Publications by authors named "Aikseng Ooi"

29 Publications

  • Page 1 of 1

Non-canonical NRF2 activation promotes a pro-diabetic shift in hepatic glucose metabolism.

Mol Metab 2021 Apr 30;51:101243. Epub 2021 Apr 30.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, USA; University of Arizona Cancer Center, University of Arizona, Tucson, AZ, 85724, USA. Electronic address:

Objective: NRF2, a transcription factor that regulates cellular redox and metabolic homeostasis, plays a dual role in human disease. While it is well known that canonical intermittent NRF2 activation protects against diabetes-induced tissue damage, little is known regarding the effects of prolonged non-canonical NRF2 activation in diabetes. The goal of this study was to determine the role and mechanisms of prolonged NRF2 activation in arsenic diabetogenicity.

Methods: To test this, we utilized an integrated transcriptomic and metabolomic approach to assess diabetogenic changes in the livers of wild type, Nrf2, p62, or Nrf2; p62 mice exposed to arsenic in the drinking water for 20 weeks.

Results: In contrast to canonical oxidative/electrophilic activation, prolonged non-canonical NRF2 activation via p62-mediated sequestration of KEAP1 increases carbohydrate flux through the polyol pathway, resulting in a pro-diabetic shift in glucose homeostasis. This p62- and NRF2-dependent increase in liver fructose metabolism and gluconeogenesis occurs through the upregulation of four novel NRF2 target genes, ketohexokinase (Khk), sorbitol dehydrogenase (Sord), triokinase/FMN cyclase (Tkfc), and hepatocyte nuclear factor 4 (Hnf4A).

Conclusion: We demonstrate that NRF2 and p62 are essential for arsenic-mediated insulin resistance and glucose intolerance, revealing a pro-diabetic role for prolonged NRF2 activation in arsenic diabetogenesis.
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http://dx.doi.org/10.1016/j.molmet.2021.101243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8164084PMC
April 2021

Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

Nat Commun 2021 03 12;12(1):1645. Epub 2021 Mar 12.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA, USA.

Anemias of chronic disease and inflammation (ACDI) result from restricted iron delivery to erythroid progenitors. The current studies reveal an organellar response in erythroid iron restriction consisting of disassembly of the microtubule cytoskeleton and associated Golgi disruption. Isocitrate supplementation, known to abrogate the erythroid iron restriction response, induces reassembly of microtubules and Golgi in iron deprived progenitors. Ferritin, based on proteomic profiles, regulation by iron and isocitrate, and putative interaction with microtubules, is assessed as a candidate mediator. Knockdown of ferritin heavy chain (FTH1) in iron replete progenitors induces microtubule collapse and erythropoietic blockade; conversely, enforced ferritin expression rescues erythroid differentiation under conditions of iron restriction. Fumarate, a known ferritin inducer, synergizes with isocitrate in reversing molecular and cellular defects of iron restriction and in oral remediation of murine anemia. These findings identify a cytoskeletal component of erythroid iron restriction and demonstrate potential for its therapeutic targeting in ACDI.
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http://dx.doi.org/10.1038/s41467-021-21938-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955080PMC
March 2021

The NRF2-LOC344887 signaling axis suppresses pulmonary fibrosis.

Redox Biol 2021 01 20;38:101766. Epub 2020 Oct 20.

Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, 85721, USA; University of Arizona Cancer Center, University of Arizona, Tucson, AZ, 85721, USA. Electronic address:

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible disease characterized by an increase in differentiation of fibroblasts to myofibroblasts and excessive accumulation of extracellular matrix in lung tissue. Pharmacological activation of NRF2 has proved to be a valuable antifibrotic approach, however the detailed mechanisms of how NRF2 mediates antifibrotic function remain unclear. In this study, we found that the antifibrotic function of sulforaphane (SFN), an NRF2 activator, was largely dependent on LOC344887, a long noncoding RNA. Two functional AREs were identified in both the promoter and intron 1 of LOC344887, which defines LOC344887 as a novel anti-fibrotic NRF2 target gene. RNA-seq analysis revealed that LOC344887 controls genes and signaling pathways associated with fibrogenesis. Deletion or downregulation of LOC344887 enhanced expression of CDH2/N-cadherin, as well as a number of other fibrotic genes and blunted the antifibrotic effects of SFN. Furthermore, LOC344887-mediated downregulation of fibrotic genes may involve the PI3K-AKT signaling pathway, as pharmacologic inhibition of PI3K activity blocked the effects of LOC344887 knockdown. Our findings demonstrate that NRF2-mediated LOC344887 upregulation contributes to the antifibrotic potential of SFN by repressing the expression of CDH2 and other fibrotic genes, providing novel insight into how NRF2 controls the regulatory networks of IPF. This study provides a better understanding of the molecular mechanisms of NRF2 activators against pulmonary fibrosis and presents a novel therapeutic axis for prevention and intervention of fibrosis-related diseases.
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http://dx.doi.org/10.1016/j.redox.2020.101766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573654PMC
January 2021

Advances in hereditary leiomyomatosis and renal cell carcinoma (HLRCC) research.

