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Role of MTH1 and MYH proteins in genotoxic effects of radiation
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
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 [en]
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: urn:nbn:se:su:diva-113835ISBN: 978-91-7649-099-0 (print)OAI: oai:DiVA.org:su-113835DiVA: diva2:788754
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
List of papers
1. Mutations and chromosomal aberrations in hMTH1-transfected and non-transfected TK6 cells after exposure to low dose rates of gamma radiation
Open this publication in new window or tab >>Mutations and chromosomal aberrations in hMTH1-transfected and non-transfected TK6 cells after exposure to low dose rates of gamma radiation
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2014 (English)In: Radiation and Environmental Biophysics, ISSN 0301-634X, E-ISSN 1432-2099, Vol. 53, no 2, 417-425 p.Article in journal (Refereed) Published
Abstract [en]

The aim of the present study was to analyse the dose rate effect of gamma radiation at the level of mutations, chromosomal aberrations, and cell growth in TK6 cells with normal as well as reduced levels of hMTH1 protein. TK6 cells were exposed to gamma radiation at dose rates ranging from 1.4 to 30.0 mGy/h (chronic exposure) as well as 24 Gy/h (acute exposure). Cell growth, frequency of thymidine kinase mutants, and of chromosomal aberrations in painted chromosomes 2, 8, and 14 were analysed. A decline in cell growth and an increase in unstable-type chromosomal aberrations with increasing dose rate were observed in both cell lines. A dose rate effect was not seen on mutations or stable-type chromosomal aberrations in any of the two cell lines. Reduction in the hMTH1 protein does not influence the sensitivity of TK6 cells to gamma radiation. This result fits well with data of others generated with the same cell line.

Keyword
Mutations, Aberrations, Oxidative stress, Gamma radiation, Dose rate effect, hMTH1
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-113327 (URN)10.1007/s00411-014-0521-1 (DOI)000334998200020 ()24549366 (PubMedID)
Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2017-12-05Bibliographically approved
2. Cooperation of MTH1 and MYH proteins in response to oxidative stress induced by chronic γ-radiation
Open this publication in new window or tab >>Cooperation of MTH1 and MYH proteins in response to oxidative stress induced by chronic γ-radiation
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Mutation in the MYH gene has been suggested as a risk factor for colorectal cancer. MYH plays an important role in mutation avoidance induced by 8-oxo-G:A mispairs which may occur during oxidative stress. Oxidative stress has been suggested to be involved in several human disorders. In the present study we have exposed human lymphoblastoid TK6 cells to very low dose rates of γ-radiation to induce chronic oxidative stress. The aim of the study is to investigate the protective role of MYH and MTH1 against mutagenicity and cytotoxicity induced by chronic radiation in the cells with knockdown MYH or knockdown MYH/MTH1. The levels of MYH and/or MTH1 were knockdown permanently in cells using shRNA. Wild type and knockdown cells were exposed to chronic γ-radiation with the dose rates ranged from 1.4 to 30 mGy/h during growth. The cells were also subjected to an acute high dose rate. Growth rate, clonogenic survival and mutant frequency were analyzed in all cell types. A reduced level of cell growth and survival as well as an increased mutant frequency were observed in cells lacking both MYH and MTH1 proteins as compared to cells lacking only MYH and wild type cells. In conclusion, our results suggest that MYH and MTH1 cooperatively respond to oxidative stress induced by chronic radiation in human cells.

Keyword
MYH, MTH1, oxidative stress, mutation rate, low dose rate radiation, ionizing radiation
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-114309 (URN)
Available from: 2015-02-26 Created: 2015-02-26 Last updated: 2016-01-29Bibliographically approved
3. Adaptive response induced by different dose rates of γ-radiation in MCF-10A cells
Open this publication in new window or tab >>Adaptive response induced by different dose rates of γ-radiation in MCF-10A cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A phenomenon in which exposure to a low adapting dose of radiation makes cells more resistant to the effects of a subsequent high dose exposure is termed radio-adaptive response. Adaptive response could hypothetically reduce the risk of late adverse effects of chronic or acute radiation exposures in humans. Understanding the underlying mechanisms of such responses are of relevance for radiation protection as well as for the clinical applications of radiation in medicine. However, due to the variability of responses depending on the model system and radiation condition, there is a need to further study under what conditions adaptive response can be induced. In this study, we analyzed if there is a dose rate dependence for the adapting dose, assuming that the adapting dose induces DNA response/repair pathways that are dose rate dependent. MCF-10A cells were exposed to a 50 mGy adapting dose administered acutely (24 Gy/h) or chronically (1.4 or 4.1 mGy/h). After 2 hours incubation time cells were exposed to a challenging dose of 1 to 5 Gy. Adaptive response was absent at the level of clonogenic survival and present at the level of mutations only at 1.4 mGy/h administration of adapting dose. Overall, no dose rate effect of the adapting dose was observed at the level of clonogenic survival, while it was seen at the frequency of mutants. On the other hand, a dose rate effect was absent at the level of mutant frequency.

Keyword
Adaptive response, low dose rate, ionizing radiation, mutation rate
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-114310 (URN)
Available from: 2015-02-26 Created: 2015-02-26 Last updated: 2016-01-29Bibliographically approved
4. 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
Open this publication in new window or tab >>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
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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.

Keyword
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:nbn:se:su:diva-66540 (URN)10.1016/j.mrfmmm.2011.07.005 (DOI)000295664200003 ()
Note

authorCount :8

Available from: 2011-12-27 Created: 2011-12-20 Last updated: 2017-12-08Bibliographically approved

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