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1
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Dolan M, St. John N, Zaidi F, Doyle F, Fasullo M. High-throughput screening of the Saccharomyces cerevisiae genome for 2-amino-3-methylimidazo [4,5-f] quinoline resistance identifies colon cancer-associated genes. G3 (BETHESDA, MD.) 2023; 13:jkad219. [PMID: 37738679 PMCID: PMC11025384 DOI: 10.1093/g3journal/jkad219] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Revised: 10/25/2022] [Accepted: 09/15/2023] [Indexed: 09/24/2023]
Abstract
Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We used Saccharomyces cerevisiae (budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. Deletion libraries expressing human CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 µM IQ for 5 or 10 generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. We identified 424 ORFs, including 337 genes of known function, in duplicate screens of the "humanized" collection for IQ resistance; resistance was further validated for a select group of 51 genes by growth curves, competitive growth, or trypan blue assays. Screens of the library not expressing human genes identified 143 ORFs conferring resistance to IQ per se. Ribosomal protein and protein modification genes were identified as IQ resistance genes in both the original and "humanized" libraries, while nitrogen metabolism, DNA repair, and growth control genes were also prominent in the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. Among DNA Repair and checkpoint genes, we identified those that function in postreplication repair (RAD18, UBC13, REV7), base excision repair (NTG1), and checkpoint signaling (CHK1, PSY2). These studies underscore the role of ribosomal protein genes in conferring IQ resistance, and illuminate DNA repair pathways for conferring resistance to activated IQ.
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Affiliation(s)
- Michael Dolan
- College of Nanotechnology, Science, and Engineering, State University of NewYork at Albany, Albany, NY 12203, USA
| | - Nick St. John
- College of Nanotechnology, Science, and Engineering, State University of NewYork at Albany, Albany, NY 12203, USA
| | - Faizan Zaidi
- College of Nanotechnology, Science, and Engineering, State University of NewYork at Albany, Albany, NY 12203, USA
| | - Francis Doyle
- College of Nanotechnology, Science, and Engineering, State University of NewYork at Albany, Albany, NY 12203, USA
| | - Michael Fasullo
- College of Nanotechnology, Science, and Engineering, State University of NewYork at Albany, Albany, NY 12203, USA
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2
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Robinson PS, Thomas LE, Abascal F, Jung H, Harvey LMR, West HD, Olafsson S, Lee BCH, Coorens THH, Lee-Six H, Butlin L, Lander N, Truscott R, Sanders MA, Lensing SV, Buczacki SJA, Ten Hoopen R, Coleman N, Brunton-Sim R, Rushbrook S, Saeb-Parsy K, Lalloo F, Campbell PJ, Martincorena I, Sampson JR, Stratton MR. Inherited MUTYH mutations cause elevated somatic mutation rates and distinctive mutational signatures in normal human cells. Nat Commun 2022; 13:3949. [PMID: 35803914 PMCID: PMC9270427 DOI: 10.1038/s41467-022-31341-0] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2021] [Accepted: 06/14/2022] [Indexed: 12/21/2022] Open
Abstract
Cellular DNA damage caused by reactive oxygen species is repaired by the base excision repair (BER) pathway which includes the DNA glycosylase MUTYH. Inherited biallelic MUTYH mutations cause predisposition to colorectal adenomas and carcinoma. However, the mechanistic progression from germline MUTYH mutations to MUTYH-Associated Polyposis (MAP) is incompletely understood. Here, we sequence normal tissue DNAs from 10 individuals with MAP. Somatic base substitution mutation rates in intestinal epithelial cells were elevated 2 to 4-fold in all individuals, except for one showing a 31-fold increase, and were also increased in other tissues. The increased mutation burdens were of multiple mutational signatures characterised by C > A changes. Different mutation rates and signatures between individuals are likely due to different MUTYH mutations or additional inherited mutations in other BER pathway genes. The elevated base substitution rate in normal cells likely accounts for the predisposition to neoplasia in MAP. Despite ubiquitously elevated mutation rates, individuals with MAP do not display overt evidence of premature ageing. Thus, accumulation of somatic mutations may not be sufficient to cause the global organismal functional decline of ageing.
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Affiliation(s)
- Philip S Robinson
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
- Department of Paediatrics, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Laura E Thomas
- Institute of Life Science, Swansea University, Swansea, SA28PP, UK
| | - Federico Abascal
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
| | - Hyunchul Jung
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
| | - Luke M R Harvey
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
| | - Hannah D West
- Institute of Medical Genetics, Division of Cancer and Genetics, Cardiff University School of Medicine, Cardiff, UK
| | - Sigurgeir Olafsson
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
| | - Bernard C H Lee
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
- Hereditary Gastrointestinal Cancer Genetic Diagnosis Laboratory, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong
| | - Tim H H Coorens
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
| | - Henry Lee-Six
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
| | - Laura Butlin
- Institute of Medical Genetics, Division of Cancer and Genetics, Cardiff University School of Medicine, Cardiff, UK
| | - Nicola Lander
- Institute of Medical Genetics, Division of Cancer and Genetics, Cardiff University School of Medicine, Cardiff, UK
| | - Rebekah Truscott
- Institute of Medical Genetics, Division of Cancer and Genetics, Cardiff University School of Medicine, Cardiff, UK
| | - Mathijs A Sanders
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
- Department of Haematology, Erasmus University Medical Centre, 3015 CN, Rotterdam, The Netherlands
| | - Stefanie V Lensing
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
| | - Simon J A Buczacki
- Nuffield Department of Surgical Sciences, Medical Sciences Division, University of Oxford, Oxford, UK
| | | | - Nicholas Coleman
- Department of Pathology, University of Cambridge, Cambridge, UK
- Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK
| | | | - Simon Rushbrook
- Norfolk and Norwich University Hospital, Norwich, UK
- Norwich Medical School, University of East Anglia, Norwich, UK
| | - Kourosh Saeb-Parsy
- Department of Surgery, University of Cambridge, Cambridge, UK
- Cambridge NIHR Biomedical Research Centre, Cambridge Biomedical Campus, Cambridge, UK
| | - Fiona Lalloo
- Manchester Centre for Genomic Medicine, Saint Mary's Hospital, Oxford Road, Manchester, UK
| | - Peter J Campbell
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
| | - Iñigo Martincorena
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK
| | - Julian R Sampson
- Institute of Medical Genetics, Division of Cancer and Genetics, Cardiff University School of Medicine, Cardiff, UK
| | - Michael R Stratton
- Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, CB10 1SA, UK.