Authors:
Aikseng Ooi

Semin Cancer Biol 2020 04 2;61:158-166. Epub 2019 Nov 2.

Department of Pharmacology and Toxicology, University of Arizona, College of Pharmacy, 1703 East Mabel Street, 85721, Tucson, AZ, United States. Electronic address:

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an autosomal dominant hereditary cancer syndrome with incomplete penetrance. It is caused by a germline amorphic allele of the FH gene, which encodes the TCA cycle enzyme, fumarate hydratase (FH). HLRCC patients are genetically predisposed to develop skin leiomyomas, uterine fibroids, and the aggressive kidney cancer of type 2 papillary morphology. Loss-of-heterozygocity at the FH locus that cause a complete loss of FH enzymatic function is always detected in these tumor tissues. Molecular pathway elucidation, genomic studies, and systematic genetics screens reported over the last two decades have identified several FH-inactivation driven pathways alterations, as well as rationally conceived treatment strategies that specifically target FH tumor cells. These treatment strategies include ferroptosis induction, oxidative stress promotion, and metabolic alteration. As the fundamental biology of HLRCC continues to be uncovered, these treatment strategies continue to be refined and may one day lead to a strategy to prevent disease onset among HLRCC patients. With a more complete picture of HLRCC biology, the safe translation of experimental treatment strategies into clinical practice is achievable in the foreseeable future.
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http://dx.doi.org/10.1016/j.semcancer.2019.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078051PMC
April 2020

Differential and overlapping targets of the transcriptional regulators NRF1, NRF2, and NRF3 in human cells.

J Biol Chem 2019 11 18;294(48):18131-18149. Epub 2019 Oct 18.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721; University of Arizona Cancer Center, University of Arizona, Tucson, Arizona 85721. Electronic address:

The nuclear factor (erythroid 2)-like (NRF) transcription factors are a subset of cap'n'collar transcriptional regulators. They consist of three members, NRF1, NRF2, and NRF3, that regulate the expression of genes containing antioxidant-response elements (AREs) in their promoter regions. Although all NRF members regulate ARE-containing genes, each is associated with distinct roles. A comprehensive study of differential and overlapping DNA-binding and transcriptional activities of the NRFs has not yet been conducted. Here, we performed chromatin immunoprecipitation (ChIP)-exo sequencing, an approach that combines ChIP with exonuclease treatment to pinpoint regulatory elements in DNA with high precision, in conjunction with RNA-sequencing to define the transcriptional targets of each NRF member. Our approach, done in three U2OS cell lines, identified 31 genes that were regulated by all three NRF members, 27 that were regulated similarly by all three, and four genes that were differentially regulated by at least one NRF member. We also found genes that were up- or down-regulated by only one NRF member, with 84, 84, and 22 genes that were regulated by NRF1, NRF2, and NRF3, respectively. Analysis of the ARE motifs identified in ChIP peaks revealed that NRF2 prefers binding to AREs flanked by GC-rich regions and that NRF1 prefers AT-rich flanking regions. Thus, sequence preference, likely in combination with upstream signaling events, determines NRF member activation under specific cellular contexts. Our analysis provides a comprehensive description of differential and overlapping gene regulation by the transcriptional regulators NRF1, NRF2, and NRF3.
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http://dx.doi.org/10.1074/jbc.RA119.009591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885608PMC
November 2019

Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor.

Mol Cell Biol 2019 07 13;39(13). Epub 2019 Jun 13.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, USA

The nuclear factor (erythroid 2)-like 2 (NRF2 or NFE2L2) transcription factor regulates the expression of many genes that are critical in maintaining cellular homeostasis. Its deregulation has been implicated in many diseases, including cancer and metabolic and neurodegenerative diseases. While several mechanisms by which NRF2 can be activated have gradually been identified over time, a more complete regulatory network of NRF2 is still lacking. Here we show through a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen that a total of 273 genes, when knocked out, will lead to sustained NRF2 activation. Pathway analysis revealed a significant overrepresentation of genes (18 of the 273 genes) involved in autophagy. Molecular validation of a subset of the enriched genes identified 8 high-confidence genes that negatively regulate NRF2 activity irrespective of cell type: , , , , , , , and the well-known negative regulator of NRF2, Of these, , , , and are known to be involved in autophagic processes. Our results present a comprehensive list of NRF2 negative regulators and reveal an intimate link between autophagy and NRF2 regulation.
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http://dx.doi.org/10.1128/MCB.00037-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580702PMC
July 2019

RPA1 binding to NRF2 switches ARE-dependent transcriptional activation to ARE-NRE-dependent repression.

