33 results on '"Mok, Myth T. S."'
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2. Clinical and molecular characteristics of hemophilia A affected individuals and carriers: A 24 years experience from three centers.
- Author
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Ho, Stephanie K. L., Ng, Samuel Y. L., Yung, Tsz‐Kwai, Mok, Myth T. S., Yiu, Wing‐Chung, Cheng, Heidi H. Y., Cheng, Shirley S. W., Luk, Ho‐ming, Lo, Ivan F. M., and Kan, Anita S. Y.
- Abstract
Hemophilia A is a rare bleeding disorder with variable expressivity and allelic heterogeneity. Despite the advancement of prenatal diagnostics and molecular studies, the number of studies reviewing the reproductive choices of hemophilia A carriers and affected individuals remains limited. Through this retrospective review, we hope to gain a deeper understanding of hemophilia A‐affected individuals' clinical and molecular characteristics, as well as the reproductive choices of the at‐risk couples. A total of 122 individuals harboring likely causative F8 gene alterations from 64 apparently unrelated families attending three centers between 3/2000 and 3/2023 were included in this study. Their clinical and molecular findings as well as reproductive choices were gathered in a clinical setting and verified through the electronic medical record database of the public health system. Forty‐seven affected males and 75 female heterozygous carriers were included in the analysis. Among 64 apparently unrelated families, 36 distinct pathogenic/likely pathogenic variants were identified, of which 30.6% (11/36) of variants were novel. While the majority of clinical findings and genotype–phenotype correlations appear to be in accordance with existing literature, female carriers who had no fertility intention were significantly more likely to have affected sons than those who had fertility intention (5/19 vs. 4/5; p = 0.047). Through this retrospective review, we summarized the clinical and molecular characteristics of 122 individuals harboring pathogenic/likely pathogenic F8 variants, as well as their fertility intentions and reproductive outcomes. Further studies are required to look into the considerations involved in reproductive decision‐making. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
3. Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis
- Author
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Zeng, Xuezhen, Zhou, Jingying, Xiong, Zhewen, Sun, Hanyong, Yang, Weiqin, Mok, Myth T. S., Wang, Jing, Li, Jingqing, Liu, Man, Tang, Wenshu, Feng, Yu, Wang, Hector Kwong-Sang, Tsang, Shun-Wa, Chow, King-Lau, Yeung, Philip Chun, Wong, John, Lai, Paul Bo-San, Chan, Anthony Wing-Hung, To, Ka Fai, Chan, Stephen Lam, Xia, Qiang, Xue, Jing, Chen, Xiao, Yu, Jun, Peng, Sui, Sung, Joseph Jao-Yiu, Kuang, Ming, and Cheng, Alfred Sze-Lok
- Published
- 2021
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4. An inflammatory-CCRK circuitry drives mTORC1-dependent metabolic and immunosuppressive reprogramming in obesity-associated hepatocellular carcinoma
- Author
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Sun, Hanyong, Yang, Weiqin, Tian, Yuan, Zeng, Xuezhen, Zhou, Jingying, Mok, Myth T. S., Tang, Wenshu, Feng, Yu, Xu, Liangliang, Chan, Anthony W. H., Tong, Joanna H., Cheung, Yue-Sun, Lai, Paul B. S., Wang, Hector K. S., Tsang, Shun-Wa, Chow, King-Lau, Hu, Mengying, Liu, Rihe, Huang, Leaf, Yang, Bing, Yang, Pengyuan, To, Ka-Fai, Sung, Joseph J. Y., Wong, Grace L. H., Wong, Vincent W. S., and Cheng, Alfred S. L.
- Published
- 2018
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5. Targeting monocyte-intrinsic enhancer reprogramming improves immunotherapy efficacy in hepatocellular carcinoma.
- Author
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Man Liu, Jingying Zhou, Xiaoyu Liu, Yu Feng, Weiqin Yang, Feng Wu, Otto Ka-Wing Cheung, Hanyong Sun, Xuezhen Zeng, Wenshu Tang, Mok, Myth T. S., Wong, John, Chun Yeung, Philip, Bo San Lai, Paul, Zhiwei Chen, Hongchuan Jin, Jie Chen, Lam Chan, Stephen, Chan, Anthony W. H., and Ka Fai To
- Subjects
HEPATOCELLULAR carcinoma ,MEDICAL sciences ,CASTRATION-resistant prostate cancer ,CYTOTOXIC T cells ,LIVER cells ,KILLER cells - Published
- 2020
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6. Epigenetic Activation of Wnt/β-Catenin Signaling in NAFLD-Associated Hepatocarcinogenesis.
- Author
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Yuan Tian, Mok, Myth T. S., Pengyuan Yang, and Cheng, Alfred S. L.
- Subjects
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HEPATOCELLULAR carcinoma , *ASIANS , *CELLULAR signal transduction , *PEOPLE with diabetes , *FATTY liver , *GENE expression , *GROWTH factors , *HISTONES , *LIVER , *GENETIC mutation , *OBESITY , *RNA , *METABOLIC syndrome , *DNA methylation , *EPIGENOMICS , *THERAPEUTICS - Abstract
Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/ β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients. [ABSTRACT FROM AUTHOR]
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- 2016
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7. Characterization of Adenomatous Polyposis Coli Protein Dynamics and Localization at the Centrosome.
- Author
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Lui, Christina, Mok, Myth T. S., and Henderson, Beric R.
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ANTINEOPLASTIC agents , *COLON tumors , *ALLELES , *AMINO acids , *TUMOR suppressor genes , *CELL cycle , *CELLULAR signal transduction , *CYTOSKELETAL proteins , *FLUORESCENT antibody technique , *HISTOLOGY , *MICROFILAMENT proteins , *MICROSCOPY , *GENETIC mutation , *RESEARCH funding , *DATA analysis software , *ADENOMATOUS polyposis coli , *DESCRIPTIVE statistics , *SEQUENCE analysis , *IN vitro studies , *GENETICS ,RECTUM tumors - Abstract
The adenomatous polyposis coli (APC) tumor suppressor is a multifunctional regulator of Wnt signaling and acts as a mobile scaffold at different cellular sites. APC was recently found to stimulate microtubule (MT) growth at the interphase centrosome; however, little is known about its dynamics and localization at this site. To address this, we analysed APC dynamics in fixed and live cells by fluorescence microscopy. In detergent-extracted cells, we discovered that APC was only weakly retained at the centrosome during interphase suggesting a rapid rate of exchange. This was confirmed in living cells by fluorescence recovery after photobleaching (FRAP), which identified two pools of green fluorescent protein (GFP)-APC: a major rapidly exchanging pool (~86%) and minor retained pool (~14%). The dynamic exchange rate of APC was unaffected by C-terminal truncations implicating a targeting role for the N-terminus. Indeed, we mapped centrosome localization to N-terminal armadillo repeat (ARM) domain amino acids 334-625. Interestingly, the rate of APC movement to the centrosome was stimulated by intact MTs, and APC dynamics slowed when MTs were disrupted by nocodazole treatment or knockdown of -tubulin. Thus, the rate of APC recycling at the centrosome is enhanced by MT growth, suggesting a positive feedback to stimulate its role in MT growth. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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8. A two-step fluorescence-activated cell sorting approach to isolate genetically modified mammalian cells with isopropyl-beta-D-thiogalactoside (IPTG)-inducible gene expression.