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3
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Caffrey PJ, Delaney S. Chromatin and other obstacles to base excision repair: potential roles in carcinogenesis. Mutagenesis 2021; 35:39-50. [PMID: 31612219 DOI: 10.1093/mutage/gez029] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2019] [Accepted: 09/13/2019] [Indexed: 12/29/2022] Open
Abstract
DNA is comprised of chemically reactive nucleobases that exist under a constant barrage from damaging agents. Failure to repair chemical modifications to these nucleobases can result in mutations that can cause various diseases, including cancer. Fortunately, the base excision repair (BER) pathway can repair modified nucleobases and prevent these deleterious mutations. However, this pathway can be hindered through several mechanisms. For instance, mutations to the enzymes in the BER pathway have been identified in cancers. Biochemical characterisation of these mutants has elucidated various mechanisms that inhibit their activity. Furthermore, the packaging of DNA into chromatin poses another obstacle to the ability of BER enzymes to function properly. Investigations of BER in the base unit of chromatin, the nucleosome core particle (NCP), have revealed that the NCP acts as a complex substrate for BER enzymes. The constituent proteins of the NCP, the histones, also have variants that can further impact the structure of the NCP and may modulate access of enzymes to the packaged DNA. These histone variants have also displayed significant clinical effects both in carcinogenesis and patient prognosis. This review focuses on the underlying molecular mechanisms that present obstacles to BER and the relationship of these obstacles to cancer. In addition, several chemotherapeutics induce DNA damage that can be repaired by the BER pathway and understanding obstacles to BER can inform how resistance and/or sensitivity to these therapies may occur. With the understanding of these molecular mechanisms, current chemotherapeutic treatment regiments may be improved, and future therapies developed.
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Affiliation(s)
- Paul J Caffrey
- Department of Chemistry, Brown University, Providence, RI
| | - Sarah Delaney
- Department of Chemistry, Brown University, Providence, RI
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4
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Abduljaleel Z, Athar M, Al-Allaf FA, Al-Dehlawi S, Vazquez JR. Association of functional variants and protein-to-protein physical interactions of human MutY homolog linked with familial adenomatous polyposis and colorectal cancer syndrome. Noncoding RNA Res 2020; 4:155-173. [PMID: 32072083 PMCID: PMC7012779 DOI: 10.1016/j.ncrna.2019.11.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2019] [Revised: 09/26/2019] [Accepted: 11/19/2019] [Indexed: 11/26/2022] Open
Abstract
The human gene MUTYH codes for a DNA glycosylase involved in the repair of oxidative DNA damage. Faulty MUTYH protein activity causes the accumulation of G→T transversions due to unrepaired 8-oxoG:A mismatches. MUTYH germ-line mutations in humans are linked with a recessive form of Familial Adenomatous Polyposis (FAP) and colorectal cancer predisposition. We studied the repair capacity of variants identified in MUTYH-associated polyposis (MAP) patients. MAP is inherited in an autosomal recessive type due to mutations in MUTYH (Y165C, G382D, P54S, A22V, Q63R, G45D, S136P and N43S), indicating that both copies of the gene become inactivated. However, the parents of an individual with an autosomal recessive condition may serve as carriers, each harboring one copy of the mutated gene without showing signs or symptoms of MAP. Six protein partners have been associated with MUTYH, four via direct physical interactions, namely, hMSH6, hPCNA, hRPA1, and hAPEX1. We examined, for the first time, specific interactions of these protein partners with MAP-associated MUTYH mutants using molecular dynamics simulations. The approach provided tools for exploration of the conformational energy landscape accessible to protein partners. The investigation also determined the impact before and after energy minimization of protein-protein interactions and binding affinities of MUTYH wild type and mutant forms, as well as the interactions with other proteins. Taken together, this study provided new insights into the role of MUTYH and its interacting proteins in MAP.
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Affiliation(s)
- Zainularifeen Abduljaleel
- Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O.Box: 715, Makkah 21955, Saudi Arabia.,Science and Technology Unit, Umm Al-Qura University, P.O. Box: 715, Makkah 21955, Saudi Arabia.,Bircham University, Av. Sierra, 2, 28691 Villanueva de la Canada, Madrid, Spain
| | - Mohammad Athar
- Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O.Box: 715, Makkah 21955, Saudi Arabia.,Science and Technology Unit, Umm Al-Qura University, P.O. Box: 715, Makkah 21955, Saudi Arabia
| | - Faisal A Al-Allaf
- Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O.Box: 715, Makkah 21955, Saudi Arabia.,Science and Technology Unit, Umm Al-Qura University, P.O. Box: 715, Makkah 21955, Saudi Arabia
| | - Saied Al-Dehlawi
- The Regional Laboratory, Ministry of Health (MOH), P.O. Box: 6251, Makkah, Saudi Arabia
| | - Jose R Vazquez
- Bircham University, Av. Sierra, 2, 28691 Villanueva de la Canada, Madrid, Spain
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5
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Nelson SR, Kathe SD, Hilzinger TS, Averill AM, Warshaw DM, Wallace SS, Lee AJ. Single molecule glycosylase studies with engineered 8-oxoguanine DNA damage sites show functional defects of a MUTYH polyposis variant. Nucleic Acids Res 2019; 47:3058-3071. [PMID: 30698731 PMCID: PMC6451117 DOI: 10.1093/nar/gkz045] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2018] [Revised: 01/03/2019] [Accepted: 01/17/2019] [Indexed: 01/09/2023] Open
Abstract
Proper repair of oxidatively damaged DNA bases is essential to maintain genome stability. 8-Oxoguanine (7,8-dihydro-8-oxoguanine, 8-oxoG) is a dangerous DNA lesion because it can mispair with adenine (A) during replication resulting in guanine to thymine transversion mutations. MUTYH DNA glycosylase is responsible for recognizing and removing the adenine from 8-oxoG:adenine (8-oxoG:A) sites. Biallelic mutations in the MUTYH gene predispose individuals to MUTYH-associated polyposis (MAP), and the most commonly observed mutation in some MAP populations is Y165C. Tyr165 is a ‘wedge’ residue that intercalates into the DNA duplex in the lesion bound state. Here, we utilize single molecule fluorescence microscopy to visualize the real-time search behavior of Escherichia coli and Mus musculus MUTYH WT and wedge variant orthologs on DNA tightropes that contain 8-oxoG:A, 8-oxoG:cytosine, or apurinic product analog sites. We observe that MUTYH WT is able to efficiently find 8-oxoG:A damage and form highly stable bound complexes. In contrast, MUTYH Y150C shows decreased binding lifetimes on undamaged DNA and fails to form a stable lesion recognition complex at damage sites. These findings suggest that MUTYH does not rely upon the wedge residue for damage site recognition, but this residue stabilizes the lesion recognition complex.