Proc Natl Acad Sci U S A 2018 10 11;115(44):E10352-E10361. Epub 2018 Oct 11.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721;

NRF2 regulates cellular redox homeostasis, metabolic balance, and proteostasis by forming a dimer with small musculoaponeurotic fibrosarcoma proteins (sMAFs) and binding to antioxidant response elements (AREs) to activate target gene transcription. In contrast, NRF2-ARE-dependent transcriptional repression is unreported. Here, we describe NRF2-mediated gene repression via a specific seven-nucleotide sequence flanking the ARE, which we term the NRF2-replication protein A1 (RPA1) element (NRE). Mechanistically, RPA1 competes with sMAF for NRF2 binding, followed by interaction of NRF2-RPA1 with the ARE-NRE and eduction of promoter activity. Genome-wide in silico and RNA-seq analyses revealed this NRF2-RPA1-ARE-NRE complex mediates negative regulation of many genes with diverse functions, indicating that this mechanism is a fundamental cellular process. Notably, repression of , which encodes the nonmuscle myosin light chain kinase, by the NRF2-RPA1-ARE-NRE complex disrupts vascular integrity in preclinical inflammatory lung injury models, illustrating the translational significance of NRF2-mediated transcriptional repression. Our findings reveal a gene-suppressive function of NRF2 and a subset of negatively regulated NRF2 target genes, underscoring the broad impact of NRF2 in physiological and pathological settings.
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http://dx.doi.org/10.1073/pnas.1812125115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217430PMC
October 2018

A catalogue of somatic NRF2 gain-of-function mutations in cancer.

Sci Rep 2018 08 27;8(1):12846. Epub 2018 Aug 27.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, 85721, USA.

Identification and characterization of somatic mutations in cancer have important prognostication and treatment implications. Genes encoding the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor and its negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), are frequently mutated in cancer. These mutations drive constitutive NRF2 activation and correlate with poor prognosis. Despite its apparent significance, a comprehensive catalogue of somatic NRF2 mutations across different tumor types is still lacking. Here, we catalogue NRF2 mutations in The Cancer Genome Atlas (TCGA) database. 226 unique NRF2-mutant tumors were identified from 10,364 cases. NRF2 mutations were found in 21 out of the 33 tumor types. A total of 11 hotspots were identified. Of these, mutation to the R34 position was most frequent. Notably, R34 and D29 mutations were overrepresented in bladder, lung, and uterine cancers. Analyses of corresponding RNA sequencing data using a de novo derived gene expression classifier showed that the R34 mutations drive constitutive NRF2 activation with a selection pressure biased against the formation of R34L. Of all R34 mutants, R34L conferred the least degree of protein stabilization, suggesting a pro-tumor NRF2 half-life threshold. Our findings offer a comprehensive catalogue of NRF2 mutations in cancer that can help prognostication and NRF2 research.
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http://dx.doi.org/10.1038/s41598-018-31281-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110754PMC
August 2018

Fumarate hydratase inactivation in hereditary leiomyomatosis and renal cell cancer is synthetic lethal with ferroptosis induction.

Cancer Sci 2018 Sep 20;109(9):2757-2766. Epub 2018 Jul 20.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, USA.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome characterized by inactivation of the Krebs cycle enzyme fumarate hydratase (FH). HLRCC patients are at high risk of developing kidney cancer of type 2 papillary morphology that is refractory to current radiotherapy, immunotherapy and chemotherapy. Hence, an effective therapy for this deadly form of cancer is urgently needed. Here, we show that FH inactivation (FH ) proves synthetic lethal with inducers of ferroptosis, an iron-dependent and nonapoptotic form of cell death. Specifically, we identified gene signatures for compound sensitivities based on drug responses for 9 different drug classes against the NCI-60 cell lines. These signatures predicted that ferroptosis inducers would be selectively toxic to FH cell line UOK262. Preferential cell death against UOK262-FH was confirmed with 4 different ferroptosis inducers. Mechanistically, the FH sensitivity to ferroptosis is attributed to dysfunctional GPX4, the primary cellular defender against ferroptosis. We identified that C93 of GPX4 is readily post-translationally modified by fumarates that accumulate in conditions of FH , and that C93 modification represses GPX4 activity. Induction of ferroptosis in FH-inactivated tumors represents an opportunity for synthetic lethality in cancer.
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http://dx.doi.org/10.1111/cas.13701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125459PMC
September 2018

Kelch-like ECH-associated protein 1 (KEAP1) differentially regulates nuclear factor erythroid-2-related factors 1 and 2 (NRF1 and NRF2).

J Biol Chem 2018 02 18;293(6):2029-2040. Epub 2017 Dec 18.

From the Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721

Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of or results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology-like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in than in isogenic cell lines, whereas NRF2 is dramatically stabilized in cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.
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http://dx.doi.org/10.1074/jbc.RA117.000428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808764PMC
February 2018

BINDING SITE ANALYSIS OF THE NR4A NUCLEAR RECEPTOR NHR-6 DURING DEVELOPMENT.