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Mok, Myth T. S. and Henderson, Beric R.
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- 2012
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9. Kinetic and Physical Characterization of the Inducible UDP-N-acetylglucosamine Pyrophosphorylase from Giardia intestinalis.
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Mok, Myth T. S. and Edwards, Michael R.
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GIARDIA lamblia , *ENZYMES , *CHEMICAL synthesis , *CHROMATOGRAPHIC analysis , *CATALYSIS , *GIARDIA - Abstract
The UDP-N-acetylglucosamine pyrophosphorylase in Giardia intestinalis (GiUAP) is one of the five inducible enzymes to synthesize UDP-GalNAc, which is an important precursor for cyst wall synthesis. The recombinant UDP-N-acetylglucosamine pyrophosphorylase (rGiUAP) and its mutants G108A and G210A were expressed and identified by SDS-PAGE, size-exclusion chromatography, Western hybridization, and MALDI mass spectrometry. Sequence comparison with other eukaryotic UAPs has identified three specific motifs. Within these motifs alanine substitution for Gly108 or Gly210 dramatically reduced the pyrophosphate synthesis, suggesting these amino acids are catalytic residues. Besides, the rGiUAP was found to have relaxed binding to other uridine-based nucleotides, suggesting the substrate binding pocket is specific to uridine rather than phosphate group(s). Moreover, thermal denaturation analysis showed a significant increase in Tm for the rGiUAP and G108A upon binding of the substrate Mg-UTP. In contrast, G210A showed a decreased Tm upon binding of Mg-UTP. These results showed that binding of Mg-UTP increases protein stability of the rGiUAP, and the catalytic residue Gly210 plays a significant role in stabilizing the protein structure. Such stabilization effect induced by substrate binding might be physiologically important as it favors the production of UDP-GlcNAc and hence the downstream GalNAc, which is crucial to survival of Giardia. These results help to define the essential amino acids for catalysis in the GiUAP and reveal the role of Mg-UTP binding in regulation of protein stability. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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10. Mitochondrial Targeting of Adenomatous Polyposis Coli Protein Is Stimulated by Truncating Cancer Mutations REGULATION OF BcI-2 AND IMPLICATIONS FOR CELL SURVIVAL.
- Author
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Brocardo, Mariana, Ying Lei, Tighe, Anthony, Taylor, Stephen S., Mok, Myth T. S., and Henderson, Beric R.
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TUMOR suppressor proteins , *COLON cancer , *GENETIC mutation , *MITOCHONDRIA , *APOPTOSIS - Abstract
The adenomatous polyposis coli (APC) protein tumor suppressor is mutated in the majority of colon cancers. Most APC gene mutations cause deletion of the C terminus and disrupt APC regulation of β-catenin turnover, microtubule dynamics, and chromosome segregation. Truncated APC mutant peptides may also gain unique properties, not exhibited by wild-type APC, which contribute to tumor cell survival and proliferation. Here we report a differential subcellular localization pattern for wild-type and mutant APC. A pool of APC truncation mutants was detected at mitochondria by cellular fractionation and confocal microscopy. In contrast, wild-type APC located poorly at mitochondria. Similar results were observed for endogenous and stably induced forms of APC, with the shortest N-terminal mutant peptides (N750, N853, N1309, N1337) displaying the strongest mitochondrial staining. The knock down of mutant APC(N1337) in SW480 tumor cells caused an increase in apoptosis and mitochondrial membrane permeability, and this correlated with reduced Bcl-2 protein levels in mitochondrial fractions. Interestingly, the silencing of APC did not alter expression of β-catenin or the apoptotic regulatory factors Bax, Bcl-xL, or survivin. APC formed a complex with Bcl-2 in mitochondrial fractions, and this may contribute to the APC-dependent regulation of Bcl-2. We propose that a subset of cancer mutations induce APC mitochondrial localization and that APC regulation of Bcl-2 at mitochondria may contribute to tumor cell survival. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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11. Targeting monocyte-intrinsic enhancer reprogramming improves immunotherapy efficacy in hepatocellular carcinoma.
- Author
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Liu M, Zhou J, Liu X, Feng Y, Yang W, Wu F, Cheung OK, Sun H, Zeng X, Tang W, Mok MTS, Wong J, Yeung PC, Lai PBS, Chen Z, Jin H, Chen J, Chan SL, Chan AWH, To KF, Sung JJY, Chen M, and Cheng AS
- Subjects
- Animals, B7-H1 Antigen antagonists & inhibitors, Carcinoma, Hepatocellular etiology, Carcinoma, Hepatocellular immunology, Cellular Reprogramming immunology, Cyclopropanes pharmacology, Cyclopropanes therapeutic use, Hepatic Stellate Cells immunology, Humans, Immune Tolerance, Liver Cirrhosis complications, Liver Cirrhosis pathology, Liver Neoplasms etiology, Liver Neoplasms immunology, Liver Neoplasms, Experimental etiology, Liver Neoplasms, Experimental immunology, Liver Neoplasms, Experimental pathology, Liver Neoplasms, Experimental therapy, Male, Mice, Inbred C57BL, Monocytes immunology, Myeloid-Derived Suppressor Cells immunology, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Pyridines pharmacology, Pyridines therapeutic use, Signal Transduction physiology, Tumor Cells, Cultured, Tumor Microenvironment, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors, p38 Mitogen-Activated Protein Kinases physiology, Carcinoma, Hepatocellular therapy, Immunotherapy methods, Liver Neoplasms therapy
- Abstract
Objective: Hepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy., Design: Functional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet and verified by clinical specimens. Mechanistic studies were conducted in human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically trialled BET bromodomain inhibitor i-BET762 was determined., Results: Accumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumour-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumour-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumour phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signalling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumour eradication and prolonged survival in the fibrotic-HCC mouse model., Conclusion: Our results signify how non-tumour-intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)
- Published
- 2020
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12. Androgen receptor drives hepatocellular carcinogenesis by activating enhancer of zeste homolog 2-mediated Wnt/β-catenin signaling.
