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Formation and repair of clustered damaged DNA sites in high LET irradiated cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
Uppsala University, The Svedberg Laboratory.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
2015 (English)In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 91, no 10, 820-826 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: Radiation with high linear energy transfer (LET) produces clustering of DNA double-strand breaks (DSB) as well as non-DSB lesions. Heat-labile sites (HLS) are non-DSB lesions in irradiated cells that may convert into DSB at elevated temperature during preparation of naked DNA for electrophoretic assays and here we studied the initial formation and repair of these clustered damaged sites after irradiation with high LET ions.

MATERIALS AND METHODS: Induction and repair of DSB were studied in normal human skin fibroblast (GM5758) after irradiation with accelerated carbon and nitrogen ions at an LET of 125 eV/nm. DNA fragmentation was analyzed by pulsed-field gel electrophoresis (PFGE) and by varying the lysis condition we could differentiate between prompt DSB and heat-released DSB.

RESULTS: Before repair (t = 0 h), the 125 eV/nm ions produced a significant fraction of heat-released DSB, which appeared clustered on DNA fragments with sizes of 1 Mbp or less. These heat-released DSB increased the total number of DSB by 30-40%. This increase is similar to what has been found in low-LET irradiated cells, suggesting that the relative biological effectiveness (RBE) for DSB induction will not be largely affected by the lysis temperature. After 1-2 hours repair, a large fraction of DSB was still unrejoined but there was essentially no heat-released DSB present.

CONCLUSIONS: These results suggest that high LET radiation, as low LET gamma radiation, induces a significant fraction of heat-labile sites which can be converted into DSB, and these heat-released DSB may affect both induction yields and estimates of repair.

Place, publisher, year, edition, pages
2015. Vol. 91, no 10, 820-826 p.
Keyword [en]
Clustered damage; DNA damage; DNA DSB repair; DNA repair; double-strand breaks; High LET
National Category
Basic Medicine Radiology, Nuclear Medicine and Medical Imaging
URN: urn:nbn:se:uu:diva-265135DOI: 10.3109/09553002.2015.1068463ISI: 000365614800007PubMedID: 26136085OAI: oai:DiVA.org:uu-265135DiVA: diva2:862579
Swedish Cancer Society
Available from: 2015-10-22 Created: 2015-10-22 Last updated: 2016-01-25Bibliographically approved
In thesis
1. Radiation response in human cells: DNA damage formation, repair and signaling
Open this publication in new window or tab >>Radiation response in human cells: DNA damage formation, repair and signaling
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ionizing radiation induces a range of different DNA lesions. In terms of mutation frequency and mammalian cell survival, the most critical of these lesions is the DNA double-strand break (DSB). DSB left unrepaired or mis-repaired may result in chromosomal aberrations that can lead to permanent genetic changes or cell death. The complexity of the DNA damage and the capacity to repair the DSB will determine the fate of the cell. This thesis focuses on the DNA damage formation, repair and signaling after irradiation of human cells.

Radiation with high linear energy transfer (LET) produces clustered damaged sites in the DNA that are difficult for the cell to repair. Within these clustered sites, non-DSB lesions are formed that can be converted into a DSB and add to the damage complexity and affect DSB repair and the measurement. Heat-labile sites in DNA are converted into DSB at elevated temperatures. We show that heat-released DSB are formed post-irradiation with high-LET ions and increase the initial yield of DSB by 30%-40%, which is similar to yields induced by low-LET radiation.

DNA-PKcs, a central player in non-homologous end-joining (NHEJ), the major mammalian DSB repair pathway, has been found to be both up- and downregulated in different tumor types. In Paper II we show that low levels of DNA-PKcs lead to extreme radiosensitivity but, surprisingly, had no effect on the DSB repair. However, the fraction of cells in G2/M phase increased two-fold in cells with low levels of DNA-PKcs. The study continued in Paper IV, where cells were synchronized to unmask potential roles of DNA-PKcs in specific cell cycle phases. Irradiation of DNA-PKcs suppressed cells in the G1/S phase caused a delay in cell cycle progression and an increase in accumulation of G2 cells. Further, these cells showed defects in DNA repair, where a significant amount of 53BP1 foci remained after 72 h. This further strengthens the hypothesis that DNA-PKcs has a role in regulation of mitotic progression.

Several cellular signaling pathways are initiated in response to radiation. One of these downstream signaling proteins is AKT. We identified an interaction between DNA-PKcs and AKT. Knockouts of both AKT1 and AKT2 impaired DSB rejoining after radiation and low levels of DNA-PKcs increased radiosensitivity and decreased DNA repair further.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 52 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1157
DNA damage, DNA repair, DSB, NHEJ, DNA-PK, ionizing radiation, heat-labile sites
National Category
Other Basic Medicine Cell and Molecular Biology Cell Biology
Research subject
Biomedical Radiation Science
urn:nbn:se:uu:diva-265137 (URN)978-91-554-9397-4 (ISBN)
Public defence
2015-12-16, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 14:00 (English)
Available from: 2015-11-24 Created: 2015-10-22 Last updated: 2016-01-13

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