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Comstock C.E.S.,Thomas Jefferson University | WonJeong K.,Gachon University | Stallcup M.R.,University of Southern California | McCue P.A.,Anatomy and Cell Biology | Knudsen K.E.,Thomas Jefferson University
Clinical Cancer Research | Year: 2013

Purpose: BAF57, a component of the switching-defective and sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex conglomerate, modulates androgen receptor activity to promote prostate cancer. However, the molecular consequences of tumor-associated BAF57 expression have remained undefined in advanced disease such as castration-resistant prostate cancer and/or metastasis. Experimental Design: Clinical human specimens of primary and metastatic prostate cancer were immunohistochemically examined for tumor-grade association of BAF57 expression. Global gene expression analyses were conducted in models mimicking tumor-associated BAF57 expression. Aberrant BAF57-dependent gene expression changes, bypass of androgen-mediated signaling, and chromatin-specific SWI/SNF complex alterations with respect to cytoskeletal remodelers such as integrins were validated. Cell migration assays were used to profile the biologic phenotypes conferred under conditions simulating tumorderived BAF57 expression. Results: Immunohistochemical quantitation of primary human specimens revealed that BAF57 was significantly and aberrantly elevated as a function of tumor grade. Critically, gene expression analyses showed that BAF57 deregulation circumvented androgen-mediated signaling, elicited α2 integrin upregulation, and altered other SWI/SNF complex components at the α2 integrin locus. BAF57-dependent α2 integrin induction conferred a prometastatic migratory advantage, which was attenuated by anti-α2 integrin antibody blockade. Furthermore, BAF57 was found to be markedly upregulated in human prostate cancer metastases of the lung, lymph node, and dura. Conclusion: The findings herein, identifying tumor-associated BAF57 perturbation as a means to bypass androgen-signaling events that facilitate novel prometastatic phenotypes, link BAF57 upregulation to tumor dissemination. These data thereby establish BAF57 as a putative marker of metastatic potential that could be leveraged for therapeutic intervention. © 2013 AACR.


PubMed | Thomas Jefferson University, University of Barcelona, Anatomy and Cell Biology. and Cooper University Hospital
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2016

A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer.