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Affiliation(s)
- Shane R Nelson
- Department of Molecular Physiology and Biophysics, Robert Larner College of Medicine, University of Vermont, Burlington, VT 05405, USA
| | - Scott D Kathe
- Department of Microbiology and Molecular Genetics, Robert Larner College of Medicine and College of Agriculture and Life Sciences, University of Vermont, Burlington, VT 05405, USA
| | - Thomas S Hilzinger
- Department of Microbiology and Molecular Genetics, Robert Larner College of Medicine and College of Agriculture and Life Sciences, University of Vermont, Burlington, VT 05405, USA
| | - April M Averill
- Department of Microbiology and Molecular Genetics, Robert Larner College of Medicine and College of Agriculture and Life Sciences, University of Vermont, Burlington, VT 05405, USA
| | - David M Warshaw
- Department of Molecular Physiology and Biophysics, Robert Larner College of Medicine, University of Vermont, Burlington, VT 05405, USA
| | - Susan S Wallace
- Department of Microbiology and Molecular Genetics, Robert Larner College of Medicine and College of Agriculture and Life Sciences, University of Vermont, Burlington, VT 05405, USA
| | - Andrea J Lee
- Department of Microbiology and Molecular Genetics, Robert Larner College of Medicine and College of Agriculture and Life Sciences, University of Vermont, Burlington, VT 05405, USA
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6
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Common Genetic Variants of MUTYH are not Associated with Cutaneous Malignant Melanoma: Application of Molecular Screening by Means of High-Resolution Melting Technique in a Pilot Case-Control Study. Int J Biol Markers 2018; 26:37-42. [DOI: 10.5301/jbm.2011.6285] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/10/2010] [Indexed: 01/09/2023]
Abstract
MUTYH glycosylase recognizes the 8-oxoG:A mismatch and is able to excise the adenine base using proofreading mechanisms. Some papers have reported a strong association between cancer development or aggressiveness and MUTYH gene mutations. The aim of this study was to find a possible association between the most frequent MUTYH mutations and melanoma in the context of a case-control pilot study. One hundred ninety-five melanoma patients and 195 healthy controls were matched for sex and age. Clinical and laboratory data were collected in a specific database and all individuals were analyzed for MUTYH mutations by high-resolution melting and direct sequencing techniques. Men and women had significantly different distributions of tumor sites and phototypes. No significant associations were observed between the Y165C, G382D and V479F MUTYH mutations and risk of melanoma development or aggressiveness. Our preliminary findings therefore do not confirm a role for MUTYH gene mutations in the melanoma risk. Further studies are necessary for the assessment of MUTYH not only in melanoma but also other cancer types with the same embryonic origin, in the context of larger arrays studies of genes involved in DNA stability or integrity.
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7
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Nuñez NN, Majumdar C, Lay KT, David SS. Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements. Methods Enzymol 2018; 599:21-68. [PMID: 29746241 DOI: 10.1016/bs.mie.2017.11.035] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S]2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease.
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Affiliation(s)
| | | | - Kori T Lay
- University of California, Davis, CA, United States
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8
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Shakeri Manesh S, Sangsuwan T, Pour Khavari A, Fotouhi A, Emami SN, Haghdoost S. MTH1, an 8-oxo-2'-deoxyguanosine triphosphatase, and MYH, a DNA glycosylase, cooperate to inhibit mutations induced by chronic exposure to oxidative stress of ionising radiation. Mutagenesis 2017; 32:389-396. [PMID: 28340109 DOI: 10.1093/mutage/gex003] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Our previous results showed that in addition to the immediate interaction of ionising radiation with DNA (direct and indirect effect), low-dose and chronic low-dose rate of irradiation induce endogenous oxidative stress. During oxidative stress, free radicals react with DNA, nucleoside triphosphates (dNTPs), proteins and lipids, and modify their structures. The MYH and MTH1 genes play important roles in preventing mutations induced by 8-hydroxy-guanine, which is an oxidised product of guanine. In this study, we used short-hairpin RNA to permanently knockdown MYH and MTH1 proteins in human lymphoblastoid TK6 cells. Knockdown and wild-type cells were chronically exposed to low dose rates of γ-radiation (between 1.4 and 30 mGy/h). The cells were also subjected to acute doses delivered at a high-dose rate. Growth rate, extracellular 8-hydroxy-2'-deoxyguanosine, clonogenic cell survival and mutant frequencies were analysed in all cell types. A reduced level of cell growth and survival as well as increased mutant frequencies were observed in cells lacking both MYH and MTH1 proteins as compared to cells lacking only MYH and wild-type cells. To sum up, our results suggest that low-dose rates elevate oxidative stress. MTH1 together with MYH plays an important role in protection against mutations induced by modified dNTPs during chronic oxidative stress. In addition, we found no dose-rate effect at the level of mutations in the wild-type TK6 and MYH-KD cells. Our data interestingly indicate a dose-rate threshold for mutation induction in MTH1/MYH double knockdown cells.
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Affiliation(s)
- Sara Shakeri Manesh
- Centre for Radiation Protection Research, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden
| | - Traimate Sangsuwan
- Centre for Radiation Protection Research, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden
| | - Ali Pour Khavari
- Centre for Radiation Protection Research, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden
| | - Asal Fotouhi
- Centre for Radiation Protection Research, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden
| | - S Noushin Emami
- Centre for Radiation Protection Research, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden
| | - Siamak Haghdoost
- Centre for Radiation Protection Research, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden
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9
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Banda DM, Nuñez NN, Burnside MA, Bradshaw KM, David SS. Repair of 8-oxoG:A mismatches by the MUTYH glycosylase: Mechanism, metals and medicine. Free Radic Biol Med 2017; 107:202-215. [PMID: 28087410 PMCID: PMC5457711 DOI: 10.1016/j.freeradbiomed.2017.01.008] [Citation(s) in RCA: 71] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/17/2016] [Revised: 01/01/2017] [Accepted: 01/04/2017] [Indexed: 12/12/2022]
Abstract
Reactive oxygen and nitrogen species (RONS) may infringe on the passing of pristine genetic information by inducing DNA inter- and intra-strand crosslinks, protein-DNA crosslinks, and chemical alterations to the sugar or base moieties of DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the most prevalent DNA lesions formed by RONS and is repaired through the base excision repair (BER) pathway involving the DNA repair glycosylases OGG1 and MUTYH in eukaryotes. MUTYH removes adenine (A) from 8-oxoG:A mispairs, thus mitigating the potential of G:C to T:A transversion mutations from occurring in the genome. The paramount role of MUTYH in guarding the genome is well established in the etiology of a colorectal cancer predisposition syndrome involving variants of MUTYH, referred to as MUTYH-associated polyposis (MAP). In this review, we highlight recent advances in understanding how MUTYH structure and related function participate in the manifestation of human disease such as MAP. Here we focus on the importance of MUTYH's metal cofactor sites, including a recently discovered "Zinc linchpin" motif, as well as updates to the catalytic mechanism. Finally, we touch on the insight gleaned from studies with MAP-associated MUTYH variants and recent advances in understanding the multifaceted roles of MUTYH in the cell, both in the prevention of mutagenesis and tumorigenesis.
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Affiliation(s)
- Douglas M Banda
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Nicole N Nuñez
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Michael A Burnside
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Katie M Bradshaw
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Sheila S David
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States.
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10
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Komine K, Shimodaira H, Takao M, Soeda H, Zhang X, Takahashi M, Ishioka C. Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia coli Strain. Hum Mutat 2016; 36:704-11. [PMID: 25820570 PMCID: PMC4682456 DOI: 10.1002/humu.22794] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2014] [Accepted: 03/25/2015] [Indexed: 12/27/2022]
Abstract
MUTYH-associated polyposis (MAP) is an adenomatous polyposis transmitted in an autosomal-recessive pattern, involving biallelic inactivation of the MUTYH gene. Loss of a functional MUTYH protein will result in the accumulation of G:T mismatched DNA caused by oxidative damage. Although p.Y179C and p.G396D are the two most prevalent MUTYH variants, more than 200 missense variants have been detected. It is difficult to determine whether these variants are disease-causing mutations or single-nucleotide polymorphisms. To understand the functional consequences of these variants, we generated 47 MUTYH gene variants via site-directed mutagenesis, expressed the encoded proteins in MutY-disrupted Escherichia coli, and assessed their abilities to complement the functional deficiency in the E. coli by monitoring spontaneous mutation rates. Although the majority of variants exhibited intermediate complementation relative to the wild type, some variants severely interfered with this complementation. However, some variants retained functioning similar to the wild type. In silico predictions of functional effects demonstrated a good correlation. Structural prediction of MUTYH based on the MutY protein structure allowed us to interpret effects on the protein stability or catalytic activity. These data will be useful for evaluating the functional consequences of missense MUTYH variants detected in patients with suspected MAP.
