376 research outputs found

    Fenretinide induces autophagic cell death in caspase-defective breast cancer cells

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    The elimination of tumor cells by apoptosis is the main mechanism of action of chemotherapeutic drugs. More recently, autophagic cell death has been shown to trigger a nonapoptotic cell death program in cancer cells displaying functional defects of caspases. Fenretinide (FenR), a synthetic derivative of retinoic acid, promotes growth inhibition and induces apoptosis in a wide range of tumor cell types. The present study was designed to evaluate the ability of fenretinide to induce caspase-independent cell death and to this aim we used the human mammary carcinoma cell line MCF-7, lacking functional caspase-3 activity. We demonstrated that in these cells fenretinide is able to trigger an autophagic cell death pathway. In particular we found that fenretinide treatment resulted in the increase in Beclin 1 expression, the conversion of the soluble form of LC3 to the autophagic vesicle-associated form LC3-II and its shift from diffuse to punctate staining and finally the increase in lysosomes/autophagosomes. By contrast, caspase-3 reconstituted MCF-7 cell line showed apoptotic cell death features in response to fenretinide treatment. These data strongly suggest that fenretinide does not invariably elicit an apoptotic response but it is able to induce autophagy when apoptotic pathway is deregulated. The understanding of the molecular mechanisms involved in fenretinide action is important for the future design of therapies employing this retinoid in breast cancer treatment

    CELL DEATH AND AUTOPHAGY: CYTOKINES, DRUGS, AND NUTRITIONAL FACTORS

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    Cellsmay use multiple pathways to commit suicide. In certain contexts, dying cells generate large amounts of autophagic vacuoles and clear large proportions of their cytoplasm, before they finally die, as exemplified by the treatment of human mammary carcinoma cells with the anti-estrogen tamoxifen (TAM, ≤1 M). Protein analysis during autophagic cell death revealed distinct proteins of the nuclear fraction including GST- and some proteasomal subunit constituents to be affected during autophagic cell death. Depending on the functional status of caspase-3, MCF-7 cells may switch between autophagic and apoptotic features of cell death [Fazi, B., Bursch,W., Fimia, G.M., Nardacci R., Piacentini, M., Di Sano, F., Piredda, L., 2008. Fenretinide induces autophagic cell death in caspase-defective breast cancer cells. Autophagy 4(4), 435–441]. Furthermore, the self-destruction of MCF-7 cells was found to be completed by phagocytosis of cell residues [Petrovski, G., Zahuczky, G., Katona, K., Vereb, G., Martinet,W., Nemes, Z., Bursch,W., Fésüs, L., 2007. Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes. Cell Death Diff. 14 (6), 1117–1128]. Autophagy also constitutes a cell’s strategy of defense upon cell damage by eliminating damaged bulk proteins/organelles. This biological condition may be exemplified by the treatment of MCF-7 cells with a necrogenic TAM-dose (10 M), resulting in the lysis of almost all cells within 24 h. However, a transient (1 h) challenge of MCF-7 cells with the same dose allowed the recovery of cells involving autophagy. Enrichment of chaperones in the insoluble cytoplasmic protein fraction indicated the formation of aggresomes, a potential trigger for autophagy. In a further experimental model HL60 cells were treated with TAM, causing dose-dependent distinct responses: 1–5 MTAM, autophagy predominant; 7–9 M, apoptosis predominant; 15 M, necrosis. These phenomena might be attributed to the degree of cell damage caused by tamoxifen, either by generating ROS, increasing membrane fluidity or forming DNA-adducts. Finally, autophagy constitutes a cell’s major adaptive (survival) strategy in response to metabolic challenges such as glucose or amino acid deprivation, or starvation in general. Notably, the role of autophagy appears not to be restricted to nutrient recycling in order to maintain energy supply of cells and to adapt cell(organ) size to given physiological needs. For instance, using a newly established hepatoma cell line HCC-1.2, amino acid and glucose deprivation revealed a pro-apoptotic activity, additive to TGF- 1. The proapoptotic action of glucose deprivation was antagonized by 2-deoxyglucose, possibly by stabilizing the mitochondrial membrane involving the action of hexokinase II. These observations suggest that signaling cascades steering autophagy appear to provide links to those regulating cell number. Taken together, our data exemplify that a given cell may flexibly respond to type and degree of (micro)environmental changes or cell death stimuli; a cell’s response may shift gradually from the elimination of damaged proteins by autophagy and the recovery to autophagic or apoptotic pathways of cell death, the failure of which eventually may result in necrosis

    Fate of conjugated natural and synthetic steroid estrogens in crude sewage and activated sludge batch studies