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Song H, Yu Z, Sun X, Feng J, Yu Q, Khan H, Zhu X, Huang L, Li M, Mok MTS, Cheng ASL, Gao Y, and Feng H
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- Animals, Carcinogenesis genetics, Carcinogenesis pathology, Carcinoma, Hepatocellular genetics, Carcinoma, Hepatocellular pathology, Cell Line, Tumor, Cell Proliferation, Enhancer of Zeste Homolog 2 Protein genetics, Epigenesis, Genetic, Female, Gene Expression Regulation, Neoplastic, Histones metabolism, Humans, Liver Neoplasms genetics, Liver Neoplasms pathology, Methylation, Mice, Nude, Models, Biological, Prognosis, Transcription, Genetic, Up-Regulation genetics, beta Catenin metabolism, Carcinoma, Hepatocellular metabolism, Enhancer of Zeste Homolog 2 Protein metabolism, Liver Neoplasms metabolism, Receptors, Androgen metabolism, Wnt Signaling Pathway
- Abstract
Background: Androgen receptor (AR) plays a crucial role as a transcription factor in promoting the development of hepatocellular carcinoma (HCC) which is prone to aberrant chromatin modifications. However, the regulatory effects of AR on epigenetic mediators in HCC remain ill-defined. Enhancer of zeste homolog 2 (EZH2), an oncogene responsible for the tri-methylation of histone H3 at lysine 27 (H3K27me3), was identified to be overexpressed in approximate 70-90% of HCC cases, which prompted us to investigate whether or how AR regulates EZH2 expression., Methods: Colony formation, soft agar assay, xenograft and orthotopic mouse models were used to determine cell proliferation and tumorigenicity of gene-manipulated HCC cells. Gene regulation was assessed by chromatin immunoprecipitation, luciferase reporter assay, quantitative RT-PCR and immunoblotting. Clinical relevance of candidate proteins in patient specimens was examined in terms of pathological parameters and postsurgical survival rates., Findings: In this study, we found that AR upregulated EZH2 expression by binding to EZH2 promoter and stimulating its transcriptional activity. EZH2 overexpression increased H3K27me3 levels and thereby silenced the expression of Wnt signal inhibitors, resulting in activation of Wnt/β-catenin signaling and subsequently induction of cell proliferation and tumorigenesis. In a cohort of human HCC patients, concordant overexpression of AR, EZH2, H3K27me3 and active β-catenin was observed in tumor tissues compared with paired non-tumor tissues, which correlated with tumor progression and poor prognosis. These findings demonstrate a novel working model in which EZH2 mediates AR-induced Wnt/β-catenin signaling activation through epigenetic modification, and support the application of EZH2-targeted reagents for treating HCC patients., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
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13. Reconstruction of enhancer-target networks in 935 samples of human primary cells, tissues and cell lines.
- Author
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Cao Q, Anyansi C, Hu X, Xu L, Xiong L, Tang W, Mok MTS, Cheng C, Fan X, Gerstein M, Cheng ASL, and Yip KY
- Subjects
- Carcinoma, Hepatocellular genetics, Cell Line, DNA Methylation, Genes, Neoplasm, Humans, K562 Cells, Liver Neoplasms genetics, Neoplasm Proteins biosynthesis, Neoplasm Proteins genetics, Phosphoproteins biosynthesis, Phosphoproteins genetics, Primary Cell Culture, RNA-Binding Proteins biosynthesis, RNA-Binding Proteins genetics, Telomerase biosynthesis, Telomerase genetics, Transcription Factors genetics, Transcription Factors metabolism, Enhancer Elements, Genetic, Epigenesis, Genetic, Gene Expression Regulation, Gene Regulatory Networks genetics, Transcription, Genetic
- Abstract
We propose a new method for determining the target genes of transcriptional enhancers in specific cells and tissues. It combines global trends across many samples and sample-specific information, and considers the joint effect of multiple enhancers. Our method outperforms existing methods when predicting the target genes of enhancers in unseen samples, as evaluated by independent experimental data. Requiring few types of input data, we are able to apply our method to reconstruct the enhancer-target networks in 935 samples of human primary cells, tissues and cell lines, which constitute by far the largest set of enhancer-target networks. The similarity of these networks from different samples closely follows their cell and tissue lineages. We discover three major co-regulation modes of enhancers and find defense-related genes often simultaneously regulated by multiple enhancers bound by different transcription factors. We also identify differentially methylated enhancers in hepatocellular carcinoma (HCC) and experimentally confirm their altered regulation of HCC-related genes.
- Published
- 2017
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14. Epigenetic Activation of Wnt/β-Catenin Signaling in NAFLD-Associated Hepatocarcinogenesis.
- Author
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Tian Y, Mok MT, Yang P, and Cheng AS
- Abstract
Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients.
- Published
- 2016
- Full Text
- View/download PDF
15. A novel role of hepatic epithelial transforming growth factor-β signaling in cholangiocarcinogenesis.
- Author
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Liu M, Mok MT, and Cheng AS
- Published
- 2016
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16. Truncated HBx-dependent silencing of GAS2 promotes hepatocarcinogenesis through deregulation of cell cycle, senescence and p53-mediated apoptosis.
- Author
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Zhu R, Mok MT, Kang W, Lau SS, Yip WK, Chen Y, Lai PB, Wong VW, To KF, Sung JJ, Cheng AS, and Chan HL
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- Animals, Binding Sites, Carcinoma, Hepatocellular genetics, Carcinoma, Hepatocellular pathology, Carcinoma, Hepatocellular virology, Female, Gene Expression Regulation, Neoplastic, Hep G2 Cells, Hepatitis B complications, Hepatitis B virus genetics, Hepatitis B virus pathogenicity, Humans, Liver Neoplasms genetics, Liver Neoplasms pathology, Liver Neoplasms virology, Mice, Inbred BALB C, Mice, Nude, Microfilament Proteins genetics, Promoter Regions, Genetic, Signal Transduction, Time Factors, Trans-Activators genetics, Transcription, Genetic, Transfection, Tumor Burden, Tumor Suppressor Protein p53 genetics, Viral Regulatory and Accessory Proteins, Apoptosis, Carcinoma, Hepatocellular metabolism, Cell Cycle, Cell Transformation, Viral, Cellular Senescence, Gene Silencing, Hepatitis B virus metabolism, Liver Neoplasms metabolism, Microfilament Proteins metabolism, Trans-Activators metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
Hepatocellular carcinoma (HCC) is a worldwide threat to public health, especially in China, where chronic hepatitis B virus (HBV) infection is found in 80-90% of all HCCs. The HBV-encoded X antigen (HBx) is a trans-regulatory protein involved in virus-induced hepatocarcinogenesis. Although the carboxyl-terminus-truncated HBx, rather than the full-length counterpart, is frequently overexpressed in human HCCs, its functional mechanisms are not fully defined. We investigated the molecular function of a naturally occurring HBx variant which has 35 amino acids deleted at the C-terminus (HBxΔ35). Genome-wide scanning analysis and PCR validation identified growth arrest-specific 2 (GAS2) as a direct target of HBxΔ35 at transcriptional level in human immortalized liver cells. HBxΔ35 was found to bind the promoter region of GAS2 and attenuate its expression to promote hepatocellular proliferation and tumourigenicity. Further functional assays demonstrated that GAS2 induces p53-dependent apoptosis and senescence to counteract HBxΔ35-mediated tumourigenesis. Notably, GAS2 expression was significantly down-regulated in HCCs compared with the corresponding normal tissues. In conclusion, our integrated study uncovered a novel viral mechanism in hepatocarcinogenesis, wherein HBxΔ35 deregulates cell growth via direct silencing of GAS2 and thereby provides a survival advantage for pre-neoplastic hepatocytes to facilitate cancer development., (Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)
- Published
- 2015
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17. The BARD1 BRCT domain contributes to p53 binding, cytoplasmic and mitochondrial localization, and apoptotic function.