Nucl Receptor Res 2017 23;4. Epub 2017 Jul 23.

Department of Basic Pharmaceutical Sciences, School of Pharmacy, and Biology Program, School of Science, University of Louisiana Monroe, Monroe, LA, USA 71209.

Members of the NR4A subfamily of nuclear receptors make up a highly conserved, functionally diverse group of transcription factors implicated in a multitude of cellular processes such as proliferation, differentiation, apoptosis, metabolism and DNA repair. The gene which encodes the sole NR4A nuclear receptor homolog, has a critical role in organogenesis and regulates the development of the spermatheca organ system. Our previous work revealed that is required for spermatheca cell divisions in late L3 and early L4 and spermatheca cell differentiation during the mid L4 stage. Here, we utilized chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify NHR-6 binding sites during both the late L3/early L4 and mid L4 developmental stages. Our results revealed 30,745 enriched binding sites for NHR-6, ~70% of which were within 3 kb upstream of a gene transcription start site. Binding sites for a cohort of candidate target genes with probable functions in spermatheca organogenesis were validated through qPCR. Reproductive and spermatheca phenotypes were also evaluated for these genes following a loss-of-function RNAi screen which revealed several genes with critical functions during spermatheca organogenesis. Our results uncovered a complex nuclear receptor regulatory network whereby NHR-6 regulates multiple cellular processes during spermatheca organogenesis.
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http://dx.doi.org/10.11131/2017/101288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634515PMC
July 2017

The effects of NRF2 modulation on the initiation and progression of chemically and genetically induced lung cancer.

Mol Carcinog 2018 02 6;57(2):182-192. Epub 2017 Nov 6.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona.

Targeting the transcription factor NRF2 has been recognized as a feasible strategy for cancer prevention and treatment, but many of the mechanistic details underlying its role in cancer development and progression are lacking. Therefore, careful mechanistic studies of the NRF2 pathway in cancer initiation and progression are needed to identify which therapeutic avenue-activation or inhibition-is appropriate in a given context. Moreover, while numerous reports confirm the protective effect of NRF2 activation against chemical carcinogenesis little is known of its role in cancer arising from spontaneous mutations. Here, we tested the effects of NRF2 modulation (activation by sulforaphane or inhibition by brusatol) in lung carcinogenesis using a chemical (vinyl carbamate) model in A/J mice and a genetic (conditional Kras oncogene expression, to simulate spontaneous oncogene mutation) model in C57BL/6J mice. Mice were treated with NRF2 modulators before carcinogen exposure or Kras expression to test the role of NRF2 in cancer initiation, or treated after tumor development to test the role of NRF2 in cancer progression. Lung tissues were analyzed to determine tumor burden, as well as status of NRF2 and KRAS pathways. Additionally, proliferation, apoptosis, and oxidative DNA damage were assessed. Overall, NRF2 activation prevents initiation of chemically induced cancer, but promotes progression of pre-existing tumors regardless of chemical or genetic etiology. Once tumors are initiated, NRF2 inhibition is effective against the progression of chemically and spontaneously induced tumors. These results have important implications for NRF2-targeted cancer prevention and intervention strategies.
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http://dx.doi.org/10.1002/mc.22745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760364PMC
February 2018

The Roles of NRF2 in Modulating Cellular Iron Homeostasis.

Antioxid Redox Signal 2018 12 21;29(17):1756-1773. Epub 2017 Sep 21.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona , Tucson, Arizona.

Significance: Iron and oxygen are intimately linked: iron is an essential nutrient utilized as a cofactor in enzymes for oxygen transport, oxidative phosphorylation, and metabolite oxidation. However, excess labile iron facilitates the formation of oxygen-derived free radicals capable of damaging biomolecules. Therefore, biological utilization of iron is a tightly regulated process. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor, which can respond to oxidative and electrophilic stress, regulates several genes involved in iron metabolism. Recent Advances: The bulk of NRF2 transcription factor research has focused on its roles in detoxification and cancer prevention. Recent works have identified that several genes involved in heme synthesis, hemoglobin catabolism, iron storage, and iron export are under the control of NRF2. Constitutive NRF2 activation and subsequent deregulation of iron metabolism have been implicated in cancer development: NRF2-mediated upregulation of the iron storage protein ferritin or heme oxygenase 1 can lead to enhanced proliferation and therapy resistance. Of note, NRF2 activation and alterations to iron signaling in cancers may hinder efforts to induce the iron-dependent cell death process known as ferroptosis.

Critical Issues: Despite growing recognition of NRF2 as a modulator of iron signaling, exactly how iron metabolism is altered due to NRF2 activation in normal physiology and in pathologic conditions remains imprecise; moreover, the roles of NRF2-mediated iron signaling changes in disease progression are only beginning to be uncovered.