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Affiliation(s)
- Keigo Komine
- Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai, Japan.,Department of Clinical Oncology, Tohoku University Hospital, Aoba-ku, Sendai, Japan
| | - Hideki Shimodaira
- Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai, Japan.,Department of Clinical Oncology, Tohoku University Hospital, Aoba-ku, Sendai, Japan
| | - Masashi Takao
- Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai, Japan
| | - Hiroshi Soeda
- Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai, Japan.,Department of Clinical Oncology, Tohoku University Hospital, Aoba-ku, Sendai, Japan
| | - Xiaofei Zhang
- Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai, Japan
| | - Masanobu Takahashi
- Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai, Japan.,Department of Clinical Oncology, Tohoku University Hospital, Aoba-ku, Sendai, Japan
| | - Chikashi Ishioka
- Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai, Japan.,Department of Clinical Oncology, Tohoku University Hospital, Aoba-ku, Sendai, Japan
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11
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Engstrom LM, Brinkmeyer MK, Ha Y, Raetz AG, Hedman B, Hodgson KO, Solomon EI, David SS. A zinc linchpin motif in the MUTYH glycosylase interdomain connector is required for efficient repair of DNA damage. J Am Chem Soc 2014; 136:7829-32. [PMID: 24841533 PMCID: PMC4063174 DOI: 10.1021/ja502942d] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Mammalian MutY glycosylases have a unique architecture that features an interdomain connector (IDC) that joins the catalytic N-terminal domain and 8-oxoguanine (OG) recognition C-terminal domain. The IDC has been shown to be a hub for interactions with protein partners involved in coordinating downstream repair events and signaling apoptosis. Herein, a previously unidentified zinc ion and its coordination by three Cys residues of the IDC region of eukaryotic MutY organisms were characterized by mutagenesis, ICP-MS, and EXAFS. In vitro kinetics and cellular assays on WT and Cys to Ser mutants have revealed an important function for zinc coordination on overall protein stability, iron-sulfur cluster insertion, and ability to mediate DNA damage repair. We propose that this "zinc linchpin" motif serves to structurally organize the IDC and coordinate the damage recognition and base excision functions of the C- and N-terminal domains.
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Affiliation(s)
- Lisa M Engstrom
- Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States
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12
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Shinmura K, Goto M, Tao H, Matsuura S, Matsuda T, Sugimura H. Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis. World J Gastroenterol 2012; 18:6935-42. [PMID: 23322991 PMCID: PMC3531677 DOI: 10.3748/wjg.v18.i47.6935] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/20/2012] [Revised: 09/19/2012] [Accepted: 09/22/2012] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion, 8-hydroxyguanine (8OHG), in human cells.
METHODS: p.R154H, p.M255V, p.L360P, and p.P377L MUTYH variants, which were previously found in patients with colorectal polyposis and cancer, were selected for use in this study. Human H1299 cancer cell lines inducibly expressing wild-type (WT) MUTYH (type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system, enabling the genomic integration of the transposon sequence for MUTYH expression. MUTYH expression was examined after cumate induction using Western blotting analysis and immunofluorescence analysis. The intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently examined. Next, the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation assay.
RESULTS: The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with cumate. All of the MUTYH variants and WT MUTYH were localized in the nucleus, and nuclear localization was also observed for FLAG-tagged MUTYH. The mutation frequency of supF was 2.2 × 10-2 in the 8OHG-containing pMY189 plasmid and 2.5 × 10-4 in WT pMY189 in empty vector cells, which was an 86-fold increase with the introduction of 8OHG. The mutation frequency (4.7 × 10-3) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells (P < 0.01). However, the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H, p.M255V, p.L360P, or p.P377L MUTYH variant were 1.84 × 10-2, 1.55 × 10-2, 1.91 × 10-2, and 1.96 × 10-2, respectively, meaning that no significant difference was observed in the mutation frequency between the empty vector cells and cells overexpressing MUTYH mutants.
CONCLUSION: The suppressive activities of p.R154H, p.M255V, p.L360P, and p.P377L MUTYH variants against mutations caused by 8OHG are thought to be severely impaired in human cells.
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13
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Gu Y, Fang TT, Chao TH. Modification of allele-specific polymerase chain reaction for hMYH mutation analysis in Taiwanese patients with colorectal cancer. J Taiwan Inst Chem Eng 2011. [DOI: 10.1016/j.jtice.2010.11.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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14
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Goto M, Shinmura K, Nakabeppu Y, Tao H, Yamada H, Tsuneyoshi T, Sugimura H. Adenine DNA glycosylase activity of 14 human MutY homolog (MUTYH) variant proteins found in patients with colorectal polyposis and cancer. Hum Mutat 2010; 31:E1861-74. [PMID: 20848659 PMCID: PMC3051265 DOI: 10.1002/humu.21363] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Biallelic inactivating germline mutations in the base excision repair MUTYH (MYH) gene have been shown to predispose to MUTYH-associated polyposis (MAP), which is characterized by multiple colorectal adenomas and carcinomas. In this study, we successfully prepared highly homogeneous human MUTYH type 2 recombinant proteins and compared the DNA glycosylase activity of the wild-type protein and fourteen variant-type proteins on adenine mispaired with 8-hydroxyguanine, an oxidized form of guanine. The adenine DNA glycosylase activity of the p.I195V protein, p.G368D protein, p.M255V protein, and p.Y151C protein was 66.9%, 15.2%, 10.7%, and 4.5%, respectively, of that of the wild-type protein, and the glycosylase activity of the p.R154H, p.L360P, p.P377L, p.452delE, p.R69X, and p.Q310X proteins as well as of the p.D208N negative control form was extremely severely impaired. The glycosylase activity of the p.V47E, p.R281C, p.A345V, and p.S487F proteins, on the other hand, was almost the same as that of the wild-type protein. These results should be of great value in accurately diagnosing MAP and in fully understanding the mechanism by which MUTYH repairs DNA in which adenine is mispaired with 8-hydroxyguanine. © 2010 Wiley-Liss, Inc.
