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Radiation response in human cells: DNA damage formation, repair and signaling
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1157
Keyword [en]
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
Identifiers
URN: urn:nbn:se:uu:diva-265137ISBN: 978-91-554-9397-4 (print)OAI: oai:DiVA.org:uu-265137DiVA: diva2:865624
Public defence
2015-12-16, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 14:00 (English)
Opponent
Supervisors
Available from: 2015-11-24 Created: 2015-10-22 Last updated: 2016-01-13
List of papers
1. Formation and repair of clustered damaged DNA sites in high LET irradiated cells
Open this publication in new window or tab >>Formation and repair of clustered damaged DNA sites in high LET irradiated cells
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.

Keyword
Clustered damage; DNA damage; DNA DSB repair; DNA repair; double-strand breaks; High LET
National Category
Basic Medicine Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-265135 (URN)10.3109/09553002.2015.1068463 (DOI)000365614800007 ()26136085 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2015-10-22 Created: 2015-10-22 Last updated: 2017-12-01Bibliographically approved
2. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair
Open this publication in new window or tab >>Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair
2014 (English)In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 769, 1-10 p.Article in journal (Refereed) Published
Abstract [en]

Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80-95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or gamma-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

Keyword
DNA repair, DNA-PKcs, Ionizing radiation, DNA-PK deficiency, NU7441
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-237292 (URN)10.1016/j.mrfmmm.2014.06.004 (DOI)000343625700001 ()
Available from: 2014-12-03 Created: 2014-12-01 Last updated: 2017-12-05Bibliographically approved
3. The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines
Open this publication in new window or tab >>The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines
Show others...
2014 (English)In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 35, no 4, 3525-3534 p.Article in journal (Refereed) Published
Abstract [en]

In response to ionizing radiation, several signaling cascades in the cell are activated to repair the DNA breaks, prevent apoptosis, and keep the cells proliferating. AKT is important for survival and proliferation and may also be an activating factor for DNA-PKcs and MRE11, which are essential proteins in the DNA repair process. AKT (PKB) is hyperactivated in several cancers and is associated with resistance to radiotherapy and chemotherapy. There are three AKT isoforms (AKT1, AKT2, and AKT3) with different expression patterns and functions in several cancer tumors. The role of AKT isoforms has been investigated in relation to radiation response and their effects on DNA repair proteins (DNA-PKcs and MRE11) in colon cancer cell lines. The knockout of AKT1 and/or AKT2 affected the radiation sensitivity, and a deficiency of both isoforms impaired the rejoining of radiation-induced DNA double strand breaks. Importantly, the active/phosphorylated forms of AKT and DNA-PKcs associate and exposure to ionizing radiation causes an increase in this interaction. Moreover, an increased expression of both DNA-PKcs and MRE11 was observed when AKT expression was ablated, yet only DNA-PKcs expression influenced AKT phosphorylation. Taken together, these results demonstrate a role for both AKT1 and AKT2 in radiotherapy response in colon cancer cells involving DNA repair capacity through the nonhomologous end joining pathway, thus suggesting that AKT in combination with DNA-PKcs inhibition may be used for radiotherapy sensitizing strategies in colon cancer.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-221446 (URN)10.1007/s13277-013-1465-9 (DOI)000334495900084 ()
Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-12-05Bibliographically approved
4. Role of DNA-PKcs in DNA damage response and cell cycle regulation
Open this publication in new window or tab >>Role of DNA-PKcs in DNA damage response and cell cycle regulation
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-265136 (URN)
Note

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. We have previously reported that suppression of DNA-PKcs sensitize cells to radiation without affecting repair. In the present study we used synchronized cells to unmask potential roles of DNA-PKcs in specific cell-cycle phases. siRNA was used to deplete DNA-PKcs to 10-15 % of normal levels and cell cycle progression and DSB repair was observed in synchronized cells irradiated at different cell cycle phases. Surprisingly, cells irradiated in G2 phase showed similar p-H3 frequency after 72 h as unirradiated control cells, irrespective of their DNA-PKcs status. When cells were irradiated in G1/S phase, a significant increase of mitotic cells in siDNA-PKcs treated cells was seen 72 h later. Further, irradiation in G1/ S phase caused initially (0-12h) the same number of DSB (53BP1 foci), however, over time (>24h) 53BP1 foci remained at relatively high levels in DNA-PKcs depleted cells, indicating presence of unrepaired DSB in the following G1. This suggest that DNA-PKcs has an important regulatory role in G1/S and a key function in mitosis.

Available from: 2015-10-22 Created: 2015-10-22 Last updated: 2016-01-13

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