(Philadelphia, PA) - Mitochondria - the energy-generating powerhouses of cells - are also a site for oxidative stress and cellular calcium regulation. The latter two functions have long been suspected of being linked mechanistically, and now new research at the Lewis Katz School of Medicine at Temple University (LKSOM) shows precisely how, with the common connection centering on a protein complex known as the mitochondrial Ca2+ uniporter (MCU). "MCU had been known for its part in driving mitochondrial calcium uptake for cellular energy production, which protects cells from bioenergetic crisis, and for its role in eliciting calcium overload-induced cell death," explained senior investigator on the study, Muniswamy Madesh, PhD, Professor in the Department of Medical Genetics and Molecular Biochemistry and Center for Translational Medicine at LKSOM. "Now, we show that MCU has a functional role in both calcium regulation and the sensing of levels of reactive oxygen species (ROS) within mitochondria." The study, published online March 2 in the journal Molecular Cell, is the first to identify a direct role for MCU in mitochondrial ROS-sensing. In previous work, Dr. Madesh and colleagues were the first to show how the MCU protein complex comes together to effect mitochondrial calcium uptake. "We know from that work, and from existing work in the field, that as calcium accumulates in mitochondria, the organelles generate increasing amounts of ROS," Dr. Madesh said. "Mitochondria have a way of dealing with that ROS surge, and because of the relationship between mitochondrial calcium uptake and ROS production, we suspected ROS-targeting of MCU was involved in that process." In the new study, Dr. Madesh and colleagues employed advanced biochemical, cell biological, and superresolution imaging to examine MCU oxidation in the mitochondrion. Critically, they discovered that MCU contains several cysteine molecules in its amino acid structure, only one of which, Cys-97, is capable of undergoing an oxidation-induced reaction known as S-glutathionylation. Structural analyses showed that oxidation-induced S-glutathionylation of Cys-97 triggers conformational changes within MCU. Those changes in turn regulate MCU activity during inflammation, hypoxia, and cardiac stimulation. They also appear to be relevant to cell survival - elimination of ROS-sensing via Cys-97 mutation resulted in persistent MCU channel activity and an increased rate of calcium-uptake, with cells eventually dying from calcium overload. Importantly, Dr. Madesh and colleagues found that S-glutathionylation of Cys-97 is reversible. "Reversible oxidation is essential to the regulation of protein function," Dr. Madesh explained. When switched on by oxidation, Cys-97 augments MCU channel activity that perpetuates cell death. Oxidation reverses when the threat has subsided. The findings could have implications for the understanding of metabolic disorders and neurological and cardiovascular diseases. "Abnormalities in ion homeostasis are a central feature of metabolic disease," Dr. Madesh said. "We plan next to explore the functional significance of ROS and MCU activity in a mouse model using genome editing technology, which should help us answer fundamental questions about MCU's biological functions in mitochondrial ROS-sensing." Other researchers involved in the study include Zhiwei Dong, Santhanam Shanmughapriya, Dhanendra Tomar, Neeharika Nemani, Sarah L. Breves, Aparna Tripathi, Palaniappan Palaniappan, Massimo F. Riitano, Alison Worth, Ajay Seelam, Edmund Carvalho, Ramasamy Subbiah, Fabia?n Jan?a, and Sudarsan Rajan, Department of Medical Genetics and Molecular Biochemistry and the Center for Translational Medicine at LKSOM; Jonathan Soboloff, Department of Medical Genetics and Molecular Biochemistry at LKSOM; Xueqian Zhang and Joseph Y. Cheung, Center for Translational Medicine at LKSOM; Naveed Siddiqui and Peter B. Stathopulos, Department of Physiology and Pharmacology, Western University, London, Ontario, Canada; Solomon Lynch and Jeffrey Caplan, Department of Biological Sciences, Delaware Biotechnology Institute, University of Delaware; Suresh K. Joseph, MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia; Yizhi Peng and Zhiwei Dong, Institute of Burn Research, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China. The research was supported in part by National Institutes of Health grants R01GM109882, R01HL086699, R01HL119306, 1S10RR027327, P01 DA037830, and RO1DK103558. Temple University Health System (TUHS) is a $1.6 billion academic health system dedicated to providing access to quality patient care and supporting excellence in medical education and research. The Health System consists of Temple University Hospital (TUH), ranked among the "Best Hospitals" in the region by U.S. News & World Report; TUH-Episcopal Campus; TUH-Northeastern Campus; Fox Chase Cancer Center, an NCI-designated comprehensive cancer center; Jeanes Hospital, a community-based hospital offering medical, surgical and emergency services; Temple Transport Team, a ground and air-ambulance company; and Temple Physicians, Inc., a network of community-based specialty and primary-care physician practices. TUHS is affiliated with the Lewis Katz School of Medicine at Temple University. The Lewis Katz School of Medicine (LKSOM), established in 1901, is one of the nation's leading medical schools. Each year, the School of Medicine educates approximately 840 medical students and 140 graduate students. Based on its level of funding from the National Institutes of Health, the Katz School of Medicine is the second-highest ranked medical school in Philadelphia and the third-highest in the Commonwealth of Pennsylvania. According to U.S. News & World Report, LKSOM is among the top 10 most applied-to medical schools in the nation. Temple Health refers to the health, education and research activities carried out by the affiliates of Temple University Health System (TUHS) and by the Katz School of Medicine. TUHS neither provides nor controls the provision of health care. All health care is provided by its member organizations or independent health care providers affiliated with TUHS member organizations. Each TUHS member organization is owned and operated pursuant to its governing documents.


Stern-Straeter J.,University of Heidelberg | Bonaterra G.A.,Anatomy and Cell Biology | Juritz S.,University of Heidelberg | Birk R.,University of Heidelberg | And 7 more authors.
International Journal of Molecular Medicine | Year: 2014

The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM-MSCs. The strongest DES expression was observed using the 30% conditioned cell culture medium. The detection of myogenic markers using different cell culture media as stimuli was only achieved in the AT-MSCs, but not in the BM-MSCs. The strongest myogenic differentiation, in terms of the markers examined, was induced by the 30% conditioned cell culture medium.