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Affiliation(s)
- Masanori Goto
- First Department of Pathology, Hamamatsu University School of Medicine, Japan
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15
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Out AA, Tops CM, Nielsen M, Weiss MM, van Minderhout IJ, Fokkema IF, Buisine MP, Claes K, Colas C, Fodde R, Fostira F, Franken PF, Gaustadnes M, Heinimann K, Hodgson SV, Hogervorst FB, Holinski-Feder E, Lagerstedt-Robinson K, Olschwang S, Ans M.W. VDO, Redeker EJ, Scott RJ, Vankeirsbilck B, Grønlund RV, Wijnen JT, Wikman FP, Aretz S, Sampson JR, Devilee P, den Dunnen JT, Hes FJ. Leiden open variation database of the MUTYH gene. Hum Mutat 2010; 31:1205-15. [DOI: 10.1002/humu.21343] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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16
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Kundu S, Brinkmeyer MK, Eigenheer RA, David SS. Ser 524 is a phosphorylation site in MUTYH and Ser 524 mutations alter 8-oxoguanine (OG): a mismatch recognition. DNA Repair (Amst) 2010; 9:1026-37. [PMID: 20724227 DOI: 10.1016/j.dnarep.2010.07.002] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2010] [Revised: 06/22/2010] [Accepted: 07/02/2010] [Indexed: 12/17/2022]
Abstract
MUTYH-associated polyposis (MAP) is a colorectal cancer predisposition syndrome that is caused by inherited biallelic mutations in the base excision repair (BER) gene, MUTYH. MUTYH is a DNA glycosylase that removes adenine (A) misinserted opposite 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG). In this work, wild type (WT) MUTYH overexpressed using a baculovirus-driven insect cell expression system (BEVS) provided significantly higher levels of enzyme compared to bacterial overexpression. The isolated MUTYH enzyme was analyzed for potential post-translational modifications using mass spectrometry. An in vivo phosphorylation site was validated at Serine 524, which is located in the C-terminal OG recognition domain within the proliferating cell nuclear antigen (PCNA) binding region. Characterization of the phosphomimetic (S524D) and phosphoablating (S524A) mutants together with the observation that Ser 524 can be phosphorylated suggest that this residue may play an important regulatory role in vivo by altering stability and OG:A mismatch affinity.
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Affiliation(s)
- Sucharita Kundu
- Department of Chemistry, University of California, Davis, Davis, CA 95616, USA
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17
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Nielsen M, Morreau H, Vasen HFA, Hes FJ. MUTYH-associated polyposis (MAP). Crit Rev Oncol Hematol 2010; 79:1-16. [PMID: 20663686 DOI: 10.1016/j.critrevonc.2010.05.011] [Citation(s) in RCA: 111] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2010] [Revised: 05/11/2010] [Accepted: 05/27/2010] [Indexed: 12/13/2022] Open
Abstract
The human mutY homologue (MUTYH) gene is responsible for inheritable polyposis and colorectal cancer. This review discusses the molecular genetic aspects of the MUTYH gene and protein, the clinical impact of mono- and biallelic MUTYH mutations and histological aspects of the MUTYH tumors. Furthermore, the relationship between MUTYH and the mismatch repair genes in colorectal cancer (CRC) families is examined. Finally, the role of other base excision repair genes in polyposis and CRC patients is discussed.
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Affiliation(s)
- Maartje Nielsen
- Department Clinical Genetics, Leiden University Medical Centre, Albinusdreef, Leiden, The Netherlands.
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18
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Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis. DNA Repair (Amst) 2010; 9:700-7. [PMID: 20418187 DOI: 10.1016/j.dnarep.2010.03.008] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2009] [Revised: 03/01/2010] [Accepted: 03/26/2010] [Indexed: 02/08/2023]
Abstract
The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G>T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.
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19
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Kundu S, Brinkmeyer MK, Livingston AL, David SS. Adenine removal activity and bacterial complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer. DNA Repair (Amst) 2010; 8:1400-10. [PMID: 19836313 DOI: 10.1016/j.dnarep.2009.09.009] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2008] [Revised: 09/17/2009] [Accepted: 09/19/2009] [Indexed: 10/20/2022]
Abstract
MUTYH-associated polyposis (MAP) is the only inherited colorectal cancer syndrome that is associated with inherited biallelic mutations in a base excision repair gene. The MUTYH glycosylase plays an important role in preventing mutations associated with 8-oxoguanine (OG) by removing adenine residues that have been misincorporated opposite OG. MAP-associated mutations are present throughout MUTYH, with a large number coding for missense variations. To date the available information on the functional properties of MUTYH variants is conflicting. In this study, a kinetic analysis of the adenine glycosylase activity of MUTYH and several variants was undertaken using a correction for active fraction to control for differences due to overexpression and purification. Using these methods, the rate constants for steps involved in the adenine removal process were determined for the MAP variants Y165C, G382D, P391L and Q324R MUTYH. Under single-turnover conditions, the rate of adenine removal for these four variants was found to be 30-40% of WT MUTYH. In addition, the ability of MUTYH and the variants to suppress mutations and complement for the absence of MutY in Escherichia coli was assessed using rifampicin resistance assays. The presence of WT and Q324R MUTYH resulted in complete suppression of the mutation frequency, while G382D MUTYH showed reduced ability to suppress the mutation frequency. In contrast, the mutation frequency observed upon expression of P391L and Y165C MUTYH were similar to the controls, suggesting no activity toward preventing DNA mutations. Notably, though all variations studied herein resulted in similar reductions in adenine glycosylase activity, the effects in the bacterial complementation are quite different. This suggests that the consequences of a specific amino acid variation on overall repair in a cellular context may be magnified.
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Affiliation(s)
- Sucharita Kundu
- Department of Chemistry, University of California Davis, One Shields Avenue, Davis, CA 95616, USA
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20
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Sidorenko VS, Zharkov DO. Role of base excision repair DNA glycosylases in hereditary and infectious human diseases. Mol Biol 2008. [DOI: 10.1134/s0026893308050166] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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21
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Baute J, Depicker A. Base excision repair and its role in maintaining genome stability. Crit Rev Biochem Mol Biol 2008; 43:239-76. [PMID: 18756381 DOI: 10.1080/10409230802309905] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10(4) events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.
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Affiliation(s)
- Joke Baute
- Department of Plant Systems Biology, Flanders Institute for Biotechnology, Gent, Belgium
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22
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Wu F, Zhang Z, Wan J, Gu S, Liu W, Jin X, Xia Z. Genetic polymorphisms in hMTH1, hOGG1 and hMYH and risk of chronic benzene poisoning in a Chinese occupational population. Toxicol Appl Pharmacol 2008; 233:447-53. [PMID: 18848840 DOI: 10.1016/j.taap.2008.09.008] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2008] [Revised: 08/25/2008] [Accepted: 09/15/2008] [Indexed: 01/01/2023]
Abstract
Oxidative damage to DNA induced by benzene is an important mechanism of its genotoxicity, which leads to chronic benzene poisoning (CBP). Therefore, genetic variation in DNA repair genes may contribute to susceptibility to CBP in the exposed population. We hypothesized that single nucleotide polymorphisms (SNPs) in hMTH1, hOGG1 and hMYH genes are associated with risk of CBP. We genotyped SNPs at codon 83 of hMTH1, codon 326 of hOGG1, and codon 324 of hMYH in 152 CBP patients and 152 healthy workers occupationally exposed to benzene without poisoning manifestations. The genotypes were determined by polymerase chain reaction-restrained fragment length polymorphism (PCR-RFLP) technique. There were 2.51-fold [adjusted odds ratio (OR(adj)), 2.51; 95% CI, 1.14-5.49; P=0.02] and 2.49-fold (OR(adj), 2.49; 95% CI: 1.52-4.07; P<0.01) increased risk of CBP for individuals carrying genotypes of hMTH1 83Val/Met+Met/Met and hOGG1 326Cys/Cys, respectively. Compared with individuals carrying genotypes of hOGG1 326Cys/Cys and hMYH 324His/His at the same time, there was a 0.33-fold (OR(adj), 0.33; 95% CI: 0.15-0.72; P<0.05) decreased risk of CBP for those with genotypes of hOGG1 326Ser/Cys+Ser/Ser and hMYH 324His/Gln+Gln/Gln. In the smoking group, there was a 0.15-fold (OR(adj), 0.15; 95% CI, 0.03-0.68; P=0.01) decreased risk of CBP for subjects carrying genotypes of hMYH 324His/Gln+Gln/Gln compared with those of genotype of hMYH 324His/His. Therefore, our results suggested that polymorphisms at codons 83 of hMTH1 and codon 326 of hOGG1 might contribute to CBP in a Chinese occupational population.
