The mission of the Georgia Cancer Center of Augusta University is to reduce cancer morbidity and mortality through the application of laboratory and clinical research discoveries to prevention, early diagnosis, control and treatment of cancer.

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  • Georgia Cancer Center Integrated Genomics Resource & HPC Server

    Chang, Chang-Shen (Sam); Georgia Cancer Center
    Georgia Cancer Center at Augusta University is home to a High Performance Computing (HPC) Server. One goal of the HPC server is to host the new Biorepository software, LabVantage. This software is a web-based laboratory information management system, which tracks samples throughout their lifespan. All specimens that the Georgia Cancer Center Biorepository receives is entered into LabVantage, which generates a unique barcode number for each sample. Chain of custody is recorded throughout the sample’s lifespan, from inception to eventual withdrawal. LabVantage organizes data such as patient demographics, diagnosis, organ site, and linked pathology reports. 
LabVantage is compliant with all regulations relevant to patient privacy and satisfies all regulations set forth by The College of American Pathologists (CAP). All Biorepository personnel are trained to maintain confidentiality of patient information according to HIPAA regulations. The HPC Server is also used for the analysis of complex data including Next-Generation Sequencing data (NGS). It is currently used to perform data analysis on datasets such as those obtained from The Cancer Genome Atlas (TCGA). The analyses that used to take several weeks can now be performed in a matter of days. Georgia Cancer Center HPC Server is composed of 544 total compute cores and an aggregated memory of 2.9TB. The system is composed of (15) PowerEdge R430 1U systems (128 GB RAM each), (1) PowerEdge R830 (1024 GB RAM) and a high-speed 10GbE interconnect for intra-node communication. The HPCC also houses 633 TB RAW storage capacity. We will also be integrating existing Cancer Center servers including our Illumina Compute system that collects data directly from the Sequencer housed in the Georgia Cancer Center Integrated Genomics Shared Resource and the existing Bioinformatics HPC (see configuration diagram below). Access to the server is available to all Augusta University employees. There is a nominal fee associated with usage and users are required to undergo training.
  • DEVELOPMENT OF TRANSGENIC ZEBRAFISH MODEL FOR INVESTIGATION OF THE FUNCTION OF MICROGLIA

    Sura, Survasha; Department of Biological Sciences; Department of Biochemical and Molecular Biology; Georgia Cancer Center; Rajpurohit, Surendra K; Augusta University (2019-02-13)
    Zebrafish have emerged as a powerful model organism for elucidating the development and function of microglia. Generation of new transgenic reporter lines and imaging tools strengthen the zebrafish model in microglia study�in-vivo. The aim is to develop a novel compound transgenic line to study the inflammatory process mediated by NF-kB in microglia cells. This novel compound transgenic line will establish a new model for microglia study. To generate the novel compound zebrafish transgenic model for microglia, we are crossbreeding microglia transgenic line zebrafish (Tg(mpeg1:mCherry) with the NF-kB Tg(6xNFkB:EGFP) transgenic progeny. We first generate a heterozygous F1 progeny which will be bred to generate an F2 homozygous progeny. Once the F1 progeny of the Microglia-NfkB transgenic line is developed, they will be crossbred to develop the Homozygous compound transgenic line. Fluorescent Microscopy will be used to screen the larvae generated from the breeding events. By developing the compound transgenic line, we are optimizing microglia isolation and sorting methodology by using the related antibodies as the marker. The NF-kB microglia transgenic line will provide a unique platform for drug screening to address microglial based ailments, thus furthering the understanding and treatment of human disease.
  • Chetomin as a Potent Hsp90 Inhibitor