Tian L.,University of Pennsylvania | Huang K.,University of Pennsylvania | Duhadaway J.B.,Lankenau Institute for Medical Research | Prendergast G.C.,Anatomy and Cell Biology | And 2 more authors.
Current Eye Research | Year: 2010

Purpose: Many miRNAs are expressed in a developmentally regulated and tissue-specific manner making them crucial for tissue development in a structure such as the eye. Since miRNA target function studies for the eye will need to be performed in ocular tissue culture cells, it is important to profile them for miRNA expression. Two commonly used human lens epithelial cell lines, HLE-B3 and SRA01/04, were profiled for miRNA. Materials and Methods: We performed miRNA profiling of two commonly used lens epithelial cell lines, HLE-B3 and SRA01/04. The differential expression levels detected for miR-184 and miR-31 were confirmed by qRT-PCR and the function of a predicted miR-184 target binding site was validated in-vitro. Results: We found that four miRNAsmiR-31, miR-124, miR-184, and miR-222were differentially expressed between the two cell lines. We show that miR-184 binds to BIN3 3′ UTR and while BIN3 mRNA expression was equal in both cell lines, the protein expression was inversely correlated with miR-184 expression. Conclusion: The differences observed with respect to miRNA expression between two different lens epithelial cell lines were minimal. Still, caution will need to be exercised when choosing one cell line over another because of the expression differences for some miRNAs. Our results also suggest that miR-184 may regulate lens BIN3 expression in lens by a miRNA-mediated translational repression mechanism. © 2010 Informa Healthcare USA, Inc.


Stern-Straeter J.,University of Heidelberg | Bonaterra G.A.,Anatomy and Cell Biology | Kassner S.S.,University of Heidelberg | Zugel S.,Anatomy and Cell Biology | And 3 more authors.
Journal of Tissue Engineering and Regenerative Medicine | Year: 2011

Tissue engineering of skeletal muscle is an encouraging possibility for the treatment of muscle loss through the creation of functional muscle tissue in vitro from human stem cells. Currently, the preferred stem cells are primary, non-immunogenic satellite cells (= myoblasts). The objective of this study was to determine the expression patterns of myogenic markers within the human satellite cell population during their differentiation into multinucleated myotubes for an accurate characterization of stem cell behaviour. Satellite cells were incubated (for 1, 4, 8, 12 or 16 days) with a culture medium containing either a low [ = differentiation medium (DM)] or high [ = growth medium (GM)] concentration of growth factors. Furthermore, we performed a quantitative gene expression analysis of well-defined differentiation makers: myogenic factor 5 (MYF5), myogenin (MYOG), skeletal muscle αactin1 (ACTA1), embryonic (MYH3), perinatal (MYH8) and adult skeletal muscle myosin heavy chain (MYH1). Additionally, the fusion indices of forming myotubes of MYH1, MYH8 and ACTA1 were calculated. We show that satellite cells incubated with DM expressed multiple characteriztic features of mature skeletal muscles, verified by time-dependent upregulation of MYOG, MYH1, MYH3, MYH8 and ACTA1. However, satellite cells incubated with GM did not reveal all morphological aspects of muscle differentiation. Immunocytochemical investigations with antibodies directed against the differentiation markers showed correlations between the gene expression and differentiation. Our data provide information about time-dependent gene expression of differentiation markers in human satellite cells, which can be used for maturation analyses in skeletal muscle tissue-engineering applications. © 2011 John Wiley & Sons, Ltd.