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    This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Environmental Science & Technology, copyright © American Chemical Society after peer review. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/es801952h.Steroids are excreted from the human body in the conjugated form but are present in sewage influent and effluent as the free steroid, the major source of estrogenic activity observed in water courses. The fate of sulfate and glucuronide conjugated steroid estrogens was investigated in batch studies using activated sludge grown on synthetic sewage in a laboratory-scale Husmann simulation and crude sewage from the field. A clear distinction between the fate of sulfate and glucuronide conjugates was observed in both matrices, with sulfated conjugates proving more recalcitrant and glucuronide deconjugation preferential in crude sewage. For each conjugate, the free steroid was observed in the biotic samples. The degree of free steroid formation was dependent on the conjugate moiety, favoring the glucuronide. Subsequent degradation of the free steroid (and sorption to the activated sludge solid phase) was evaluated. Deconjugation followed the first order reaction rate with rate constants for 17α-ethinylestradiol 3-glucuronide, estriol 16α-glucuronide, and estrone 3-glucuronide determined as 0.32, 0.24, and 0.35 h respectively. The activated sludge solid retention time over the range of 3−9 days had 74 to 94% of sulfate conjugates remaining after 8 h. In contrast, a correlation between increasing temperature and decreasing 17α-ethinylestradiol 3-glucuronide concentrations in the activated sludge observed no conjugate present in the AS following 8 h at 22 °C Based on these batch studies and literature excretion profiles, a hypothesis is presented on which steroids and what form (glucuronide, sulfate, or free) will likely enter the sewage treatment plant.EPSR

    Dbf4-dependent kinase (DDK)-mediated proteolysis of CENP-A prevents mislocalization of CENP-A in Saccharomyces cerevisiae

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    The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4)byE3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1D strains display synthetic dosage lethality (SDL) with GALCSE4. We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4. We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4. Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7 psh1D strain were similar to that of cdc7-7 and psh1D strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1. Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.Fil: Eisenstatt, Jessica R.. National Institutes of Health; Estados UnidosFil: Boeckmann, Lars. National Institutes of Health; Estados UnidosFil: Au, Wei Chun. National Institutes of Health; Estados UnidosFil: Garcia, Valerie. National Institutes of Health; Estados UnidosFil: Bursch, Levi. National Institutes of Health; Estados UnidosFil: Ocampo, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. National Instituto of Child Health & Human Development; Estados UnidosFil: Costanzo, Michael. National Institutes of Health; Estados Unidos. University of Toronto; CanadáFil: Weinreich, Michael. Van Andel Research Institute; Estados UnidosFil: Sclafani, Robert A.. University of Colorado; Estados UnidosFil: Baryshnikova, Anastasia. University of Princeton; Estados UnidosFil: Myers, Chad L.. University of Minnesota; Estados UnidosFil: Boone, Charles. University of Toronto; Canadá. National Institutes of Health; Estados UnidosFil: Clark, David J.. National Institutes of Health; Estados UnidosFil: Baker, Richard. University of Massachusetts; Estados UnidosFil: Basrai, Munira A.. National Institutes of Health; Estados Unido

    Mafb lineage tracing to distinguish macrophages from other immune lineages reveals dual identity of Langerhans cells

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    Current systems for conditional gene deletion within mouse macrophage lineages are limited by ectopic activity or low efficiency. In this study, we generated a Mafb-driven Cre strain to determine whether any dendritic cells (DCs) identified by Zbtb46-GFP expression originate from a Mafb-expressing population. Lineage tracing distinguished macrophages from classical DCs, neutrophils, and B cells in all organs examined. At steady state, Langerhans cells (LCs) were lineage traced but also expressed Zbtb46-GFP, a phenotype not observed in any other population. After exposure to house dust mite antigen, Zbtb46-negative CD64(+) inflammatory cells infiltrating the lung were substantially lineage traced, but Zbtb46-positive CD64(−) cells were not. These results provide new evidence for the unique identity of LCs and challenge the notion that some inflammatory cells are a population of monocyte-derived DCs

    Sphingosine 1-phosphate modulates antigen capture by murine langerhans cells via the S1P2 receptor subtype

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    Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P2 receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P2 not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions

    Cannibalism, cell survival, and endocrine resistance in breast cancer

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    Breast cancer cells often respond to an endocrine therapy by altering expression of specific estrogen-responsive genes and inducing autophagy, a cannibalistic lysosomal pathway. Autophagy eliminates damaged or other organelles, allowing the recovery of the energy stored in their macromolecules to attempt restoration of metabolic homeostasis. Induction of autophagy can result from activation of the unfolded protein response following metabolic stress, the final cell fate often being determined by the extent and duration of autophagy. A study by Gonzalez-Malerva and colleagues builds upon this extensive knowledge, adding HSPB8 to the list of altered genes associated with endocrine resistance in breast cancer and describing the ability of HSPB8 to regulate autophagy and confer tamoxifen resistance
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