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Tembe V, Martino-Echarri E, Marzec KA, Mok MT, Brodie KM, Mills K, Lei Y, DeFazio A, Rizos H, Kettle E, Boadle R, and Henderson BR
- Subjects
- Amino Acid Sequence, Breast Neoplasms genetics, Breast Neoplasms pathology, Cytoplasm genetics, DNA Breaks, Female, Humans, MCF-7 Cells, Mitochondria genetics, Mitochondria pathology, Protein Structure, Tertiary, Protein Transport genetics, Proto-Oncogene Proteins c-bcl-2 genetics, Proto-Oncogene Proteins c-bcl-2 metabolism, Sequence Deletion, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Proteins genetics, Ubiquitin-Protein Ligases genetics, bcl-2-Associated X Protein genetics, bcl-2-Associated X Protein metabolism, Apoptosis, Breast Neoplasms metabolism, Cytoplasm metabolism, Mitochondria metabolism, Tumor Suppressor Protein p53 metabolism, Tumor Suppressor Proteins metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
BARD1 is a breast cancer tumor suppressor with multiple domains and functions. BARD1 comprises a tandem BRCT domain at the C-terminus, and this sequence has been reported to target BARD1 to distinct subcellular locations such as nuclear DNA breakage sites and the centrosome through binding to regulatory proteins such as HP1 and OLA1, respectively. We now identify the BRCT domain as a binding site for p53. We first confirmed previous reports that endogenous BARD1 binds to p53 by immunoprecipitation assay, and further show that BARD1/p53 complexes locate at mitochondria suggesting a cellular location for p53 regulation of BARD1 apoptotic activity. We used a proximity ligation assay to map three distinct p53 binding sequences in human BARD1, ranging from weak (425-525) and modest (525-567) to strong (551-777 comprising BRCT domains). Deletion of the BRCT sequence caused major defects in the ability of BARD1 to (1) bind p53, (2) localize to the cytoplasm and mitochondria, and (3) induce Bax oligomerization and apoptosis. Our data suggest that BARD1 can move to mitochondria independent of p53, but subsequently associates with p53 to induce Bax clustering in part by decreasing mitochondrial Bcl-2 levels. We therefore identify a role for the BRCT domain in stimulating BARD1 nuclear export and mitochondrial localization, and in assembling mitochondrial BARD1/p53 complexes to regulate specific activities such as apoptotic function., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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18. CUL4B: a novel epigenetic driver in Wnt/β-catenin-dependent hepatocarcinogenesis.
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Mok MT and Cheng AS
- Subjects
- Humans, Carcinoma, Hepatocellular enzymology, Cullin Proteins metabolism, Liver Neoplasms enzymology, Wnt Signaling Pathway, beta Catenin metabolism
- Abstract
Emerging evidence indicates that Cullin 4B (CUL4B), a major component of ubiquitin ligase complexes, is over-expressed in diverse cancer types with pro-tumourigenic effects. In this issue of the Journal of Pathology, Yuan and colleagues [6] elucidated the oncogenic activity of CUL4B in hepatocellular carcinoma (HCC) and delineated its role in driving Wnt/β-catenin signalling. In addition to the stabilization of β-catenin protein against proteasomal degradation, CUL4B also acts in concert with enhancer of Zeste homologue 2 (EZH2) to concordantly silence multiple Wnt inhibitors. These findings provide significant mechanistic insights into the epigenetic activation of the Wnt/β-catenin pathway in HCC and shed light on the functional importance of ubiquitination in this intricate regulatory system., (Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)
- Published
- 2015
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19. The ubiquitin ligases RNF8 and RNF168 display rapid but distinct dynamics at DNA repair foci in living cells.
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Mok MT, Cheng AS, and Henderson BR
- Subjects
- Cell Line, Cell Nucleus metabolism, DNA Damage, Fluorescence Recovery After Photobleaching, HEK293 Cells, Humans, MCF-7 Cells, DNA Repair, DNA-Binding Proteins metabolism, Ligases metabolism, Ubiquitin metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
Rapid assembly of DNA damage response (DDR) proteins at nuclear "repair" foci is a hallmark response of ionizing radiation (IR)-treated cells. The ubiquitin E3 ligases RNF8 and RNF168 are critical for foci formation, and here we aim to determine their dynamic mobility and abundance at individual foci in living cells. To this end, YFP-tagged RNF8 and RNF168 were expressed at physiological levels in MCF-7 cells, then analyzed by fluorescence recovery after photobleaching (FRAP) assays, nuclear retention measurement, and virus-like particles (VLPs)-based quantification. The results showed that RNF8 and RNF168 were both highly dynamic at IR-induced foci. Intriguingly, RNF8 displayed remarkably faster in vivo association/dissociation rates than RNF168, and RNF8-positive IR-foci were less resistant to detergent extraction. In addition, copy number assay revealed that RNF168 was two-fold more abundant than RNF8 at foci. Collectively, we show for the first time that RNF8 moves on-and-off nuclear DNA repair foci more than six-fold as quickly as RNF168. The faster kinetics of RNF8 recruitment explains why RNF8 is generally observed at DNA-breaks prior to RNF168. Moreover, our finding that RNF8 is less abundant than RNF168 identifies RNF8 as a rate-limiting determinant of focal repair complex assembly., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
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20. Dissecting the pleiotropic actions of HBx mutants against hypoxia in hepatocellular carcinoma.
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Lee YY, Mok MT, and Cheng AS
- Abstract
Error-prone integration of the hepatitis B virus X protein (HBx) into the hepatocellular genome generates a multitude of mutants exerting diverse effects on the development and progression of hepatocellular carcinoma (HCC). A recent study by Lai and colleagues revealed the disparate regulatory activity of clinically-predominant HBx mutants towards hypoxia-inducible factor-1α (HIF-1α), a central regulator of tumor angiogenesis, proliferation, metastasis and differentiation. These findings have shed insight into specific viral contribution of hypoxic response during hepatocarcinogenesis.