Future Directions: Further studies are necessary to connect NRF2 activation with physiological and pathological changes to iron signaling and oxidative stress. Antioxid. Redox Signal. 00, 000-000.
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http://dx.doi.org/10.1089/ars.2017.7176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208163PMC
December 2018

Fumarate Mediates a Chronic Proliferative Signal in Fumarate Hydratase-Inactivated Cancer Cells by Increasing Transcription and Translation of Ferritin Genes.

Mol Cell Biol 2017 06 16;37(11). Epub 2017 May 16.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, USA

Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase () cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how inactivation can endow cells with a growth advantage.
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http://dx.doi.org/10.1128/MCB.00079-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440649PMC
June 2017

Brusatol overcomes chemoresistance through inhibition of protein translation.

Mol Carcinog 2017 05 8;56(5):1493-1500. Epub 2017 Feb 8.

Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona.

The NRF2 pathway activates a cell survival response when cells are exposed to xenobiotics or are under oxidative stress. Therapeutic activation of NRF2 can also be used prior to insult as a means of disease prevention. However, prolonged expression of NRF2 has been shown to protect cancer cells by inducing the metabolism and efflux of chemotherapeutics, leading to both intrinsic and acquired chemoresistance to cancer drugs. This effect has been termed the "dark side" of NRF2. In an effort to combat this chemoresistance, our group discovered the first NRF2 inhibitor, the natural product brusatol, however the mechanism of inhibition was previously unknown. In this report, we show that brusatol's mode of action is not through direct inhibition of the NRF2 pathway, but through the inhibition of both cap-dependent and cap-independent protein translation, which has an impact on many short-lived proteins, including NRF2. Therefore, there is still a need to develop a new generation of specific NRF2 inhibitors with limited toxicity and off-target effects that could be used as adjuvant therapies to sensitize cancers with high expression of NRF2.
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http://dx.doi.org/10.1002/mc.22609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404829PMC
May 2017

Whole-Exome Sequencing Validates a Preclinical Mouse Model for the Prevention and Treatment of Cutaneous Squamous Cell Carcinoma.

Cancer Prev Res (Phila) 2017 Jan 6;10(1):67-75. Epub 2016 Dec 6.

Division of Cancer Research, School of Medicine, University of Dundee, Scotland, United Kingdom.

Cutaneous squamous cell carcinomas (cSCC) are among the most common and highly mutated human malignancies. Solar UV radiation is the major factor in the etiology of cSCC. Whole-exome sequencing of 18 microdissected tumor samples (cases) derived from SKH-1 hairless mice that had been chronically exposed to solar-simulated UV (SSUV) radiation showed a median point mutation (SNP) rate of 155 per Mb. The majority (78.6%) of the SNPs are C.G>T.A transitions, a characteristic UVR-induced mutational signature. Direct comparison with human cSCC cases showed high overlap in terms of both frequency and type of SNP mutations. Mutations in Trp53 were detected in 15 of 18 (83%) cases, with 20 of 21 SNP mutations located in the protein DNA-binding domain. Strikingly, multiple nonsynonymous SNP mutations in genes encoding Notch family members (Notch1-4) were present in 10 of 18 (55%) cases. The histopathologic spectrum of the mouse cSCC that develops in this model resembles very closely the spectrum of human cSCC. We conclude that the mouse SSUV cSCCs accurately represent the histopathologic and mutational spectra of the most prevalent tumor suppressors of human cSCC, validating the use of this preclinical model for the prevention and treatment of human cSCC. Cancer Prev Res; 10(1); 67-75. ©2016 AACR.
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http://dx.doi.org/10.1158/1940-6207.CAPR-16-0218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408961PMC
January 2017

Fumarate Hydratase-deficient Cell Line NCCFH1 as a New In Vitro Model of Hereditary Papillary Renal Cell Carcinoma Type 2.

Anticancer Res 2015 Dec;35(12):6639-53

Division of Medical Sciences, National Cancer Centre Singapore, Singapore, Republic of Singapore Division of Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Republic of Singapore

Background/aim: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare autosomal dominant disorder characterized by fumarate hydratase (FH) gene mutation. It is associated with the development of very aggressive kidney tumors, characterized by early onset and high metastatic potential, and has no effective therapy. The aim of the study was to establish a new preclinical platform for investigating morphogenetic and metabolic features, and alternative therapy of metastatic hereditary papillary renal cell carcinoma type 2 (PRCC2).

Materials And Methods: Fresh cells were collected from pleural fluid of a patient with metastatic hereditary PRCC2. Morphogenetic and functional characteristics were evaluated via microscopy, FH gene sequencing analysis, real-time polymerase chaine reaction and enzymatic activity measurement. We performed bioenergetic analysis, gene-expression profiling, and cell viability assay with 19 anti-neoplastic drugs.

