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Reduction of 8-oxodGTP in the nucleotide pool by hMTH1 leads to reduction in mutations in the human lymphoblastoid cell line TK6 exposed to UVA
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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2011 (English)In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 715, no 1-2, 13-18 p.Article in journal (Refereed) Published
Abstract [en]

UVA has been suggested to play an important role in UV-induced mutagenesis. The mechanisms by which UVA induces mutations are still a matter of debate. Our aim was to investigate the protective capacity of hMTH1, a nucleotide pool sanitization enzyme with 8-oxodGTPase activity. Human B lymphoblastoid cells were stably transfected with shRNA directed against hMTH1. Clonogenic survival, mutations, intracellular and extracellular levels of 8-oxodG (8-oxo-7, 8-dihydro-2'-deoxyguanosine) and dG in the nucleotide pool of UVA-irradiated transfected and non-transfected cells were investigated. Mutations were determined in the thymidine kinase locus. Intracellular 8-oxodG and dG were measured using a modified ELISA and HPLC, respectively, after extraction of the nucleotide pool and conversion of nucleotides to their corresponding nucleosides. 8-oxodG in the medium was measured using ELISA. UVA-induced mutations were significantly higher while the survival was slightly lower in transfected compared to non-transfected cells. The increased mutation rate in transfected cells at increased exposure correlated with enhanced levels of 8-oxodG in the nucleotide pool, and a somewhat reduced level of 8-oxodG in the medium. The results indicate that the nucleotide pool is a significant target for UVA-induced mutations and implicates that hMTH1 plays an important role in protecting cells from UVA-induced oxidative stress.

Place, publisher, year, edition, pages
2011. Vol. 715, no 1-2, 13-18 p.
Keyword [en]
Oxidative stress, hMTH1, Mutagenesis, UVA, 8-oxo-dG, 8-OH-dG, 8-oxo-dGTP, Reactive oxygen species
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-66540DOI: 10.1016/j.mrfmmm.2011.07.005ISI: 000295664200003OAI: oai:DiVA.org:su-66540DiVA: diva2:469784
Note

authorCount :8

Available from: 2011-12-27 Created: 2011-12-20 Last updated: 2017-12-08Bibliographically approved
In thesis
1. The role of MTH1 in ultraviolet radiation-induced mutagenesis
Open this publication in new window or tab >>The role of MTH1 in ultraviolet radiation-induced mutagenesis
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ultraviolet radiation (UVR) is known to be highly mutagenic. What types of DNA lesions that are induced by different UVR wavelengths are still a matter of debate. UVR induces mutagenesis mostly by the formation of photoproducts and the induction of reactive oxygen species (ROS). ROS can give rise to mutations via oxidation of nucleotides in the DNA or the nucleotide pool. Oxidized nucleotides in the nucleotide pool can thereby be incorporated into the DNA during replication and ultimately give rise to mutations. MTH1 however, dephosphorylates oxidized nucleotides in the nucleotide pool, in particular 8-oxo-dGTP and 2-OH-dATP, and inhibits their incorporation into the DNA.The aim of the present study was to investigate the role of MTH1 in mutagenesis and cytogenetic damage induced by UVR in a human lymphoblastoid TK6 cell line. The clonogenic survival, mutant frequency and micronucleus frequency were measured following exposure to UVA, UVB and UVC in MTH1-knockdown and wild-type TK6 cells. As a biomarker for oxidative damage the level of intracellular and extracellular 8-oxo-dG was measured in TK6 cells exposed to UVA. The mutational spectra of UVA-induced mutations at the thymidine kinase gene in MTH1-knockdown and wild-type TK6 cells were investigated.The results show that MTH1 protects against UVA and UVB mutagenesis significantly. MTH1, however, has been shown to offer no protection against UVR-induced cytogenetic damage and is therefore suggested to mainly inhibit mutagenesis. The mutational spectra show that GC>AT and AT>GC transitions are the dominant mutation types in cells exposed to UVA.In conclusion, MTH1 protects TK6 cells against mutagenesis induced by longer wavelengths of UVR. This indicates that the nucleotide pool is a significant target in mutagenesis for longer wavelengths of UVR. The type of mutations induced by UVA, GC>AT and AT>GC, can be formed by the incorporation of 2-OH-dATP from nucleotide pool into the DNA. UVA is therefore suggested to induce mutations by induction of oxidized nucleotides such as 2-OH-dATP.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2015. 46 p.
Keyword
ultraviolet radiation, MTH1, reactive oxygen species, 8-oxo-dGTP, 2-OH-dATP, micronucleus
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-113320 (URN)978-91-7649-096-9 (ISBN)
Public defence
2015-03-06, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2015-02-12 Created: 2015-01-28 Last updated: 2016-11-02Bibliographically approved
2. Role of MTH1 and MYH proteins in genotoxic effects of radiation
Open this publication in new window or tab >>Role of MTH1 and MYH proteins in genotoxic effects of radiation
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Humans are constantly exposed to different types of radiations. It has been suggested that low dose and low dose rate of γ-radiation as well as ultra violet A (UVA) induce oxidative stress in cells that may promote mutations. The mechanisms behind radiation-induced oxidative stress and its relation to genotoxicity and cancer induction are not well understood. In the majority of investigations, the DNA molecule has been studied as the target for mutations, however the results obtained in our group point out that DNA bases in the cytoplasm could also be a significant target. MTH1 and MYH are two of the key proteins of the repair pathway that prevent mutations arising from oxidized DNA bases. In this thesis, we studied the role of MTH1 and MYH in genotoxicity of UVA and γ-radiation. The adaptive response to low dose rates of γ-radiation was also investigated. MTH1 and/or MYH were knockdown in human lymphoblastoid TK6 cells. The clonogenic survival, mutant frequency and chromosomal aberration assays were performed following UVA or γ-radiation exposure. Our results indicated that acute exposure to UVA or γ-radiation affects cell survival and also increases the mutant frequency above the background. The mutant frequency in MTH1 deficient cells was higher than that in wild types after UVA exposure. Following γ-radiation exposure, a higher mutant frequency was observed in the MYH and MTH1 deficient cells, in comparison to either MYH or MTH1 deficient or wild type cells. No dose rate effect of γ-radiation for mutations was observed. An adaptive response to γ-radiation was observed at the mutation level in MCF-10A cells but not at the survival level. In summary, our results suggest that; a) MYH and MTH1 cooperatively protect cells against genotoxic effects of γ-radiation; b) MTH1 protects cells from UVA-induced mutations; c) low dose rates of γ-radiation may induce an adaptive response at the mutation level; d) there is no dose rate effect for γ-radiation at the mutation level.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2015. 45 p.
Keyword
MTH1, MYH, gamma radiation, UV radiation, oxidative stress, low dose rate ionizing radiation
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-113835 (URN)978-91-7649-099-0 (ISBN)
Public defence
2015-03-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Available from: 2015-03-09 Created: 2015-02-12 Last updated: 2015-03-18Bibliographically approved

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Fotouhi, AsalSkiöld, SaraShakeri Manesh, SaraOsterman-Golkar, SivWojcik, AndrzejJenssen, DagHarms-Ringdahl, MatsHaghdoost, Siamak
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