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- 2014
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21. Stimulation of in vivo nuclear transport dynamics of actin and its co-factors IQGAP1 and Rac1 in response to DNA replication stress.
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Johnson MA, Sharma M, Mok MT, and Henderson BR
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- Actins antagonists & inhibitors, Actins genetics, Active Transport, Cell Nucleus, Blotting, Western, DNA Repair drug effects, Fluorescence Recovery After Photobleaching, Humans, Hydroxyurea pharmacology, Immunoprecipitation, Protein Binding, Protein Transport, RNA, Small Interfering genetics, Signal Transduction, Thymidine pharmacology, Actins metabolism, Cell Nucleus genetics, Cell Nucleus metabolism, DNA Replication drug effects, rac1 GTP-Binding Protein metabolism, ras GTPase-Activating Proteins metabolism
- Abstract
Actin, a constituent of the cytoskeleton, is now recognized to function in the nucleus in gene transcription, chromatin remodeling and DNA replication/repair. Actin shuttles in and out of the nucleus through the action of transport receptors importin-9 and exportin-6. Here we have addressed the impact of cell cycle progression and DNA replication stress on actin nuclear localization, through study of actin dynamics in living cells. First, we showed that thymidine-induced G1/S phase cell cycle arrest increased the nuclear levels of actin and of two factors that stimulate actin polymerization: IQGAP1 and Rac1 GTPase. When cells were exposed to hydroxyurea to induce DNA replication stress, the nuclear localization of actin and its regulators was further enhanced. We employed live cell photobleaching assays and discovered that in response to DNA replication stress, GFP-actin nuclear import and export rates increased by up to 250%. The rate of import was twice as fast as export, accounting for actin nuclear accumulation. The faster shuttling dynamics correlated with reduced cellular retention of actin, and our data implicate actin polymerization in the stress-dependent uptake of nuclear actin. Furthermore, DNA replication stress induced a nuclear shift in IQGAP1 and Rac1 with enhanced import dynamics. Proximity ligation assays revealed that IQGAP1 associates in the nucleus with actin and Rac1, and formation of these complexes increased after hydroxyurea treatment. We propose that the replication stress checkpoint triggers co-ordinated nuclear entry and trafficking of actin, and of factors that regulate actin polymerization., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
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22. Inhibitory role of Smad7 in hepatocarcinogenesis in mice and in vitro.
- Author
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Wang J, Zhao J, Chu ES, Mok MT, Go MY, Man K, Heuchel R, Lan HY, Chang Z, Sung JJ, and Yu J
- Subjects
- Animals, Apoptosis, Apoptosis Regulatory Proteins metabolism, Carcinoma, Hepatocellular chemically induced, Carcinoma, Hepatocellular genetics, Carcinoma, Hepatocellular metabolism, Carcinoma, Hepatocellular pathology, Cell Cycle Proteins metabolism, Cell Proliferation, Diethylnitrosamine, G1 Phase, Genes, Reporter, Genetic Predisposition to Disease, Hep G2 Cells, Hepatocytes pathology, Humans, Immunoprecipitation, Liver Neoplasms, Experimental chemically induced, Liver Neoplasms, Experimental genetics, Liver Neoplasms, Experimental metabolism, Liver Neoplasms, Experimental pathology, Mice, Mice, Inbred C57BL, Mice, Knockout, NF-kappa B metabolism, Phenotype, Primary Cell Culture, S Phase, Signal Transduction, Smad7 Protein deficiency, Smad7 Protein genetics, Time Factors, Transfection, Transforming Growth Factor beta metabolism, Carcinoma, Hepatocellular prevention & control, Hepatocytes metabolism, Liver Neoplasms, Experimental prevention & control, Smad7 Protein metabolism
- Abstract
Smad7 is a principal inhibitor of the TGFβ-Smad signalling pathway. We have investigated the functional significance of Smad7 in hepatocellular carcinoma (HCC). Smad7 knockout (KO) and wild-type (WT) mice were injected with diethylnitrosamine (DEN) to induce HCC. The effects of Smad7 on cellular features were examined in HCC cells, using a Smad7 over-expression or deletion approach. Signalling pathway components modulated by Smad7 in HCC were evaluated using luciferase reporter assay and co-immunoprecipitation. Smad7 was down-regulated in human HCCs compared with the adjacent normal tissues (p < 0.001). Smad7 KO mice were more susceptible to DEN-induced HCC than WT mice (78% versus 22%, p < 0.05). HCCs from KO mice displayed a greater proliferation activity (p < 0.05) and a reduced apoptotic index compared with WT littermates (p < 0.05). Deletion of Smad7 promoted cell proliferation in primary cultured HCC cells. In addition, over-expression of Smad7 in HCC cell lines markedly suppressed cell growth (p < 0.0001) and colony formation (p < 0.01). Cell cycle analysis revealed an increase in the G1 phase and a reduction in the S-phase populations, accompanied by up-regulation of p27(Kip1) and down-regulation of cyclin D1. Smad7 increased cell apoptosis (p < 0.01) by mediating an intrinsic [caspase-9, caspase-3 and poly(ADP-ribose) polymerase] apoptotic pathway. Moreover, Smad7 inhibited NF-κB signalling by interacting with TAB2, an upstream activator of NF-κB, and inhibited TGFβ signalling by suppressing phosphorylation of Smad3. In conclusion, loss of Smad7 enhances susceptibility to HCC. Smad7 suppresses HCC cell growth by inhibiting proliferation and G1 -S phase transition and inducing apoptosis through attenuation of NF-κB and TGFβ signalling. Smad7 acts as a potential tumour suppressor in liver., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)
- Published
- 2013
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23. The in vivo dynamic interplay of MDC1 and 53BP1 at DNA damage-induced nuclear foci.