    Leibou, Stav; Lu, Sumin; Debbab, Abdssamad; Chadli, Ahmed; Georgia Cancer Center (2017-03)
    Molecular chaperones have been the focus of intense research for their important role in cancer cell homeostasis. Heat shock protein 90 (Hsp90) promotes metastasis, evasion of apoptosis, and proliferative angiogenesis in tumors through preserving the stability and functionality of its client proteins [1]. While the first generation of Hsp90 inhibitors has proven effective in hindering Hsp90 function, they have shown low clinical efficacy in part due to the induction of anti-apoptotic proteins Hsp27, Hsp40, and Hsp70 [2,3]. It is therefore our objective to develop novel efficacious Hsp90 inhibitors without these detrimental effects. During our screen for novel Hsp90 inhibitors, we found that the natural product, Chetomin, is a potent inhibitor of the Hsp90 machine chaperoning activity. Our in vitro data using human and murine mammary carcinoma cell lines suggest that Chetomin is effective in causing degradation of several known Hsp90 physiological client proteins that are crucial to cancer cell proliferation and survival. While the molecular mechanism by which Chetomin inhibits the Hsp90 function is still unclear, our data suggests that Chetomin is highly efficacious in killing cancer cells without induction of the anti-apoptotic proteins as does the first generation of Hsp90 inhibitors making Chetomin a promising new therapeutic agent.
  • T Cell Immune Response in Persistent Infection of Lymphocytic Choriomeningitis Virus (LCMV)

    Ou, Rong; Georgia Cancer Center (2004-07)
    The m urine LCMV system provides a ciassic model to study the mechanism of immunological tolerance, an efficient strategy used by virus to establish a persistent infection by selective down-regulation of virus-specific T lymphocytes. High viral burden in the onset o f infection drives responding cells into functional unresposiveness (anergy) that can, be followed by their physical elimination. In this study, the downregulation o f the virus-specific CD8^-T-ceil response was studied during a persistent infection o f adult mice, with particular emphasis on the contribution of the interferon response in promoting host defense, or perforin-, Fas/FasL-, or TN FR l-m ediated cytolysis in regulating T-cell homeostasis. Since LCMV infects a broad range o f host tissues, the functional properties o f virus-specific CD8'^ T cells in different tissues during LCMV infection were also evaluated. Infection of mice deficient in receptor for type I (IFN-a/p), type II (IFN-y), or both type I and II IFNs with LCMV isolates that vary in their capacity to induce T-celi exhaustion, revealed a critical role for IFN -a/p in restricting LCMV spread at the onset o f infection while IFN-y has impact on effector cells. The production o f IF N -a/p and/or IFN-y critically regulates the virus-host balance during the acute phase o f infection, such that a high viral burden drives responding cells into different programs o f exhaustion. Infection o f mice deficient in perferin, FasL or TNFRl with the Docile or Aggressive strains of LCMV revealed comparable kinetics of expansion and functional inactivation o f virusspecific C D ^ T cells in the early phase o f Infection in C57BL/6 controls. However, the data underscore a critical role for these molecules in the persistence o f the virus-specific CD8"‘-T-ceil population once it has become anergic. Study o f the functional properties of virus-specific CD8'^ T cells in different tissues during LCMV infections showed that a centra! role for the viral load in lymphoid tissue in the induction and maintenance of clonal exhaustion. The data strongly suggest that CD8^ T ceils may be differentially regulated in the environments o f lymphoid versus nonlymphoid tissues, and the pattern of T cell exhaustion observed with mice is likely a common feature o f the immune response during chronic infections in humans.
  • Molecular Biology of Amino Acid and Peptide Transport Systems

    Li, Huiwu; Georgia Cancer Center (1999)
    (First Paragraph) Amino acids are essential components in cellular metabolism. Some of these amino acids can be synthesized within the cells from other biological molecules and these amino acids are termed ‘nonessential’. These ‘nonessential’ amino acids are alanine, aspartate, cysteine, glutamate, glycine, pro line, serine, tyrosine, glutamine and asparagine. In contrast, some amino acids cannot be synthesized endogenously and have to be supplied in the diet (1). These amino acids are termed ‘essential’. These ‘essential’ amino acids are histidine, arginine, leucine, isoleucine, lysine, methionine, threonine, phenylalanine, tryptophan, and valine. Mammalian cells require ‘essential’ as well as ‘nonessential’ amino acids for their metabolic activity. Even though the cells can synthesize the ‘nonessential’ amino acids to some extent, most of the amino acids have to be supplied to the cells via specific membrane transport mechanisms.
  • Regulation and Function of the Major Stress-Induced HSP70 Molecular Chaperone in vivo: Analysis of Mice with Targeted Gene Disruption of the HSP70.1 or HSP70A1