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Affiliation(s)
- Fen Wu
- Department of Occupational Health and Toxicology, School of Public Health, Fudan University, Shanghai, China
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23
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Ali M, Kim H, Cleary S, Cupples C, Gallinger S, Bristow R. Characterization of mutant MUTYH proteins associated with familial colorectal cancer. Gastroenterology 2008; 135:499-507. [PMID: 18534194 PMCID: PMC2761659 DOI: 10.1053/j.gastro.2008.04.035] [Citation(s) in RCA: 80] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2008] [Revised: 04/07/2008] [Accepted: 04/30/2008] [Indexed: 01/04/2023]
Abstract
BACKGROUND & AIMS The human mutyh gene encodes a base excision repair protein that prevents G:C to T:A transversions in DNA. Biallelic mutations in this gene are associated with recessively inherited familial colorectal cancer. The aim of this study was to characterize the functional activity of mutant-MUTYH and single-nucleotide polymorphism (SNP)-MUTYH proteins involving familial colorectal cancer. METHODS MUTYH variants were cloned and assayed for their glycosylase and DNA binding activities using synthetic double-stranded oligonucleotide substrates by analyzing cleavage products by polyacrylamide gel electrophoresis. RESULTS In this study, we have characterized 9 missense/frameshift mutants and 2 SNPs for their DNA binding and repair activity in vitro. Two missense mutants (R260Q and G382D) were found to be partially active in both glycosylase and DNA binding, whereas 3 other missense mutants (Y165C, R231H, and P281L) were severely defective in both activities. All of the frameshift mutants (Y90X, Q377X, E466X, and 1103delC) were completely devoid of both glycosylase and DNA binding activities. One SNP (V22M) showed the same activity as wild-type MUTYH protein, but the other SNP (Q324H) was partially impaired in adenine removal. CONCLUSIONS This study of MUTYH mutants suggests that certain SNPs may be as partially dysfunctional in base excision repair as missense-MUTYH mutants and lead to colorectal carcinogenesis.
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Affiliation(s)
- Mohsin Ali
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada, Applied Molecular Oncology, Ontario Cancer Institute/Princess Margaret Hospital (University Health Network) and Departments of Radiation Oncology and Medical Biophysics, University of Toronto, Toronto, ON, Canada
| | - Hyeja Kim
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
| | - Sean Cleary
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
| | - Claire Cupples
- Dept. of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
| | - Steven Gallinger
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada,Corresponding author: Robert Bristow MD PhD FRCPC, Radiation Medicine Program, Princess Margaret Hospital (UHN), 610 University Avenue, Toronto, Ontario, CANADA M5G2M9, Tel: 416-946-2223; Fax: 416-946-4586;
| | - Robert Bristow
- Applied Molecular Oncology, Ontario Cancer Institute/Princess Margaret Hospital (University Health Network) and Departments of Radiation Oncology and Medical Biophysics, University of Toronto, Toronto, ON, Canada,Corresponding author: Robert Bristow MD PhD FRCPC, Radiation Medicine Program, Princess Margaret Hospital (UHN), 610 University Avenue, Toronto, Ontario, CANADA M5G2M9, Tel: 416-946-2223; Fax: 416-946-4586;
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24
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Küry S, Buecher B, Robiou-Du-Pont S, Scoul C, Colman H, Lelièvre B, Olschwang S, Houérou CL, Neel TL, Faroux R, Ollivry J, Lafraise B, Chupin LD, Bézieau S. The Thorough Screening of the MUTYH Gene in a Large French Cohort of Sporadic Colorectal Cancers. ACTA ACUST UNITED AC 2007; 11:373-9. [DOI: 10.1089/gte.2007.0029] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Affiliation(s)
- Sébastien Küry
- Laboratoire d'Etude de l'ADN, Faculté de Médecine de Nantes, 44035 France
- Institut des Maladies de l'Appareil Digestif et CIC INSERM, Centre Hospitalier Universitaire de Nantes 44093, France
| | - Bruno Buecher
- Institut des Maladies de l'Appareil Digestif et CIC INSERM, Centre Hospitalier Universitaire de Nantes 44093, France
- Service d'Hépato-Gastroentérologie, Centre Hospitalier Universitaire de Nantes 44093, France
| | - Sébastien Robiou-Du-Pont
- Laboratoire d'Etude de l'ADN, Faculté de Médecine de Nantes, 44035 France
- Institut des Maladies de l'Appareil Digestif et CIC INSERM, Centre Hospitalier Universitaire de Nantes 44093, France
| | - Catherine Scoul
- Laboratoire d'Etude de l'ADN, Faculté de Médecine de Nantes, 44035 France
- Institut des Maladies de l'Appareil Digestif et CIC INSERM, Centre Hospitalier Universitaire de Nantes 44093, France
| | - Hélène Colman
- Laboratoire d'Etude de l'ADN, Faculté de Médecine de Nantes, 44035 France
- Institut des Maladies de l'Appareil Digestif et CIC INSERM, Centre Hospitalier Universitaire de Nantes 44093, France
| | - Bénédicte Lelièvre
- Laboratoire d'Etude de l'ADN, Faculté de Médecine de Nantes, 44035 France
- Institut des Maladies de l'Appareil Digestif et CIC INSERM, Centre Hospitalier Universitaire de Nantes 44093, France
| | - Sylviane Olschwang
- Département d'Oncologie Génétique, Prévention et Dépistage, Institut Paoli-Calmettes, Marseille 13273, France
| | - Claire Le Houérou
- Institut des Maladies de l'Appareil Digestif et CIC INSERM, Centre Hospitalier Universitaire de Nantes 44093, France
| | - Tanguy Le Neel
- Laboratoire d'Etude de l'ADN, Faculté de Médecine de Nantes, 44035 France
- Biofortis, Nantes 44200, France
| | - Roger Faroux
- Service d'Hépato-Gastroentérologie, Centre Hospitalier Universitaire de la Roche-Sur-Yon, 85000 France
| | - Jean Ollivry
- Association des Gastroentérologues de Vendée, 85300 Challans, France
| | | | | | - Stéphane Bézieau
- Laboratoire d'Etude de l'ADN, Faculté de Médecine de Nantes, 44035 France
- Institut des Maladies de l'Appareil Digestif et CIC INSERM, Centre Hospitalier Universitaire de Nantes 44093, France
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Abstract
Maintaining the chemical integrity of DNA in the face of assault by oxidizing agents is a constant challenge for living organisms. Base-excision repair has an important role in preventing mutations associated with a common product of oxidative damage to DNA, 8-oxoguanine. Recent structural studies have shown that 8-oxoguanine DNA glycosylases use an intricate series of steps to locate and excise 8-oxoguanine lesions efficiently against a high background of undamaged bases. The importance of preventing mutations associated with 8-oxoguanine is shown by a direct association between defects in the DNA glycosylase MUTYH and colorectal cancer. The properties of other guanine oxidation products and the associated DNA glycosylases that remove them are now also being revealed.