Sue Menko A.,Anatomy and Cell Biology | Andley U.P.,University of Washington
Experimental Eye Research | Year: 2010

α-Crystallins are small heat-shock proteins important to lens transparency that provide the lens with its refractive properties. In their role as molecular chaperones, these crystallins also prevent protein aggregation, affect cytoskeletal remodeling, enhance resistance to cell stress, and provide lens cells with protection against apoptosis. While many of the functions assigned to αA-crystallin are attributable to its presence in the cytoplasm of lens cells, αA-crystallin also has been detected at the lens plasma membrane. However, how αA-crystallin becomes linked to the plasma membrane or what its functions are at this site has remained unknown. In this study, we examined the mechanisms by which αA-crystallin becomes associated with the lens membrane, focusing specifically on its interaction with membrane receptors, and the differentiation-specificity of these interactions. We also determined how the long-term absence of αA-crystallin alters receptor-linked signaling pathways. αA-crystallin association with membrane receptors was determined by co-immunoprecipitation analysis; its membrane localization was examined by confocal imaging; and the effect of αA-crystallin loss-of-function on the activation state of signaling molecules in pathways linked to membrane receptors was determined by immunoblot analysis. The results show that, in lens epithelial cells, plasma membrane αA-crystallin was primarily localized to apicolateral borders, reflecting the association of αA-crystallin with E-cadherin complexes. These studies also provide the first evidence that αA-crystallin maintained its association with the plasma membrane in lens cortical fiber cells, where it was localized to lateral interfaces, and further show that this association was mediated, in part, by αA-crystallin interaction with α6 integrin receptor complexes. We report that the absence of αA-crystallin led to constitutive activation of the stress kinases p38 and JNK, classical inducers of apoptotic cell death, and the loss of the phospho-Bad pro-survival signal, effects that were greatest in differentiating lens fiber cells. Concurrent with this, activation of FAK and ERK kinases was increased, demonstrating that these receptor-linked pathways also were dysregulated in the absence of αA-crystallin. These data link αA-crystallin plasma membrane association to its differentiation-state-specific interaction with E-cadherin and α6 integrin receptor complexes. The changes in cell signaling in αA-crystallin-null lenses suggest that dysregulation of receptor-linked cell-signaling pathways that accompany the failure of αA-crystallin to associate with membrane receptors may be responsible for the induction of apoptosis. The observed changes in lens cell signaling likely reflect long-term functional adaptations to the absence of the αA-crystallin chaperone/small heat-shock protein. © 2010 Elsevier Ltd.


Simen B.B.,Thomas Jefferson University | Yin L.,Anatomy and Cell Biology | Goswami C.P.,Thomas Jefferson University | Davis K.O.,Thomas Jefferson University | And 6 more authors.
Archives of Pathology and Laboratory Medicine | Year: 2015

Context.-Next-generation sequencing allows for highthroughput processing and sensitive variant detection in multiple genes from small samples. For many diseases, including cancer, a comprehensive mutational profile of a targeted list of genes can be used to simultaneously inform patient care, establish eligibility for ongoing clinical trials, and further research. Objective.-To validate a pan-cancer, next-generation-sequencing assay for use in the clinical laboratory. Design.-DNA was extracted from 68 clinical specimens (formalin-fixed, paraffin-embedded; fine-needle aspirates; peripheral blood; or bone marrow) and 5 normal controls. Sixty-four DNA samples (94%; 64 of 68) were successfully processed with the TruSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) and sequenced in 4 sequencing runs. The data were analyzed at 4 different filter settings for sequencing coverage and variant frequency cutoff. Results.-Libraries created from 40 specimens could be successfully sequenced in a single run and still yield sufficient coverage for robust data analysis of individual samples. Sensitivity for mutation detection down to 5% was demonstrated using dilutions of clinical specimens and control samples. The test was highly repeatable and reproducible and showed 100% concordance with clinically validated Sanger sequencing results. Comparison to an alternate next-generation sequencing technology was performed by also processing 9 of the specimens with the AmpliSeq Cancer Hotspot Panel (version 2; Life Technologies, Grand Island, New York). Thirty of the 31 (97%) TruSeq-detected variants covered by the designs of both panels were confirmed. Conclusions.-A sensitive, high-throughput, pan-cancer mutation panel for sequencing of cancer hot-spot mutations in 42 genes was validated for routine use in clinical testing. Copyright © 2015 College of American Pathologists.