Results: We established a new in vitro model of hereditary PRCC2 - the NCCFH1 cell line. The cell line possesses a c.1162 delA - p.Thr375fs frameshift mutation in the FH gene. Our findings indicate severe attenuation of oxidative phosphorylation and glucose-dependent growth of NCCFH1 cells that is consistent with the Warburg effect. Furthermore, gene-expression profiling identified that the most prominent molecular features reflected a high level of apoptosis, cell adhesion, and cell signaling. Drug screening revealed a marked sensitivity of FH(-/-) cells to mitoxantrone, epirubicin, topotecan and a high sensitivity to bortezomib.

Conclusion: We demonstrated that the NCCFH1 cell line is a very interesting preclinical model for studying the metabolic features and testing new therapies for hereditary PRCC2, while bortezomib may be a potential efficient therapeutic option.
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December 2015

Sustained NRF2 activation in hereditary leiomyomatosis and renal cell cancer (HLRCC) and in hereditary tyrosinemia type 1 (HT1).

Biochem Soc Trans 2015 Aug 3;43(4):650-6. Epub 2015 Aug 3.

Department of Toxicology and Pharmacology, College of Pharmacy, University of Arizona. Tucson, Arizona 85721, U.S.A.

The nuclear erythroid 2-like 2 transcription factor (NRF2), is a major regulator of cellular redox balance. Although NRF2 activation is generally regarded as beneficial to human health, recent studies have identified that sustained NRF2 activation is over-represented in many cancers. This raises the question regarding the role of NRF2 activation in the development and progression of those cancers. This review focuses on the mechanisms and the effects of NRF2 activation in two hereditary cancer predisposition syndromes: hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary tyrosinemia type 1 (HT1). Because the cancer initiating mutations in these hereditary syndromes are well defined, they offer a unique opportunity to explore the roles of NRF2 activation in the early stages of carcinogenesis. Over the years, a variety of approaches have been utilized to study the biology of HLRCC and HT1. In HLRCC, in vitro studies have demonstrated the importance of NRF2 activation in sustaining cancer cell proliferation. In the mouse model of HT1 however, NRF2 activation seems to protect cells from malignant transformation. In both HT1 and HLRCC, NRF2 activation promotes the clearance of electrophilic metabolites, enabling cells to survive cancer-initiating mutations. Biological insights gained from the hereditary syndromes' studies may shed light on to the roles of NRF2 activation in sporadic tumours.
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http://dx.doi.org/10.1042/BST20150041DOI Listing
August 2015

Exome-wide Sequencing Shows Low Mutation Rates and Identifies Novel Mutated Genes in Seminomas.

Eur Urol 2015 Jul 14;68(1):77-83. Epub 2015 Jan 14.

Centre for Computational Biology, Duke-NUS Graduate Medical School, Singapore; Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore. Electronic address:

Background: Testicular germ cell tumors are the most common cancer diagnosed in young men, and seminomas are the most common type of these cancers. There have been no exome-wide examinations of genes mutated in seminomas or of overall rates of nonsilent somatic mutations in these tumors.

Objective: The objective was to analyze somatic mutations in seminomas to determine which genes are affected and to determine rates of nonsilent mutations.

Design, Setting, And Participants: Eight seminomas and matched normal samples were surgically obtained from eight patients.

Intervention: DNA was extracted from tissue samples and exome sequenced on massively parallel Illumina DNA sequencers. Single-nucleotide polymorphism chip-based copy number analysis was also performed to assess copy number alterations.

Outcome Measurements And Statistical Analysis: The DNA sequencing read data were analyzed to detect somatic mutations including single-nucleotide substitutions and short insertions and deletions. The detected mutations were validated by independent sequencing and further checked for subclonality.

Results And Limitations: The rate of nonsynonymous somatic mutations averaged 0.31 mutations/Mb. We detected nonsilent somatic mutations in 96 genes that were not previously known to be mutated in seminomas, of which some may be driver mutations. Many of the mutations appear to have been present in subclonal populations. In addition, two genes, KIT and KRAS, were affected in two tumors each with mutations that were previously observed in other cancers and are presumably oncogenic.

Conclusions: Our study, the first report on exome sequencing of seminomas, detected somatic mutations in 96 new genes, several of which may be targetable drivers. Furthermore, our results show that seminoma mutation rates are five times higher than previously thought, but are nevertheless low compared to other common cancers. Similar low rates are seen in other cancers that also have excellent rates of remission achieved with chemotherapy.

Patient Summary: We examined the DNA sequences of seminomas, the most common type of testicular germ cell cancer. Our study identified 96 new genes in which mutations occurred during seminoma development, some of which might contribute to cancer development or progression. The study also showed that the rates of DNA mutations during seminoma development are higher than previously thought, but still lower than for other common solid-organ cancers. Such low rates are also observed among other cancers that, like seminomas, show excellent rates of disease remission after chemotherapy.
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http://dx.doi.org/10.1016/j.eururo.2014.12.040DOI Listing
July 2015

Oncogenic KRAS confers chemoresistance by upregulating NRF2.

Cancer Res 2014 Dec 22;74(24):7430-41. Epub 2014 Oct 22.

Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona.