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Affiliation(s)
- Sheila S David
- Department of Chemistry, University of California at Davis, 1 Shields Avenue, Davis, California 95616, USA.
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26
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Bai H, Grist S, Gardner J, Suthers G, Wilson TM, Lu AL. Functional characterization of human MutY homolog (hMYH) missense mutation (R231L) that is linked with hMYH-associated polyposis. Cancer Lett 2006; 250:74-81. [PMID: 17081686 PMCID: PMC1907362 DOI: 10.1016/j.canlet.2006.09.016] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2006] [Accepted: 09/22/2006] [Indexed: 12/23/2022]
Abstract
The MutY homolog (MYH) can excise adenines misincorporated opposite to guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG) during DNA replication; thereby preventing G:C to T:A transversions. Germline mutations in the human MYH gene are associated with recessive inheritance of colorectal adenomatous polyposis (MAP). Here, we characterize one newly identified MAP-associated MYH missense mutation (R231L) that lies adjacent to the putative hMSH6 binding domain. The R231L mutant protein has severe defects in A/GO binding and in adenine glycosylase activities. The mutant fails to complement mutY-deficiency in Escherichia coli, but does not affect binding to hMSH6. These data support the role of the hMYH pathway in carcinogenesis.
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Affiliation(s)
- Haibo Bai
- Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD 21201, USA
| | - Scott Grist
- Department of Haematology and Genetic Pathology, Flinders Medical Centre and Flinders University, Adelaide, South Australia, 5042
| | - Justin Gardner
- Department of Haematology and Genetic Pathology, Flinders Medical Centre and Flinders University, Adelaide, South Australia, 5042
| | - Graeme Suthers
- South Australian Familial Cancer Service, Women’s and Children’s Hospital, Adelaide, South Australia, 5042
| | - Teresa M. Wilson
- Department of Radiation Oncology, University of Maryland, Baltimore, MD 21201, USA
- The University of Maryland Greenebaum Cancer Center, Baltimore, MD 21201, USA
| | - A-Lien Lu
- Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD 21201, USA
- The University of Maryland Greenebaum Cancer Center, Baltimore, MD 21201, USA
- * Corresponding author. Tel.: +1-410-706-4356; Fax: 410-706-1787. E-mail address: (A-L. Lu)
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Berti PJ, McCann JAB. Toward a detailed understanding of base excision repair enzymes: transition state and mechanistic analyses of N-glycoside hydrolysis and N-glycoside transfer. Chem Rev 2006; 106:506-55. [PMID: 16464017 DOI: 10.1021/cr040461t] [Citation(s) in RCA: 213] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Affiliation(s)
- Paul J Berti
- Department of Chemistry, McMaster University, Hamilton, Ontario, Canada.
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Takao M, Yasui A. DNA repair initiated by glycosylases in the nucleus and mitochondria of mammalian cells; how our cells respond to a flood of oxidative DNA damage. ACTA ACUST UNITED AC 2005. [DOI: 10.1016/j.descs.2005.06.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Nohmi T, Kim SR, Yamada M. Modulation of oxidative mutagenesis and carcinogenesis by polymorphic forms of human DNA repair enzymes. Mutat Res 2005; 591:60-73. [PMID: 16081110 DOI: 10.1016/j.mrfmmm.2005.03.033] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2005] [Revised: 03/24/2005] [Accepted: 03/25/2005] [Indexed: 11/15/2022]
Abstract
Chromosome DNA is continuously exposed to various endogenous and exogenous mutagens. Among them, oxidation is one of the most common threats to genetic stability, and multiple DNA repair enzymes protect chromosome DNA from the oxidative damage. In Escherichia coli, three repair enzymes synergistically reduce the mutagenicity of oxidized base 8-hydroxy-guanine (8-OH-G). MutM DNA glycosylase excises 8-OH-G from 8-OH-G:C pairs in DNA and MutY DNA glycosylase removes adenine incorporated opposite template 8-OH-G during DNA replication. MutT hydrolyzes 8-OH-dGTP to 8-OH-dGMP in dNTP pool, thereby reducing the chance of misincorporation of 8-OH-dGTP by DNA polymerases. Simultaneous inactivation of MutM and MutY dramatically increases the frequency of spontaneous G:C to T:A mutations, and the deficiency of MutT leads to the enhancement of T:A to G:C transversions more than 1000-fold over the control level. In humans, the functional homologues of MutM, MutY and MutT, i.e., OGG1, MUTYH (MYH) and MTH1, contribute to the protection of genomic DNA from oxidative stress. Interestingly, several polymorphic forms of these proteins exist in human populations, and some of them are suggested to be associated with cancer susceptibility. Here, we review the polymorphic forms of OGG1, MUTYH and MTH1 involved in repair of 8-OH-G and 8-OH-dGTP, and discuss the significance of the polymorphisms in the maintenance of genomic integrity. We also summarize the polymorphic forms of human DNA polymerase eta, which may be involved in damage tolerance and mutagenesis induced by oxidative stress.
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Affiliation(s)
- Takehiko Nohmi
- Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
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Pope MA, Chmiel NH, David SS. Insight into the functional consequences of hMYH variants associated with colorectal cancer: distinct differences in the adenine glycosylase activity and the response to AP endonucleases of Y150C and G365D murine MYH. DNA Repair (Amst) 2005; 4:315-25. [PMID: 15661655 DOI: 10.1016/j.dnarep.2004.10.003] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2004] [Indexed: 02/08/2023]
Abstract
Escherichia coli MutY and its eukaryotic homologues play an important role in preventing mutations by removing adenine from 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A mismatches. It has recently been demonstrated that inherited biallelic mutations in the genes encoding the human homologue of MutY (hMYH) are correlated with a genetic predisposition for multiple colorectal adenomas and carcinomas. The two most common hMYH variants found in patients with colorectal cancer are Y165C and G382D. In this study, we examined the equivalent variants in the murine MutY homologue (mMYH), Y150C and G365D. The Y150C mMYH enzyme showed a large decrease in the rate of adenine removal from both OG:A- and G:A-containing substrates, while G365D mMYH showed a decrease in the ability to catalyze adenine removal only with a G:A-containing substrate. Both mMYH variants exhibit a significantly decreased affinity for duplexes containing noncleavable 2'-deoxyadenosine analogues. In addition, the human apurinic/apyrimidinic endonuclease (Ape1) stimulated product formation by wild-type and G365D mMYH with an OG:A substrate under conditions of multiple-turnover ([E]<[S]). In contrast, the presence of Ape1 nearly completely inhibited adenine removal by Y150C mMYH from the OG:A mismatch substrate. The more deleterious effect of Ape1 on the glycosylase activity of Y150C relative to G365D mMYH correlated with the more compromised binding affinity of Y150C to substrate analogue duplexes. These results suggest that the equivalent hMYH variants may be significantly compromised in substrate targeting in vivo due to a decrease in binding to substrate DNA; moreover, competition with other DNA binding proteins may further reduce the effective adenine glycosylase activity in vivo.
