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1.  Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5 
Oncotarget  2013;4(9):1349-1364.
TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. We have previously shown that deficiency of DR4 and DR5 on the surface membrane is a critical mechanism of cancer cell resistance to the recombinant human TRAIL and its receptor agonistic antibodies, which are being evaluated clinically for treating cancers. In certain cancer cells, DR4 and DR5 were found to be mislocalized in intracellular compartments yet to be characterized. Here, we report a novel role of autophagy in the regulation of dynamics of TRAIL death receptors. We first assessed basal levels of autophagosomes in a panel of 11 breast cancer cell lines using complementary approaches (LC3 immunoblotting, RFP-LC3 fluorescence microscopy, and electron microscopy). We found high levels of basal autophagosomes in TRAIL resistant breast cancer cell lines (e.g. BT474 and AU565) and relevant mouse xenograft models under nutrition-rich conditions. Notably, DR4 and DR5 co-localized with LC3-II in the autophagosomes of TRAIL-resistant cells. Disruption of basal autophagosomes successfully restored the surface expression of the death receptors which was accompanied by sensitization of TRAIL-resistant cells to TRAIL induced apoptosis. By contrast, TRAIL-sensitive cell lines (MDA-MB-231) are characterized by high levels of surface DR4/DR5 and an absence of basal autophagosomes. Inhibition of lysosomal activity induced an accumulation of autophagosomes and a decrease in surface DR4 and DR5, and the cells became less sensitive to TRAIL-induced apoptosis. These findings demonstrate a novel role for the basal autophagosomes in the regulation of TRAIL death receptors. Further studies are warranted to explore the possibility of using autophagosome markers such as LC3-II/LC3-I ratios for prediction of tumor resistance to TRAIL related therapies. The results also provide a rationale for future non-clinical and clinical studies testing TRAIL agonists in combination with agents that directly inhibit autophagosome assembly.
PMCID: PMC3824535  PMID: 23988408
TRAIL resistance; basal autophagosomes; death receptors; cell surface expression
2.  Defective Ribosomal Products Are the Major Source of Antigenic Peptides Endogenously Generated from Influenza A Virus Neuraminidase 
The defective ribosomal product (DRiP) hypothesis of endogenous Ag processing posits that rapidly degraded forms of nascent proteins are a major source of peptide ligands for MHC class I molecules. Although there is broad experimental support for the DRiP hypothesis, careful kinetic analysis of the generation of defined peptide class I complexes has been limited to studies of recombinant vaccinia viruses expressing genes derived from other organisms. In this study, we show that insertion of the SIIN-FEKL peptide into the stalk of influenza A virus neuraminidase (NA) does not detectably modify NA folding, degradation, transport, or sp. act. when expressed in its natural context of influenza A virus infection. Using the 25-D1.16 mAb specific for Kb-SIINFEKL to precisely quantitate cell surface complexes by flow cytometry, we demonstrate that SIINFEKL is generated in complete lockstep with initiation and abrogation of NA biosynthesis in both L-Kb fibroblast cells and DC2.4 dendritic/monocyte cells. SIINFEKL presentation requires active proteasomes and TAP, consistent with its generation from a cytosolic DRiP pool. From the difference in the shutoff kinetics of Kb-SIINFEKL complex expression following protein synthesis versus proteasome inhibition, we estimate that the t1/2 of the biosynthetic source of NA peptide is ~5 min. These observations extend the relevance of the DRiP hypothesis to viral proteins generated in their natural context.
PMCID: PMC2940057  PMID: 20038640
3.  Cutting Edge: Sympathetic Nervous System Increases Proinflammatory Cytokines and Exacerbates Influenza A Virus Pathogenesis 
Although the sympathetic nervous system innervates the lung, little is known about its participation in host immunity to pulmonary pathogens. In this study, we show that peripheral sympathectomy reduces mouse morbidity and mortality from influenza A virus-induced pneumonia due to reduced inflammatory influx of monocytes, neutrophils, and NK cells. Mortality was also delayed by treating mice with an α-adrenergic antagonist. Sympathectomy diminished the immediate innate cytokine responses, particularly IL-1, which was profoundly reduced. These findings demonstrate an unexpected role for the sympathetic nervous system in innate antiviral immunity and in exacerbating the pathology of a virus of great significance to human and animal health.
PMCID: PMC2941093  PMID: 20018617
4.  Conditional Ablation of Nonmuscle Myosin II-B Delineates Heart Defects in Adult Mice 
Circulation research  2009;105(11):1102-1109.
Germline ablation of the cytoskeletal protein nonmuscle myosin II-B (NMII-B) results in embryonic lethality with defects in both the brain and heart. Tissue specific ablation of NMII-B by a Cre-recombinase strategy should avoid embryonic lethality and permit study of the function of NMII-B in adult hearts.
To understand the function of NMII-B in adult mouse hearts and to see if the brain defects found in germline ablated mice influence cardiac development.