Oncogenic KRAS mutations found in 20% to 30% of all non-small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically, KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in a regulatory region in exon 1 of NRF2. In a mouse model of mutant KrasG12D-induced lung cancer, we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Cotreatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS mutations with NRF2 pathway inhibitors.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-1439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268230PMC
December 2014

CUL3 and NRF2 mutations confer an NRF2 activation phenotype in a sporadic form of papillary renal cell carcinoma.

Cancer Res 2013 Apr 30;73(7):2044-51. Epub 2013 Jan 30.

Interdisciplinary Renal Oncology Laboratory, Van Andel Research Institute, Grand Rapids, MI 49503, USA.

Sustained activation of the stress-regulated transcription factor NRF2 (NFE2L2) is a prominent feature of many types of cancer, implying that mutations driving NRF2 may be important to tumor progression. In hereditary type 2 papillary renal cell carcinoma (PRCC2, also known as hereditary leiomyomatosis and renal cell cancer), NRF2 activation is a direct consequence of the accumulation of intracellular fumarate, a result of fumarate hydratase (FH) inactivation, but it is not clear how NRF2 may be activated in sporadic forms of PRCC2. Here we show that somatic mutations in NRF2, CUL3, and SIRT1 are responsible for driving the NRF2 activation phenotype in sporadic PRCC2. Transcriptome sequencing revealed the expression pattern of mutant alleles of NRF2, CUL3, and SIRT1 and also confirmed NRF2 activation in clinical specimens. Our results show a convergence in somatic mutations in sporadic PRCC2 with FH mutation in hereditary PRCC2.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-3227DOI Listing
April 2013

Expression of the PTTG1 oncogene is associated with aggressive clear cell renal cell carcinoma.

Cancer Res 2012 Sep 17;72(17):4361-71. Epub 2012 Jul 17.

Laboratories of Cancer Genetics, Cytogenetics, and Computational Biology, Van Andel Research Institute; Department of Urology, Spectrum Health Hospital, Grand Rapids, MI 49503, USA.

The pituitary tumor transforming gene (PTTG1) is a recently discovered oncogene implicated in malignant progression of both endocrine and nonendocrine malignancies. Clear cell renal cell carcinoma (ccRCC) is cytogenetically characterized by chromosome 3p deletions that harbor the ccRCC-related von Hippel-Lindau, PBRM1, BAP1, and SETD2 tumor suppressor genes, along with chromosome 5q amplifications where the significance has been unclear. PTTG1 localizes to the chromosome 5q region where amplifications occur in ccRCC. In this study, we report a functional role for PTTG1 in ccRCC tumorigenesis. PTTG1 was amplified in ccRCC, overexpressed in tumor tissue, and associated with high-grade tumor cells and poor patient prognosis. In preclinical models, PTTG1 ablation reduced tumorigenesis and invasion. An analysis of gene expression affected by PTTG1 indicated an association with invasive and metastatic disease. PTTG1-dependent expression of the RhoGEF proto-oncogene ECT2 was observed in a number of ccRCC cell lines. Moreover, ECT2 expression correlated with PTTG1 expression and poor clinical features. Together, our findings reveal features of PTTG1 that are consistent with its identification of an oncogene amplified on chromsome 5q in ccRCC, where it may offer a novel therapeutic target of pathologic significance in this disease.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-2330DOI Listing
September 2012

Fumarate hydratase inactivation in renal tumors: HIF1α, NRF2, and "cryptic targets" of transcription factors.

Chin J Cancer 2012 Sep 2;31(9):413-20. Epub 2012 Jul 2.

Laboratory of Interdisciplinary Urological Oncology, Van Andel Research Institute, 333 Bostwick Ave NE, Grand Rapids, MI 49503, USA.

Biallelic inactivation of fumarate hydratase(FH) causes type 2 papillary renal cell carcinoma (PRCC2), uterine fibroids, and cutaneous leimyomas, a condition known as hereditary leiomyomatosis and renal cell cancer(HLRCC). The most direct effect of FH inactivation is intracellular fumarate accumulation. A majority of studies on FH inactivation over the past decade have focused on the theory that intracellular fumarate stabilizes hypoxia-inducible factor 1α(HIF1A) through competitive inhibition of HIF prolyl hydroxylases. Recently, a competing theory that intracellular fumarate activates nuclear factor (erythroid-derived 2)-like 2(NRF2) through post-translational modification of its negative regulator. Kelch-like ECH-associated protein 1(KEAP1) has emerged from a computational modeling study and mouse model studies. This review dissects the origin of these two governing theories and highlights the presence of chromatin-structure-regulated targets of transcription factors, which we refer to as "cryptic targets" of transcription factors. One such cryptic target is heme oxygenase I(HMOX1), the expression of which is known to be modulated by the gene product of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4, also known as BRG1).
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http://dx.doi.org/10.5732/cjc.012.10102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777506PMC
September 2012

Exome sequencing of liver fluke-associated cholangiocarcinoma.