Gulati G.,Anatomy and Cell Biology | Gulati G.,Thomas Jefferson University | Hevelow M.,Foundation Medicine | George M.,Anatomy and Cell Biology | And 5 more authors.
Archives of Pathology and Laboratory Medicine | Year: 2011

Context.-The key question when managing patients on warfarin therapy who present with life-threatening bleeding is how to use the international normalized ratio (INR) to best direct corrective therapy. The corollary question for the clinical laboratory is at what level will the INR reflect a critical value that requires notifying the clinician. Objective.-To determine the levels of vitamin K-dependent factors over a range of INR values. Design.-Evaluation of the vitamin K-dependent coagulation factor levels on plasma remnants from patients in whom a prothrombin time and INR was ordered to monitor warfarin therapy. There were a total of 83 specimens evaluated with an INR range from 1.0 to 8.26. Results.-The mean activity levels of all 4 factors remained near or above 50% when the INR was less than 1.5. The average factor X level was 23% when the INR range was 1.6 to 2.5, but levels of factors II, VII, and IX did not drop below the hemostatic range until the INR was greater than 2.5. At an INR of 3.6 or more, the activity levels of all 4 factors were less than 30% in more than 90% of the specimens. Conclusion.-Levels of factors II, VII, IX, and X declined with increasing INR but not at the same rate and not to the same level at a given INR. However, most of the values were below the hemostatic value once the INR was 3.6 or more, the level that was also considered critical for physician notification.


News Article | February 27, 2017
Site: www.rdmag.com

Scientists have identified another symptom of the Zika virus, this time focusing on the damage it causes to vision. A research team from the Wayne State University School of Medicine Department of Ophthalmology at the Kresge Eye Institute are the first to show that the Zika virus (ZIKV) can replicate in the eye’s retinal cells, causing severe tissue damage and even blindness in some cases. Ashok Kumar, Ph.D., a microbiologist and an assistant professor in the departments of Ophthalmology and of Anatomy and Cell Biology, led the research team in investigating whether Zika causes abnormalities in the eye. Zika is a significant emergent threat to global health, particularly during pregnancy. While it is not life-threatening, Zika has severe health implications beyond microcephaly—a condition where an infant’s head is significantly smaller than what is expected. According to Kumar, infants born with congenital Zika virus also have pathology in their eyes, ears, limbs and possibly other organs. Clinical studies show that Zika in the eye mainly impacts the retina—a layer of cells at the back of the eye that sends signals to the brain to visualize an object. “We studied the interaction of ZIKV with retinal cells. We observed that ZIKV can replicate and survive in retinal cells and ultimately kill them,” Kumar said in a statement. “Next, we tested whether ZIKV can cause retinal damage using an animal model. “To our surprise, we discovered that ZIKV infection of a mouse eye resulted in retinal lesions referred to as ‘chorioretinal atrophy’,” he added. “Interestingly, the ZIKV-infected mouse eyes showed some features of ZIKV-infected human eye. We believe we have a unique model to study molecular mechanisms of ocular ZIKV infection, and perhaps to test drugs or new anti-viral molecules to treat this blinding eye disease.” The eye is an immune-privileged organ, meaning Zika could hide there for months, Kumar said. The virus also has been shown to cause uveitis, another ophthalmic complication, in adults. More studies into the impact of Zika will continue. “Indeed, animal and human studies have shown the presence of ZIKV in tears, and there is ongoing research to determine how long, where and at what concentration the virus can survive in the eye,” Kumar said. “Similarly, we do not know the long-term impact of ZIKV-induced ocular abnormalities in infants born with congenital ZIKV infection. “Will they have a normal vision? What kind of vision rehab might they need? What about the social and economic impact in raising these children with a vision disability, if any?” Kumar’s team is now looking at how Zika modulates a cell to its advantage, while collaborating with another university to identify key genes and molecules that they think can attenuate Zika. The study was published in JCI Insight.

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