCH 3 is a potent inducer of apoptosis in tumor cells, especially in leukemic cells, sparing normal cells. 7,10,11 Recent evidence has shown that ET-18-OCH 3 -induced apoptosis is mediated by the intracellular activation of Fas/CD95 cell death receptor, independently of its ligand FasL, leading to clustering and subsequent capping of Fas. 12 ET-18-OCH 3 must be incorporated into the cell to exert its apoptotic action, 7 and the ether lipid has been reported to be accumulated in the plasma membrane. 13 Because ET-18-OCH 3 is a phospholipid and acts through plasma membrane-related processes, involving activation of cell surface Fas receptor, we investigated whether the Fas-mediated apoptotic effect of ET-18-OCH 3 on leukemic cells involved lipid rafts. Study design Cell culture and apoptosisThe human leukemic cell lines HL-60 (acute myeloid leukemia) and Jurkat (acute T-cell leukemia) were grown as described previously in RPMI-1640 culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS). 7 Bone marrow aspirates, obtained from patients at the initial diagnosis and after signing informed consent, were kindly provided by the Hematology Department of the Rio Hortega Hospital (Valladolid, Spain). Mononuclear cells isolated by Ficoll-Hypaque density gradient centrifugation, consisting mostly of leukemia blasts (Ͼ 90%), were washed in phosphate-buffered saline (PBS), resuspended in cell culture medium, and used immediately for experimentation. ET-18-OCH 3 (INKEYSA, Barcelona, Spain) was prepared as described previously. 7 To disrupt lipid rafts, cells (5 ϫ 10 5 ) were pretreated with methyl-␤-cyclodextrin (MCD) (Sigma Chemical, St Louis, MO; 15 g/mL) or filipin (Sigma; 1 g/mL) for 1 hour at 37°C in serum-free medium before ether lipid addition.Apoptosis was assessed by ...

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