    Huang, Lei; Georgia Cancer Center (6/3/2002)
    (First Paragraph) The cellular response to stress, including exposure to environmental (UV radiation, heat shock, heavy metals), pathological (infection, fever, inflammation, malignancy, ischemia) or physiological (growth factor, hormonal stimulation, tissue development) stimuli is represented at the molecular level by synthesis of groups of protein named heat shock proteins [hsp(s)] (Benjamin 1998; Feder and others 1992; Jolly and Morimoto 2000; Li and Mivechi 1986; Lindquist 1986; Smith 1998). The presence of hsp(s) protect host cells from the damage caused by thermal stress, and after induction of hsp expression, cells are protected well from higher temperatures than they can normally tolerate. This phenomenon is defined as themiotoleranee (Gemer 1975; Li and Mivechi 1986). The protective role of hsp(s) is attributed to several functional properties, including active participation in maintaining proteins in their native correctly folded states, promoting degradation and refolding of misfolded proteins, and minimizing aggregation and incorrect interactions between proteins (Agashe and Hartl 2000; Gething and Sambrook 1992). In addition, hsp(s) can function in cellular protection by modulating the engagement and progression of apoptosis induced by a variety of stress stimuli (Beere and Green 2001). Besides the recognition of the cytoprotective function of hsp(s) under stress conditions, widespread clinical interests exist in their chaperone function during a range of human pathologies, including neurodegenerative conditions, such as amyloidosis, prion disease, and Alzheimer's disease, and cardiovascular diseases, such as myocardial ischemia, cardiac hypertrophy, stroke, and blood vessel injury (Benjamin 1998; Planas and others 1997; Smith 1998).
  • Protection Against Colonic Inflammation and Colon Cancer by Commensal Bacterial Metabolites: An Obligatory Role for the Short- Chain Fatty Acid Transporter Slc5a8

    Gurav, Ashish; Georgia Cancer Center (2014-11)
    Dietary fiber consumption has long been known to protect against inflammatory bowel diseases and colorectal carcinogenesis. In mammals, large intestinal microorganisms ferment dietary fiber to generate energy, while releasing short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate. Interestingly, SCFAs are also known to protect against intestinal inflammation and colorectal carcinogenesis, although the molecular mechanisms behind these actions are still being investigated. For most of their biological effects, SCFAs must be transported from lumen into the intestinal tissue, where they activate multiple biological processes. We and others have reported Slc5a8 as a high affinity transport mechanism for SCFAs, which would remain fully functional, even when SCFA concentration drops to sub-millimolar range, whereas other transport mechanisms are rendered inefficient. The aim of the current study was to test protective role of Slc5a8 against intestinal inflammation and colorectal carcinogenesis during suboptimal intake of dietary fiber. We observed that Slc5a8 is obligatory for HDAC-inhibition in colonic epithelium and intestinal barrier function, only when the animals were fed a dietary fiber-free diet (FF diet), and not when the animals were fed diet containing optimal amounts of fibers (FC Diet). Compared to WT, Slc5a8-/- animals demonstrated higher susceptibility to AOMDSS- mediated intestinal inflammation and colorectal carcinogenesis under FF dietary conditions, but not under FC dietary conditions. At molecular level, we found that butyrate and propionate could induce potent immunosuppressive enzymes Indoleamine 2,3-dioxygenase and Aldehyde Dehydrogenase 1A2 in dendritic cells obtained from WT animals, but not from Slc5a8-/- animals. Butyrate, transported via Slc5a8 enabled DCs to suppress conversion of naïve T cells to interferon-γ secreting pro-inflammatory T cells and Slc5a8-/- animals harbored higher proportion of interferon-γ+ CD4+ T cells in vivo. Taken together, our data provide crucial evidence for critical role of Slc5a8 mediating protective effects of dietary fiber metabolites, SCFAs in protecting against intestinal inflammation and colorectal carcinogenesis.
  • B-Lymphoid Cells with Attributes of Dendritic Cells Regulate T Cells via Indoleamine 2,3 Dioxygenase