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Affiliation(s)
- Mary Ann Pope
- Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112, USA
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Parker AR, Sieber OM, Shi C, Hua L, Takao M, Tomlinson IP, Eshleman JR. Cells with pathogenic biallelic mutations in the human MUTYH gene are defective in DNA damage binding and repair. Carcinogenesis 2005; 26:2010-8. [PMID: 15987719 DOI: 10.1093/carcin/bgi166] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Inherited biallelic mutations in the human MUTYH gene are responsible for the recessive syndrome--adenomatous colorectal polyposis (MUTYH associated polyposis, MAP)--which significantly increases the risk of colorectal cancer (CRC). Defective MUTYH activity causes G:C to T:A transversions in tumour APC and other genes thereby altering genomic integrity. We report that of the four established cell lines, derived from patients with the MAP phenotype and containing biallelic MUTYH mutations, three contain altered expressions of MUTYH protein (MUTYH Y165C(-/-), MUTYH 1103delC/G382D and MUTYH Y165C/G382D but not MUTYH G382D(-/-)), but that all four cell lines have wild type levels of MUTYH mRNA. Mutant MUTYH proteins in these four cell lines possess significantly lowered binding and cleavage activities with heteroduplex oligonucleotides containing A.8-oxoG and 8-oxoA.G mispairs. Transfection of mitochondrial or nuclear MUTYH cDNAs partially correct altered MUTYH expression and activity in these defective cell lines. Finally, we surprisingly find that defective MUTYH may not alter cell survival after hydrogen peroxide and menadione treatments. The Y165C and 1103delC mutations significantly reduce MUTYH protein stability and thus repair activity, whereas the G382D mutation produces dysfunctional protein only suggesting different functional molecular mechanisms by which the MAP phenotype may contribute to the development of CRC.
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Affiliation(s)
- Antony R Parker
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA.
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Bai H, Jones S, Guan X, Wilson TM, Sampson JR, Cheadle JP, Lu AL. Functional characterization of two human MutY homolog (hMYH) missense mutations (R227W and V232F) that lie within the putative hMSH6 binding domain and are associated with hMYH polyposis. Nucleic Acids Res 2005; 33:597-604. [PMID: 15673720 PMCID: PMC548354 DOI: 10.1093/nar/gki209] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2005] [Accepted: 01/06/2005] [Indexed: 12/12/2022] Open
Abstract
The base excision repair DNA glycosylase MutY homolog (MYH) is responsible for removing adenines misincorporated into DNA opposite guanine or 7,8-dihydro-8-oxo-guanine (8-oxoG), thereby preventing G:C to T:A mutations. Biallelic germline mutations in the human MYH gene predispose individuals to multiple colorectal adenomas and carcinoma. We have recently demonstrated that hMYH interacts with the mismatch repair protein hMSH6, and that the hMSH2/hMSH6 (hMutSalpha) heterodimer stimulates hMYH activity. Here, we characterize the functional effect of two missense mutations (R227W and V232F) associated with hMYH polyposis that lie within, or adjacent to, the putative hMSH6 binding domain. Neither missense mutation affects the physical interaction between hMYH and hMSH6. However, hMYH(R227W) has a severe defect in A/8-oxoG binding and glycosylase activities, while hMYH(V232F) has reduced A/8-oxoG binding and glycosylase activities. The glycosylase activity of the V232F mutant can be partially stimulated by hMutSalpha but cannot be restored to the wild-type level. Both mutants also fail to complement mutY-deficiency in Escherichia coli. These data define the pathogenic mechanisms underlying two further hMYH polyposis-associated mutations.
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Affiliation(s)
- Haibo Bai
- Department of Biochemistry and Molecular Biology, University of MarylandBaltimore, MD, USA
| | - Siân Jones
- Institute of Medical Genetics, Cardiff UniversityHeath Park, Cardiff, CF14 4XN, UK
| | - Xin Guan
- Department of Biochemistry and Molecular Biology, University of MarylandBaltimore, MD, USA
| | - Teresa M. Wilson
- Department of Radiation Oncology, University of MarylandBaltimore, MD 21201, USA
- The University of Maryland Greenebaum Cancer CenterBaltimore, MD, USA
| | - Julian R. Sampson
- Institute of Medical Genetics, Cardiff UniversityHeath Park, Cardiff, CF14 4XN, UK
| | - Jeremy P. Cheadle
- Institute of Medical Genetics, Cardiff UniversityHeath Park, Cardiff, CF14 4XN, UK
| | - A-Lien Lu
- Department of Biochemistry and Molecular Biology, University of MarylandBaltimore, MD, USA
- The University of Maryland Greenebaum Cancer CenterBaltimore, MD, USA
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Zhou HJ, Lin JS, Zhou XM, Li PY, Tao LW. Expression of DNA repair enzyme hMTH1 mRNA and its protein in hepatocellular carcinoma. Shijie Huaren Xiaohua Zazhi 2004; 12:2057-2060. [DOI: 10.11569/wcjd.v12.i9.2057] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the expression of DNA repair enzyme hMTH1 mRNA and protein in hepatocellular carcinoma (HCC) tissues, normal liver cell and hepatoma cell lines, and to investigate their function in the progress of HCC.
METHODS: Expression of hMTH1 mRNA in matched HCC tissues(33 cases)/surrounding tissues of HCC (33 cases), normal liver cell L-02 and hepatoma cell lines SMMC7721 HepG2 were detected by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). hMTH1 protein was detected in corresponding HT tissues (17 cases HT) as well as their surrounding liver tissues (17 cases HST) by immunohistochemistry.
RESULTS: The expression level of hMTH1 mRNA in HT was higher than that in HST (t = 2.424 , P = 0.021<0.05). The expression level of hMTH1 mRNA in two hepatoma cell lines was higher than that in normal liver cell line (F = 6.810, P = 0.009<0.01). The expression of hMTH1 mRNA in SMMC7721 was similar to that in HepG2. hMTH1 protein was 88.2% (15 of 17) positive in HT and 82.4% (14 of 17) in HST and was mainly distributed in hepatocytesi cytoplasm. The protein level of hMTH1 in HCC tissues was correspondingly higher than in their surrounding tissues (t = 2.618, P = 0.019<0.05).
CONCLUSION: Overexpression of hMTH1 mRNA and its protein in the liver tissues of HCC and hepatoma cell lines is found.
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