- Author
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Mok MT and Henderson BR
- Subjects
- Adaptor Proteins, Signal Transducing, Cell Cycle Proteins, Cell Line, Tumor, Cell Nucleus metabolism, Cell Nucleus radiation effects, DNA Replication genetics, DNA Replication radiation effects, Humans, Intracellular Signaling Peptides and Proteins chemistry, Tumor Suppressor p53-Binding Protein 1, Cell Nucleus genetics, DNA Damage, Intracellular Signaling Peptides and Proteins metabolism, Nuclear Proteins metabolism, Trans-Activators metabolism
- Abstract
MDC1 (NFBD1) and 53BP1 are critical mediators of the mammalian DNA damage response (DDR) at nuclear foci. Here we show by quantitative imaging assays that MDC1 and 53BP1 are similar in total copy number (~1200 copies per focus), but differ substantially in dynamics at both replication-associated nuclear bodies in normal cells and DNA repair foci in ionizing radiation (IR)-damaged cells. The majority of MDC1 (~80%) is extremely mobile and under continuous exchange, with only a small fraction (~20%) remaining immobile at foci irrespective of IR treatment. By contrast, 53BP1 has a smaller mobile fraction (~35%) and a larger immobile fraction (~65%) at nuclear bodies, and becomes more dynamic (~20% increase in mobile pool) upon IR-induced DNA damage. More specifically, the dynamics of 53BP1 is dependent on a minimal foci-targeting region (1231-1709), and differentially regulated by its N-terminus (1-1231) and C-terminal tBRCT domain (1709-1972). Furthermore, MDC1 knockdown, or disruption of 53BP1-MDC1 interaction, reduced the number of 53BP1 molecules at foci by ~60%, but only modestly affected 53BP1 retention. This novel in vivo evidence reveals distinct dynamics of MDC1 and 53BP1 at different types of nuclear structures, and shows that MDC1 directly recruits and retains a subset of 53BP1 for DNA repair., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
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24. Three-dimensional imaging reveals the spatial separation of γH2AX-MDC1-53BP1 and RNF8-RNF168-BRCA1-A complexes at ionizing radiation-induced foci.
- Author
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Mok MT and Henderson BR
- Subjects
- Adaptor Proteins, Signal Transducing, Cell Cycle Proteins, Cell Line, Tumor, Cells, Cultured, DNA End-Joining Repair, DNA Repair, DNA-Binding Proteins metabolism, Female, Fluorescent Antibody Technique, Histones metabolism, Humans, Imaging, Three-Dimensional, Intracellular Signaling Peptides and Proteins metabolism, Microscopy, Confocal, Neoplasm Proteins radiation effects, Nuclear Proteins radiation effects, Radiation, Ionizing, Recombinational DNA Repair, Trans-Activators metabolism, Transfection, Tumor Suppressor p53-Binding Protein 1, Ubiquitin-Protein Ligases metabolism, Breast Neoplasms metabolism, DNA Damage, Multiprotein Complexes metabolism, Neoplasm Proteins metabolism, Nuclear Proteins metabolism
- Abstract
Background and Purpose: Ionizing radiation (IR)-induced DNA damage causes the accumulation of DNA damage response (DDR) proteins as visible foci in cell nuclei. Despite the identified functional roles in DNA repair, the spatial relationships of different DDR proteins at foci have not been explicitly examined. This study aims to systematically compare the distribution of DDR proteins at IR-induced foci., Materials and Methods: MCF-7 cells were treated with IR, stained for γH2AX, MDC1, RNF8, RNF168, 53BP1, Abraxas (CCDC98), BRCA1, BRCC36, Merit40 (NBA1) and RAP80, and then imaged using high-resolution three-dimensional (3-D) confocal microscopy to assess the relative localization of proteins at foci., Results: All BRCA1-A complex components displayed strong co-localization, which overlapped significantly with RNF8 and RNF168, but not with γH2AX and MDC1. Intriguingly, 53BP1 co-located well with γH2AX and MDC1, but remained separate from RNF8 and RNF168. These co-localization patterns were consistent for at least 3h after IR., Conclusions: The foci formations of γH2AX-MDC1-53BP1 and RNF8-RNF168-BRCA1-A complexes are spatially independent. Such divergence was not anticipated from prior studies on the recruitment of these proteins to foci. This information indicates that individual foci may represent distinct sites of DNA repair facilitated by a specific subset of DDR proteins., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2012
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25. The in vivo dynamic organization of BRCA1-A complex proteins at DNA damage-induced nuclear foci.
- Author
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Mok MT and Henderson BR
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Carrier Proteins metabolism, Cell Line, Tumor, Cytoplasm metabolism, DNA Repair, DNA-Binding Proteins, Deubiquitinating Enzymes, Histone Chaperones, Humans, Membrane Proteins metabolism, Models, Theoretical, Nuclear Proteins metabolism, RNA, Small Interfering metabolism, Radiation, Ionizing, Tumor Suppressor Proteins metabolism, Ubiquitin-Protein Ligases metabolism, BRCA1 Protein metabolism, Cell Nucleus metabolism, DNA Damage, Genes, BRCA1
- Abstract
The breast cancer associated gene 1 (BRCA1)-A protein complex assembles at DNA damage-induced nuclear foci to facilitate repair of double-stranded breaks. Here, we describe the first systematic comparison of the dynamics, copy number and organization of its core components at foci. We show that the protein pools at individual foci generally comprise a small immobile fraction (∼20%) and larger mobile fraction (∼80%), which together occupy the same focal space but exist at different densities. In the mobile fraction, Abraxas (CCDC98) and the heterodimer BARD1-BRCA1 share similar rates of dynamic exchange (complete turnover in ∼500 seconds). In contrast, RAP80, which is required for initial foci assembly, was more dynamic with 25-fold faster turnover at mature foci. In addition, Abraxas, BARD1, BRCA1 and Merit40 (NBA1) were stably retained in the immobile fraction of foci under conditions causing loss of BRCC36 and RAP80, suggesting a shift to RAP80-independent localization after foci formation. These results, combined with our finding that RAP80 (∼1200 copies per focus) is twofold more abundant than Abraxas/BARD1/BRCA1 at foci, suggest new models defining the dynamic organization of BRCA1-A complex at mature foci, wherein the unusually fast turnover of RAP80 may contribute to its regulation of BRCA1-dependent DNA repair., (© 2012 John Wiley & Sons A/S.)
- Published
- 2012
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26. Characterization of BARD1 targeting and dynamics at the centrosome: the role of CRM1, BRCA1 and the Q564H mutation.