    Johnson, Burles Avner III; Cancer Research Center (2012-04)
    A rare subset of murine dendritic cells expressing the B cell marker CD19 are specialized to express the T cell regulatory enzyme indoleamine 2,3 dioxygenase (IDO-competent DCs). Here we show that IDO-competent DCs expressed Pax5, a transcription factor that maintains B cell lineage commitment, and drives expression of CD19 and surface immunoglobulin (slg). However IDO-competent DCs also exhibited multiple attributes of DCs including DC marker expression and potent T cell stimulatory properties when IDO was not induced. Unexpectedly, DCs expressing IDO were present in B cell deficient mice following TLR9 ligation, indication that B cell receptor (BCR) expression was not required for IDO function. Conversely, DCs from CD19 deficient mice did not express IDO after in vivo TLR9 ligation. This defect was not caused by blockade of IDO-competent DC development in CD19-deficient mice because IDO expression was incduced in these cells by in vitro interferon gamma treatment. Even though DCs from B cell deficient mice expressed IDO following TLR9 ligation, regulatory T cells (Tregs) from B cell deficient mice had impaired suppressor activity. IDO-competent DCs expressed high levels of CD1d-deficient mice. IDO-competent DCs also expressed IL-10 deficient mice to express IDO. Finally we demonstrated that DCs from draining lymph nodes (dLNs) of four week old prediabetic female non obese diabetic (NOD) mice expressed functional IDO following topical treatment with phorbol myristate acetate (PMA). However DCs from dLNs of six week old prediabetic NOD female mice did not express IDO following topical PMA treatment, indicating a critical defect in a specific immunosuppressive mechanism in NOD female mice that coincides with the appearance of insulitis. These data identify IDO competent DCs as a unique B lymphoid lineage cell type that has tightly controlled regulatory properties, and a DC subset whose acquired defect may contribute to autoimmune disease in NOD mice.
  • De novo transcriptome sequencing in a songbird, the dark-eyed junco (Junco hyemalis): genomic tools for an ecological model system

    Peterson, Mark P; Whittaker, Danielle J; Ambreth, Shruthi; Sureshchandra, Suhas; Buechlein, Aaron; Podicheti, Ram; Choi, Jeong-Hyeon; Lai, Zhao; Mockatis, Keithanne; Colbourne, John K.; et al. (2012-07-9)
    Background: Though genomic-level data are becoming widely available, many of the metazoan species sequenced are laboratory systems whose natural history is not well documented. In contrast, the wide array of species with very well-characterized natural history have, until recently, lacked genomics tools. It is now possible to address significant evolutionary genomics questions by applying high-throughput sequencing to discover the majority of genes for ecologically tractable species, and by subsequently developing microarray platforms from which to investigate gene regulatory networks that function in natural systems. We used GS-FLX Titanium Sequencing (Roche/454-Sequencing) of two normalized libraries of pooled RNA samples to characterize a transcriptome of the dark-eyed junco (Junco hyemalis), a North American sparrow that is a classically studied species in the fields of photoperiodism, speciation, and hormone-mediated behavior.
  • Novel Somatic Mutations to PI3K Pathway Genes in Metastatic Melanoma

    Shull, Austin Y.; Latham-Schwark, Alicia; Ramasamy, Poornema; Leskoske, Kristin; Oroian, Dora; Birtwistle, Marc R.; Buckhaults, Phillip J.; GHSU Cancer Center (2012-08-17)
    Background: BRAFV600 inhibitors have offered a new gateway for better treatment of metastatic melanoma. However, the overall efficacy of BRAFV600 inhibitors has been lower than expected in clinical trials, and many patients have shown resistance to the drugâ s effect. We hypothesized that somatic mutations in the Phosphoinositide 3-Kinase (PI3K) pathway, which promotes proliferation and survival, may coincide with BRAFV600 mutations and contribute to chemotherapeutic resistance.
  • Towards Curative Cancer Immunotherapy: Overcoming Posttherapy Tumor Escape