Nat Genet 2012 May 6;44(6):690-3. Epub 2012 May 6.

National Cancer Centre Singapore-Van Andel Research Institute Translational Research Laboratory, Division of Medical Sciences, Singapore.

Opisthorchis viverrini-related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini-related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8-3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini-related CCA.
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http://dx.doi.org/10.1038/ng.2273DOI Listing
May 2012

An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma.

Cancer Cell 2011 Oct;20(4):511-23

Laboratory of Cancer Genetics, Van Andel Research Institute, Grand Rapids, MI 49503, USA.

Fumarate hydratase (FH) mutation causes hereditary type 2 papillary renal cell carcinoma (PRCC2). The main effect of FH mutation is fumarate accumulation. The current paradigm posits that the main consequence of fumarate accumulation is HIF-α stabilization. Paradoxically, FH mutation differs from other HIF-α stabilizing mutations, such as VHL and SDH mutations, in its associated tumor types. We identified that fumarate can directly up-regulate antioxidant response element (ARE)-controlled genes. We demonstrated that aldo-keto reductase family 1 member B10 (AKR1B10) is an ARE-controlled gene and is up-regulated upon FH knockdown as well as in FH null cell lines. AKR1B10 overexpression is also a prominent feature in both hereditary and sporadic PRCC2. This phenotype better explains the similarities between hereditary and sporadic PRCC2.
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http://dx.doi.org/10.1016/j.ccr.2011.08.024DOI Listing
October 2011

Serglycin is a theranostic target in nasopharyngeal carcinoma that promotes metastasis.

Cancer Res 2011 Apr 2;71(8):3162-72. Epub 2011 Feb 2.

State Key Laboratory of Oncology in South China, Departments of Pathology and Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.

Nasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparative genomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed the serglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibition of serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducing metastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motile behavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorly metastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting a critical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression of vimentin but not other epithelial-mesenchymal transition proteins. In clinical specimens, serglycin expression was elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated the prognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patients followed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorable independent indicator of distant metastasis-free and disease-free survival. Our findings establish that glycosylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as an independent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-3557DOI Listing
April 2011

p38delta/MAPK13 as a diagnostic marker for cholangiocarcinoma and its involvement in cell motility and invasion.

Int J Cancer 2010 May;126(10):2353-61

Department of General Surgery, Singapore General Hospital, Singapore.

Cholangiocarcinoma (CC) and hepatocellularcarcinoma (HCC) are two main forms of liver malignancies, which exhibit differences in drug response and prognosis. Immunohistotochemical staining for cytokeratin markers has been used to some success in the differential diagnosis of CC from HCC. However, there remains a need for additional markers for increased sensitivity and specificity of diagnosis. In this study, we have identified a p38 MAP kinase, p38delta (also known as MAPK13 or SAPK4) as a protein that is upregulated in CC relative to HCC and to normal biliary tract tissues. We performed microarray gene expression profiling on 17 cases of CC, 12 cases of adjacent normal liver tissue, and three case of normal bile duct tissue. p38delta was upregulated in 16 out of 17 cases of CC relative to normal tissue. We subsequently performed immunohistochemical staining of p38delta in 54 cases of CC and 54 cases of HCC. p38delta staining distinguished CC from HCC with a sensitivity of 92.6% and a specificity of 90.7%. To explore the possible functional significance of p38delta expression in CC, we examined the effects of overexpression and knockdown of p38delta expression in human CC cell lines. Our results indicate that p38delta is important for motility and invasion of CC cells, suggesting that p38delta may play an important role in CC metastasis. In summary, p38delta may serve as a novel diagnostic marker for CC and may also serve as a new target for molecular based therapy of this disease.
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http://dx.doi.org/10.1002/ijc.24944DOI Listing
May 2010

The full-length clone of cucumber green mottle mosaic virus and its application as an expression system for Hepatitis B surface antigen.

J Biotechnol 2006 Feb 3;121(4):471-81. Epub 2005 Nov 3.

Institute of Biological Science, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

A cucumber green mosaic mottle virus (CGMMV) full-length clone was developed for the expression of Hepatitis B surface antigen (HBsAg). The expression of the surface displayed HBsAg by the chimeric virus was confirmed through a double antibody sandwich ELISA. Assessment of the coat protein composition of the chimeric virus particles by SDS-PAGE analysis showed that 50% of the coat proteins were fused to the HBsAg. Biological activity of the expressed HBsAg was assessed through the stimulation of in vitro antibody production by cultured peripheral blood mononuclear cells (PBMC). PBMC that were cultured in the presence of the chimeric virus showed up to an approximately three-fold increase in the level of anti HBsAg immunoglobulin thus suggesting the possible use of this new chimeric virus as an effective Hepatitis B vaccine.
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http://dx.doi.org/10.1016/j.jbiotec.2005.08.032DOI Listing
February 2006