- Author
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Brodie KM, Mok MTS, and Henderson BR
- Subjects
- Active Transport, Cell Nucleus genetics, Active Transport, Cell Nucleus radiation effects, BRCA1 Protein genetics, Breast Neoplasms genetics, Breast Neoplasms pathology, Cell Line, Tumor, Cell Nucleus genetics, Cell Nucleus metabolism, Centrosome metabolism, Centrosome radiation effects, Colonic Neoplasms genetics, Colonic Neoplasms pathology, DNA Damage radiation effects, DNA Repair radiation effects, Dimerization, Fluorescence, Fluorescence Recovery After Photobleaching, Gamma Rays, Humans, Karyopherins genetics, Mitosis radiation effects, Mutation, Plasmids, Receptors, Cytoplasmic and Nuclear genetics, Transfection, Tumor Suppressor Proteins chemistry, Tumor Suppressor Proteins genetics, Ubiquitin-Protein Ligases chemistry, Ubiquitin-Protein Ligases genetics, Exportin 1 Protein, BRCA1 Protein metabolism, Breast Neoplasms metabolism, Colonic Neoplasms metabolism, Karyopherins metabolism, Receptors, Cytoplasmic and Nuclear metabolism, Signal Transduction radiation effects, Tumor Suppressor Proteins metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
BARD1 heterodimerizes with BRCA1, forming an E3 ubiquitin ligase that functions at nuclear foci to repair DNA damage and the centrosome to regulate mitosis. We compared BARD1 recruitment at these structures using fluorescence recovery after photobleaching assays to measure YFP-BARD1 dynamics in live cells. In nuclei at ionizing radiation-induced foci, 20% of the BARD1 pool was immobile and 80% of slow mobility exhibiting a recovery time >500 s. In contrast, at centrosomes 83% of BARD1 was rapidly mobile with extremely fast turnover (recovery time ~20s). The ~25-fold faster exchange of BARD1 at centrosomes correlated with BRCA1-independent recruitment. We mapped key targeting sequences to a combination of the N and C-termini, and showed that mutation of the nuclear export signal reduced centrosome localization by 50%, revealing a role for CRM1. Deletion of the sequence 128-550 increased BARD1 turnover at the centrosome, consistent with a role in transient associations. Conversely, the cancer mutation Q564H reduced turnover by 25%. BARD1 is one of the most highly mobile proteins yet detected at the centrosome, and in contrast to its localization at DNA repair foci, which requires dimerization with BRCA1, targeting of BARD1 to the centrosome occurs prior to heterodimerization and its rapid turnover may provide a mechanism to regulate dimer formation., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
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27. A comparison of BRCA1 nuclear localization with 14 DNA damage response proteins and domains: identification of specific differences between BRCA1 and 53BP1 at DNA damage-induced foci.
- Author
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Mok MT and Henderson BR
- Subjects
- Active Transport, Cell Nucleus radiation effects, BRCA1 Protein genetics, Cell Cycle, Cell Line, Humans, RNA Splicing, Tumor Suppressor p53-Binding Protein 1, BRCA1 Protein metabolism, DNA Damage, Intracellular Signaling Peptides and Proteins metabolism
- Abstract
BRCA1 is an important mediator of the DNA damage response pathway. Previous studies have identified a number of proteins that associate with BRCA1 at nuclear foci after ionizing radiation (IR)-induced DNA damage. However, the co-localization patterns of BRCA1 and various DNA damage response proteins have not yet been systematically quantified and compared within the same experimental system. In this study, a new inducible human cell line was established to allow unambiguous detection of YFP-BRCA1 at nuclear foci. Quantitative 2-D microscopic analysis was performed to compare the intranuclear co-localization of YFP-BRCA1 with 10 cellular proteins and 4 cellular domains before and after IR. Intriguingly, YFP-BRCA1 displayed significantly better focal co-localization with BARD1, RAP80 and Abraxas than with the upstream foci-initiating proteins gamma H2AX and MDC1. In contrast to previous reports, we found that the co-localization between YFP-BRCA1 and 53BP1 foci was surprisingly weak. Quantitative analyses of 3-D confocal images showed that approximately 60% of 53BP1 foci were unrelated to YFP-BRCA1 foci, approximately 35% of foci were abutting and only approximately 5% of foci co-localized. The YFP-BRCA1 and 53BP1 nuclear foci were distinctively separated within the first 3h after IR. In addition, in situ nuclear retention analysis revealed YFP-BRCA1 and BARD1 are less mobile than 53BP1 at IR-induced nuclear foci. Our findings indicate that BRCA1-BARD1 and 53BP1 are proximal but not overlapping at DNA break sites and are consistent with recent evidence for distinct roles of these proteins in the DNA damage response pathway.
- Published
- 2010
- Full Text
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28. Regulation of beta-catenin trafficking to the membrane in living cells.
- Author
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Johnson M, Sharma M, Jamieson C, Henderson JM, Mok MT, Bendall L, and Henderson BR
- Subjects
- Amino Acid Substitution, Animals, Cell Adhesion, Cell Line, Cell Movement, Fluorescence Recovery After Photobleaching, Glycogen Synthase Kinase 3 antagonists & inhibitors, Glycogen Synthase Kinase 3 metabolism, Glycogen Synthase Kinase 3 beta, Lithium Chloride pharmacology, Mice, Mutagenesis, Site-Directed, NIH 3T3 Cells, Phosphorylation, Protein Transport, Time Factors, ras GTPase-Activating Proteins metabolism, Cell Membrane metabolism, beta Catenin metabolism
- Abstract
Beta-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3beta. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of beta-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged beta-catenin, and found that a small mobile pool of beta-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t(0.5) of approximately 30 s. Thus, beta-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of beta-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t(0.5) approximately 5 s) is indicative of high turnover and transient association. In contrast, beta-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t(0.5) approximately 8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of beta-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than beta-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3beta increased beta-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of beta-catenin. In summary, beta-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.
- Published
- 2009
- Full Text
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29. Mouse mammary tumor virus-like env sequences in human breast cancer.
- Author
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Mok MT, Lawson JS, Iacopetta BJ, and Whitaker NJ
- Subjects
- Animals, Base Sequence, Cell Line, Tumor, DNA, Viral, Humans, Mice, Mice, Inbred C3H, Molecular Sequence Data, Mutation, RNA, Messenger genetics, Sequence Homology, Nucleic Acid, Tumor Suppressor Protein p53 genetics, Breast Neoplasms genetics, Genes, env, Mammary Tumor Virus, Mouse genetics
- Abstract
Mouse mammary tumor virus (MMTV) has long been known as a causal agent of breast cancer in mice. To date, varied MMTV-like envelope gene (env) sequences have been identified in up to 74% of human breast cancers. However, the role and origin of these MMTV-like sequences in human breast cancer remain uncertain. Our study was initiated to study the integration of MMTV-like env sequences in human breast cancer. PCR screening has identified 28 (56%) Australian breast cancer specimens and 7 (87.5%) human breast cancer cell lines to be positive for MMTV-like env sequence. In the MCF-7 genome, a fragment containing an MMTV-like env sequence of approximately 1.9 kb plus a downstream rodent-like sequence of approximately 200 bp was found to be integrated into a bacterial-like beta-lactamase sequence by insertional mutagenesis. The identified MMTV-rodent fragment is present in some MCF-7 sublines but absent in the screened specimens and other cell lines. Sporadic mutations found in this fragment indicate it has multiple copies in the MCF-7 genome. Sequence analysis has identified a novel ORF of approximately 1.6 kb which is 94-99% identical to MMTV env genes. RT-PCR was performed on the MCF-7 cDNA but no MMTV-like env transcript was detected. This is the first report to reveal the locus of MMTV-like env sequence in human cells. The MMTV-like env sequence was shown to be distinct from the human endogenous retroviral sequences and is closely related to rodents., ((c) 2008 Wiley-Liss, Inc.)