    Zhou, Gang; Levitsky, Hyam; GHSU Cancer Center; Department of Medicine (2012-05-31)
    The past decade has witnessed the evolvement of cancer immunotherapy as an increasingly effective therapeutic modality, evidenced by the approval of two immune-based products by the FDA, that is, the cancer vaccine Provenge (sipuleucel-T) for prostate cancer and the antagonist antibody against cytotoxic T-lymphocyte antigen-4 (CTLA-4) ipilimumab for advanced melanoma. In addition, the clinical evaluations of a variety of promising immunotherapy drugs are well under way. Benefiting from more efficacious immunotherapeutic agents and treatment strategies, a number of recent clinical studies have achieved unprecedented therapeutic outcomes in some patients with certain types of cancers. Despite these advances, however, the efficacy of most cancer immunotherapies currently under clinical development has been modest. A recurring scenario is that therapeutic maneuvers initially led to measurable antitumor immune responses in cancer patients but ultimately failed to improve patient outcomes. It is increasingly recognized that tumor cells can antagonize therapy-induced immune attacks through a variety of counterregulation mechanisms, which represent a fundamental barrier to the success of cancer immunotherapy. Herein we summarize the findings from some recent preclinical and clinical studies, focusing on how tumor cells advance their survival and expansion by hijacking therapy-induced immune effector mechanisms that would otherwise mediate their destruction.
  • Acute Progression of BCR-FGFR1 Induced Murine B-Lympho/Myeloproliferative Disorder Suggests Involvement of Lineages at the Pro-B Cell Stage

    Ren, MingQiang; Tidwell, Josephine A.; Sharma, Suash; Cowell, John K.; GHSU Cancer Center; Department of Pathology (2012-06-6)
    Constitutive activation of FGFR1, through rearrangement with various dimerization domains, leads to atypical myeloproliferative disorders where, although T cell lymphoma are common, the BCR-FGFR1 chimeric kinase results in CML-like leukemia. As with the human disease, mouse bone marrow transduction/transplantation with BCR-FGFR1 leads to CML-like myeloproliferation as well as B-cell leukemia/lymphoma. The murine disease described in this report is virtually identical to the human disease in that both showed bi-lineage involvement of myeloid and B-cells, splenomegaly, leukocytosis and bone marrow hypercellularity. A CD19+ IgMâ CD43+ immunophenotype was seen both in primary tumors and two cell lines derived from these tumors. In all primary tumors, subpopulations of these CD19+ IgMâ CD43+ were also either B220+ or B220â , suggesting a block in differentiation at the pro-B cell stage. The B220â phenotype was retained in one of the cell lines while the other was B220+. When the two cell lines were transplanted into syngeneic mice, all animals developed the same B-lymphoblastic leukemia within 2-weeks. Thus, the murine model described here closely mimics the human disease with bilineage myeloid and B-cell leukemia/lymphoma which provides a representative model to investigate therapeutic intervention and a better understanding of the etiology of the disease.
  • Toward integrative cancer immunotherapy: targeting the tumor microenvironment