Methods and Results
We used a loxP/Cre-recombinase strategy to specifically ablate NMII-B in the brains or hearts of mice. Mice ablated for NMII-B in neural tissues, die between postnatal day 12 and 22 without showing cardiac defects. Mice deficient in NMII-B only in cardiac myocytes (BαMHC/BαMHC mice) do not show brain defects. However BαMHC/BαMHC mice display novel cardiac defects not seen in NMII-B germline ablated mice. Most of the BαMHC/BαMHC mice are born with enlarged cardiac myocytes some of which are multinucleated, reflecting a defect in cytokinesis. Between 6–10 months they develop a cardiomyopathy which includes interstitial fibrosis and infiltration of the myocardium and pericardium with inflammatory cells. Four of five BαMHC/BαMHC hearts develop marked widening of intercalated discs.
By avoiding the embryonic lethality found in germline-ablated mice we were able to study the function of NMII-B in adult mice and show that absence of NMII-B in cardiac myocytes results in cardiomyopathy in the adult heart. We also define a role for NMII-B in maintaining the integrity of intercalated discs.
PMCID: PMC2792753  PMID: 19815823
Nonmuscle myosin II-B; Cardiomyopathy; Intercalated discs
5.  The Exception that Reinforces the Rule: Cross-Priming by Cytosolic Peptides that Escape Degradation 
Immunity  2008;28(6):787-798.
The nature of cross-priming immunogens for CD8+ T cell responses is highly controversial. Using a panel of T cell receptor-like antibodies specific for viral peptides bound to mouse Db major histocompatibility complex class I molecules, we show that an exceptional peptide (PA224-233) expressed as a viral minigene product formed a sizeable cytosolic pool continuously presented for hours after protein synthesis was inhibited. PA224-233 pool formation required active cytosolic heat shock protein 90 but not ER g96, and uniquely enabled cross-priming by this peptide. These findings demonstrate that exceptional class I binding oligopeptides that escape proteolytic degradation are potent cross-priming agents. Thus, the feeble immunogenicity of natural proteasome products in cross-priming can be attributed to their evanescence in donor cells, and not an absolute inability of cytosolic oligopeptides to be transferred to and presented by professional antigen presenting cells..
PMCID: PMC2587262  PMID: 18549799
6.  Role for Mitochondrial Oxidants as Regulators of Cellular Metabolism 
Molecular and Cellular Biology  2000;20(19):7311-7318.
Leakage of mitochondrial oxidants contributes to a variety of harmful conditions ranging from neurodegenerative diseases to cellular senescence. We describe here, however, a physiological and heretofore unrecognized role for mitochondrial oxidant release. Mitochondrial metabolism of pyruvate is demonstrated to activate the c-Jun N-terminal kinase (JNK). This metabolite-induced rise in cytosolic JNK1 activity is shown to be triggered by increased release of mitochondrial H2O2. We further demonstrate that in turn, the redox-dependent activation of JNK1 feeds back and inhibits the activity of the metabolic enzymes glycogen synthase kinase 3β and glycogen synthase. As such, these results demonstrate a novel metabolic regulatory pathway activated by mitochondrial oxidants. In addition, they suggest that although chronic oxidant production may have deleterious effects, mitochondrial oxidants can also function acutely as signaling molecules to provide communication between the mitochondria and the cytosol.
PMCID: PMC86285  PMID: 10982848
7.  Gene dosage affects the cardiac and brain phenotype in nonmuscle myosin II-B–depleted mice 
Journal of Clinical Investigation  2000;105(5):663-671.
Complete ablation of nonmuscle myosin heavy chain II-B (NMHC-B) in mice resulted in cardiac and brain defects that were lethal during embryonic development or on the day of birth. In this paper, we report on the generation of mice with decreased amounts of NMHC-B. First, we generated BΔI/BΔI mice by replacing a neural-specific alternative exon with the PGK-Neo cassette. This resulted in decreased amounts of NMHC-B in all tissues, including a decrease of 88% in the heart and 65% in the brain compared with B+/B+ tissues. BΔI/BΔI mice developed cardiac myocyte hypertrophy between 7 months and 11 months of age, at which time they reexpressed the cardiac β-MHC. Serial sections of BΔI/BΔI brains showed abnormalities in neural cell migration and adhesion in the ventricular wall. Crossing BΔI/BΔI with B+/B– mice generated BΔI/B– mice, which showed a further decrease of approximately 55% in NMHC-B in the heart and brain compared with BΔI/BΔI mice. Five of 8 BΔI/B– mice were born with a membranous ventricular septal defect. Moreover, 5 of 5 BΔI/B– mice developed myocyte hypertrophy by 1 month; BΔI/B– mice also reexpressed the cardiac β-MHC. More than 60% of BΔI/B– mice developed overt hydrocephalus and showed more severe defects in neural cell migration and adhesion than did BΔI/BΔI mice. These data on BΔI/BΔI and BΔI/B– mice demonstrate a gene dosage effect of the amount of NMHC-B on the severity and time of onset of the defects in the heart and brain.
PMCID: PMC289177  PMID: 10712438
8.  Adult Murine Skeletal Muscle Contains Cells That Can Differentiate into Beating Cardiomyocytes In Vitro 
PLoS Biology  2005;3(4):e87.
It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.
A population of primitive cells from adult murine skeletal muscle can develop into beating cardiomyocytes in vitro and can contribute to the repair of damaged heart in vivo
PMCID: PMC1064849  PMID: 15757365

Results 1-8 (8)