- Published
- 2008
- Full Text
- View/download PDF
30. Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?
- Author
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Lovelock PK, Spurdle AB, Mok MT, Farrugia DJ, Lakhani SR, Healey S, Arnold S, Buchanan D, Couch FJ, Henderson BR, Goldgar DE, Tavtigian SV, Chenevix-Trench G, and Brown MA
- Subjects
- Alanine, Arginine, Breast Neoplasms pathology, Centrosome, DNA, Complementary, Factor Analysis, Statistical, Female, Genetic Predisposition to Disease, Glutamic Acid, Glutamine, Glycine, Humans, Immunohistochemistry, Keratins analysis, Nucleic Acid Amplification Techniques, Plasmids, Polymorphism, Single Nucleotide, Predictive Value of Tests, Receptors, Estrogen analysis, Risk Assessment, Risk Factors, Sequence Analysis, DNA, Valine, Biomarkers, Tumor analysis, Breast Neoplasms chemistry, Breast Neoplasms genetics, Genes, BRCA1, Mutation, Missense
- Abstract
Introduction: Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited., Methods: We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V., Results: Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification., Conclusion: These data highlight the need for a range of functional studies to be performed in order to identify variants with partially compromised function. The results also raise the possibility that A1708V and R1699Q may be associated with a low or moderate risk of cancer. While data pooling strategies may provide more information for multifactorial analysis to improve the interpretation of the clinical significance of these variants, it is likely that the development of current multifactorial likelihood approaches and the consideration of alternative statistical approaches will be needed to determine whether these individually rare variants do confer a low or moderate risk of breast cancer.
- Published
- 2007
- Full Text
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31. Giardia intestinalis: molecular characterization of UDP-N-acetylglucosamine pyrophosphorylase.
- Author
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Mok MT, Tay E, Sekyere E, Glenn WK, Bagnara AS, and Edwards MR
- Subjects
- 3' Untranslated Regions genetics, 5' Untranslated Regions genetics, Acetylgalactosamine biosynthesis, Animals, Base Sequence, Cell Wall enzymology, Cell Wall genetics, Gene Dosage, Giardia lamblia genetics, Giardiasis enzymology, Giardiasis genetics, Glucose metabolism, Humans, Introns genetics, Molecular Sequence Data, Nucleotidyltransferases metabolism, Polyadenylation physiology, Polymerase Chain Reaction methods, Protozoan Proteins metabolism, Gene Expression Regulation, Enzymologic physiology, Giardia lamblia enzymology, Nucleotidyltransferases genetics, Open Reading Frames physiology, Protozoan Proteins genetics
- Abstract
The flagellated protozoan Giardia intestinalis is one of the most prevalent human-infective parasites with a worldwide distribution. This parasite must encyst to complete the life cycle and N-acetylgalactosamine is produced from endogenous glucose for cyst wall synthesis during the transformation. UDP-N-acetylglucosamine pyrophosphorylase in G. intestinalis (GiUAP, EC 2.7.7.23) is the fourth enzyme in the inducible pathway of N-acetylgalactosamine biosynthesis, catalysing the conversion of N-acetylglucosamine-1-P to UDP-N-acetylglucosamine. In this study the gene GiUAP was cloned and sequenced from the Portland 1 strain using PCR techniques. It has an ORF of approximately 1.3 kb and contains no introns. BLAST and ClustalW analysis of the deduced amino acid sequence revealed significant similarities to other eukaryotic UAPs with putative active sites identified. Southern hybridization showed that GiUAP exists as a single-copy gene and it was shown to have two transcripts by RT-PCR and Northern hybridization. RLM-RACE identified both 5' and 3' untranslated regions and suggested the transcripts exist as a 5'-capped mRNA, with the use of two tandem polyadenylation sites to generate two unusually long giardial 3' untranslated regions of approximately 522 bp and approximately 3 kb. Moreover, a recombinant protein (rGiUAP) was expressed in E. coli and subjected to physical characterizations. Surprisingly the results obtained in this study were significantly different from those reported for the GiUAP in MR4 strain, suggesting this gene is under different transcription control in different strains of G. intestinalis. This report describes the molecular characterization of GiUAP and provides an opportunity to explore the control of gene expression during encystation of the parasite.
- Published
- 2005
- Full Text
- View/download PDF
32. Protein kinase B from Giardia intestinalis.
- Author
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Kim KT, Mok MT, and Edwards MR
- Subjects
- Amino Acid Sequence, Animals, Molecular Sequence Data, Molecular Weight, Protein Serine-Threonine Kinases analysis, Proto-Oncogene Proteins analysis, Proto-Oncogene Proteins c-akt, Recombinant Proteins analysis, Recombinant Proteins chemistry, Sequence Homology, Amino Acid, Giardia lamblia genetics, Giardia lamblia metabolism, Protein Serine-Threonine Kinases chemistry, Proto-Oncogene Proteins chemistry
- Abstract
A novel serine/threonine protein kinase from Giardia intestinalis (GiPKB) was isolated by a combination of PCR techniques. Analysis of the GiPKB sequence indicated that the encoded protein appears to be a member of a novel subgroup of serine/threonine protein kinases known as protein kinase B. Reverse transcription PCR and Northern hybridization showed that the transcription of GiPKB is developmentally regulated. The GiPKB was expressed as a recombinant protein, which was characterized and shown to have a protein kinase activity. The preferred substrate for the GiPKB was histone H1, while histone H2A, GSK3 peptide, GS peptide, and Kemptide were phosphorylated at about 96, 73, 51, and 40% of the activity with histone H1, respectively. Neither cAMP, Ca(2+), nor Ca(2+)/calmodulin stimulated the enzyme activity. The GiPKB utilized ATP rather than GTP as a phosphate donor with an apparent K(m) of 20 microM. The identification and characterization of this differentially and constitutively expressed GiPKB should allow further analysis of the regulation and signal transduction pathways in Giardia.
- Published
- 2005
- Full Text
- View/download PDF
33. Critical sources of error in colorimetric assay for UDP-N-acetylglucosamine pyrophosphorylase.
- Author
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Mok MT and Edwards MR
- Subjects
- Animals, Buffers, Giardia lamblia enzymology, Glucosephosphates chemistry, Kinetics, Nucleotidyltransferases chemistry, Reproducibility of Results, Research Design, Rosaniline Dyes analysis, Substrate Specificity, Uridine Triphosphate chemistry, Colorimetry methods, Nucleotidyltransferases analysis
- Published
- 2005
- Full Text
- View/download PDF
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