    Emens, Leisha A; Silverstein, Samuel C; Khleif, Samir; Marincola, Francesco M; Galon, Jérôme; GHSU Cancer Center (2012-04-10)
    The development of cancer has historically been attributed to genomic alterations of normal host cells. Accordingly, the aim of most traditional cancer therapies has been to destroy the transformed cells themselves. There is now widespread appreciation that the progressive growth and metastatic spread of cancer cells requires the cooperation of normal host cells (endothelial cells, fibroblasts, other mesenchymal cells, and immune cells), both local to, and at sites distant from, the site at which malignant transformation occurs. It is the balance of these cellular interactions that both determines the natural history of the cancer, and influences its response to therapy. This active tumor-host dynamic has stimulated interest in the tumor microenvironment as a key target for both cancer diagnosis and therapy. Recent data has demonstrated both that the presence of CD8+ T cells within a tumor is associated with a good prognosis, and that the eradication of all malignantly transformed cells within a tumor requires that the intra-tumoral concentration of cytolytically active CD8+ effector T cells remain above a critical concentration until every tumor cell has been killed. These findings have stimulated two initiatives in the field of cancer immunotherapy that focus on the tumor microenvironment. The first is the development of the immune score as part of the routine diagnostic and prognostic evaluation of human cancers, and the second is the development of combinatorial immune-based therapies that reduce tumor-associated immune suppression to unleash pre-existing or therapeutically-induced tumor immunity. In support of these efforts, the Society for the Immunotherapy of Cancer (SITC) is sponsoring a workshop entitled "Focus on the Target: The Tumor Microenvironment" to be held October 24-25, 2012 in Bethesda, Maryland. This meeting should support development of the immune score, and result in a position paper highlighting opportunities for the development of integrative cancer immunotherapies that sculpt the tumor microenvironment to promote definitive tumor rejection.
  • Cytotoxic Chemotherapy and CD4+ Effector T Cells: An Emerging Alliance for Durable Antitumor Effects

    Ding, Zhi-Chun; Zhou, Gang; GHSU Cancer Center; Department of Medicine (2012-02-6)
    Standard cytotoxic chemotherapy can initially achieve high response rates, but relapses often occur in patients and represent a severe clinical problem. As increasing numbers of chemotherapeutic agents are found to have immunostimulatory effects, there is a growing interest to combine chemotherapy and immunotherapy for synergistic antitumor effects and improved clinical benefits. Findings from recent studies suggest that highly activated, polyfunctional CD4+ effector T cells have tremendous potential in strengthening and sustaining the overall host antitumor immunity in the postchemotherapy window. This review focuses on the latest progresses regarding the impact of chemotherapy on CD4+ T-cell phenotype and function and discusses the prospect of exploiting CD4+ T cells to control tumor progression and prevent relapse after chemotherapy.
  • Loss of Zebrafish lgi1b Leads to Hydrocephalus and Sensitization to Pentylenetetrazol Induced Seizure-Like Behavior

    Teng, Yong; Xie, Xiayang; Walker, Steven L.; Saxena, Meera T.; Kozlowski, David J.; Mumm, Jeff S.; Cowell, John K.; GHSU Cancer Center; Department of Cellular Biology and Anatomy; Vision Discovery Institute; et al. (2011-09-16)
    Mutations in the LGI1 gene predispose to a hereditary epilepsy syndrome and is the first gene associated with this disease which does not encode an ion channel protein. In zebrafish, there are two paralogs of the LGI1 gene, lgi1a and lgi1b. Knockdown of lgi1a results in a seizure-like hyperactivity phenotype with associated developmental abnormalities characterized by cellular loss in the eyes and brain. We have now generated knockdown morphants for the lgi1b gene which also show developmental abnormalities but do not show a seizure-like behavior. Instead, the most striking phenotype involves significant enlargement of the ventricles (hydrocephalus). As shown for the lgi1a morphants, however, lgi1b morphants are also sensitized to PTZ-induced hyperactivity. The different phenotypes between the two lgi1 morphants support a subfunctionalization model for the two paralogs.
  • Linear Approaches to Intramolecular Forster Resonance Energy Transfer Probe Measurements for Quantitative Modeling

    Birtwistle, Marc R.; von Kriegsheim, Alexander; Kida, Katarzyna; Schwarz, Juliane P.; Anderson, Kurt I.; Kolch, Walter; GHSU Cancer Center (2011-11-16)
    Numerous unimolecular, genetically-encoded Forster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted Ralt) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on Ralt are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.
  • Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing

    Lee, Eun-Joon; Pei, Lirong; Srivastava, Gyan; Joshi, Trupti; Kushwaha, Garima; Choi, Jeong-Hyeon; Robertson, Keith D.; Wang, Xinguo; Colbourne, John K.; Zhang, Lu; et al. (2011-10-23)
    We applied a solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of ~1 million CpGs in more than 21â 408 CGIs and more than 15â 946 transcriptional regulatory regions. Of the CpGs analyzed, 77â 84% fell on or near capture probe sequences; 69â 75% fell within CGIs. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. Differentially methylated regions (DMRs) were identified in the 5â ²-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X-chromosome among the three breast cancer cell lines analyzed. We chose 46 candidate loci (762 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. Targeted bisulfite sequencing of three DNA methyltransferase (DNMT) knockout cell lines and the wild-type HCT116 colon cancer cell line revealed a significant decrease in CpG methylation for the DNMT1 knockout and DNMT1, 3B double knockout cell lines, but not in DNMT3B knockout cell line. We demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer epigenome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples.
  • The temporal and spatial expression pattern of the LGI1 epilepsy predisposition gene during mouse embryonic cranial development

    Silva, Jeane; Wang, Guanghu; Cowell, John K.; GHSU Cancer Center; Department of Neurology; Institute of Molecular Medicine and Genetics (2011-05-13)
    Background: Mutations in the LGI1 gene predispose to a rare, hereditary form of temporal epilepsy. Currently, little is known about the temporal and spatial expression pattern of Lgi1 during normal embryogenesis and so to define this more clearly we used a transgenic mouse line that expresses GFP under the control of Lgi1 cis-regulatory elements.
  • Functional Dissection of HOXD Cluster Genes in Regulation of Neuroblastoma Cell Proliferation and Differentiation

    Zha, Yunhong; Ding, Emily; Yang, Liqun; Mao, Ling; Wang, Xiangwei; McCarthy, Brian A.; Huang, Shuang; Ding, Han-Fei; GHSU Cancer Center; Department of Pathology; et al. (2012-08-7)
    Retinoic acid (RA) can induce growth arrest and neuronal differentiation of neuroblastoma cells and has been used in clinic for treatment of neuroblastoma. It has been reported that RA induces the expression of several HOXD genes in human neuroblastoma cell lines, but their roles in RA action are largely unknown. The HOXD cluster contains nine genes (HOXD1, HOXD3, HOXD4, and HOXD8-13) that are positioned sequentially from 3â ² to 5â ², with HOXD1 at the 3â ² end and HOXD13 the 5â ² end. Here we show that all HOXD genes are induced by RA in the human neuroblastoma BE(2)-C cells, with the genes located at the 3â ² end being activated generally earlier than those positioned more 5â ² within the cluster. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 is sufficient to induce both growth arrest and neuronal differentiation, which is associated with downregulation of cell cycle-promoting genes and upregulation of neuronal differentiation genes. However, induction of other HOXD genes either has no effect (HOXD1) or has partial effects (HOXD3, HOXD4, HOXD11 and HOXD13) on BE(2)-C cell proliferation or differentiation. We further show that knockdown of HOXD8 expression, but not that of HOXD9 expression, significantly inhibits the differentiation-inducing activity of RA. HOXD8 directly activates the transcription of HOXC9, a key effector of RA action in neuroblastoma cells. These findings highlight the distinct functions of HOXD genes in RA induction of neuroblastoma cell differentiation.
  • NF-kB2 mutation targets survival, proliferation and differentiation pathways in the pathogenesis of plasma cell tumors

    McCarthy, Brian A.; Yang, Liqun; Ding, Jane; Ren, MingQiang; King, William; ElSalanty, Mohammed; Zakhary, Ibrahim; Sharawy, Mohamed; Cui, Hongjuan; Ding, Han-Fei; et al. (2012-05-29)
    Background: Abnormal NF-κB2 activation has been implicated in the pathogenesis of multiple myeloma, a cancer of plasma cells. However, a causal role for aberrant NF-κB2 signaling in the development of plasma cell tumors has not been established. Also unclear is the molecular mechanism that drives the tumorigenic process. We investigated these questions by using a transgenic mouse model with lymphocyte-targeted expression of p80HT, a lymphoma-associated NF-κB2 mutant, and human multiple myeloma cell lines.

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