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Induction and repair of clustered DNA damage sites after exposure to ionizing radiation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. (Bo Stenerlöw)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The mechanisms that maintain genomic stability safeguard cells from constant DNA damage produced by endogenous and external stressors. Therefore, this thesis aimed to specifically address questions regarding the requirement and involvement of DNA repair proteins in the repair of various types of radiation-induced DNA damage.

The first aim was to determine whether the phosphorylation of DNA-PKcs, a major kinase involved in non-homologous end joining pathway, can be utilized to score the DNA double-strand break (DSB) content in cells. DNA-PKcs phosphorylated (pDNA-PKcs) at T2609 was more sensitive to the cellular DSB content than ɣH2AX, as analyzed by flow cytometry. Further, pDNA-PKcs at T2609 could discriminate between DSB repair-compromised and normal cells, confirming that the pDNA-PKcs can be used as a DSB repair marker. In paper II, the DSB repair was assessed in cells with reduced levels of DNA-PKcs. The reduction in DNA-PKcs resulted in decreased cell survival and unaffected DSB repair. These results clearly indicate that DNA-PKcs plays an additional role in promoting cell survival in addition to its function in DSB repair.

The second part of the thesis focused on the characterization of complex DNA damage. DNA damage was investigated after exposure to α-particles originating from Ra-223. The Ra-223 treatment induced a nonrandom DSB distribution consistent with damage induced by high-linear energy transfer radiation. The exposure to Ra-223 significantly reduced cell survival in monolayers and 3D cell structures. The last paper unraveled the fate of heat-sensitive clustered DNA damage site (HSCS) repair in cells. HSCS repair was independent of DSB repair, and these lesions did not contribute to the generation of additional DSBs during repair. Prolonged heating of DNA at relatively low temperatures induced structural changes in the DNA that contributed to the production of DNA artifacts.

In conclusion, these results demonstrate that DNA-PKcs can be used to monitor DSB repair in cells after exposure to ionizing radiation. However, the functions of DNA-PKcs are not limited to DSB repair, as it can promote cell survival through other mechanisms. The complexity of the DNA damage produced by high-LET radiation is a major contributor to cell death. However, not all clusters produced in irradiated cells are converted into DSBs during repair.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 54
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1548
Keywords [en]
NHEJ, DSB repair, clustered DNA damage, DNA repair, DNA-PKcs, HSCS, Ra-223, ionizing radiation
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-378721ISBN: 978-91-513-0591-2 (print)OAI: oai:DiVA.org:uu-378721DiVA, id: diva2:1294853
Public defence
2019-04-29, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-04-04 Created: 2019-03-08 Last updated: 2019-05-07
List of papers
1. Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity
Open this publication in new window or tab >>Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity
2017 (English)In: Radiation Research, ISSN 0033-7587, E-ISSN 1938-5404, Vol. 188, no 6, p. 597-604Article in journal (Refereed) Published
Abstract [en]

Uncontrolled generation of DNA double-strand breaks (DSBs) in cells is regarded as a highly toxic event that threatens cell survival. Radiation-induced DNA DSBs are commonly measured by pulsed-field gel electrophoresis, microscopic evaluation of accumulating DNA damage response proteins (e.g., 53BP1 or gamma-H2AX) or flow cytometric analysis of gamma-H2AX. The advantage of flow cytometric analysis is that DSB formation and repair can be studied in relationship to cell cycle phase or expression of other proteins. However, gamma-H2AX is not able to monitor repair kinetics within the first 60 min postirradiation, a period when most DSBs undergo repair. A key protein in non-homologous end joining repair is the catalytic subunit of DNA-dependent protein kinase. Among several phosphorylation sites of DNA-dependent protein kinase, the threonine at position 2609 (T2609), which is phosphorylated by ataxia telangiectasia mutated (ATM) or DNA-dependent protein kinase catalytic subunit itself, activates the end processing of DSB. Using flow cytometry, we show here that phosphorylation at T2609 is faster in response to DSBs than gamma-H2AX. Furthermore, flow cytometric analysis of T2609 resulted in a better representation of fast repair kinetics than analysis of gamma-H2AX. In cells with reduced ligase IV activity, and wild-type cells where DNA-dependent protein kinase activity was inhibited, the reduced DSB repair capacity was observed by T2609 evaluation using flow cytometry. In conclusion, flow cytometric evaluation of DNA-dependent protein kinase T2609 can be used as a marker for early DSB repair and gives a better representation of early repair events than analysis of gamma-H2AX.

Place, publisher, year, edition, pages
RADIATION RESEARCH SOC, 2017
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-343567 (URN)10.1667/RR14693.1 (DOI)000416744600001 ()
Funder
Swedish Cancer SocietySwedish Radiation Safety Authority
Available from: 2018-03-02 Created: 2018-03-02 Last updated: 2019-03-08Bibliographically 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, p. 1-10Article 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.

Keywords
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: 2019-03-08Bibliographically approved
3. The α-emitter Ra-223 induces clustered DNA damage and significantly reduces cell survival
Open this publication in new window or tab >>The α-emitter Ra-223 induces clustered DNA damage and significantly reduces cell survival
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The bone-seeking radiopharmaceutical Xofigo (Radium-223 dichloride) has demonstrated both extended survival and palliative effects in treatment of bone metastases in patients with prostate cancer. The alpha-particle emitter Ra-223, administered as Ra-223 dichloride, targets regions undergoing active bone remodeling and strongly binds hydroxyapatite found in bone. However, the mechanisms mediating toxicity and properties of Ra-223 binding to hydroxyapatite are not fully understood. In the current study, we show that the alpha-particles originating from the Ra-223 decay chain produce a track-like distribution of the DNA damage response proteins 53BP1 and ɣH2AX and induce high amounts of clustered DNA double-strand breaks in prostate cancer cell nuclei. The Ra-223 treatment inhibited growth of prostate cancer cells, grown in 2D- and 3D- models in vitro, independent of prostate cancer cell type and androgen receptor variant 7 (ARv7) expression. The rapid binding with a high affinity of Ra-223 to bone structures was verified in an in silico assay (KD= 19.2 ± 6.5 e-18) and almost no dissociation was detected within 24 hours. Importantly, there was no significant uptake of Ra-223 in cells. Further, we demonstrate the importance of the local dose-distribution of this treatment; there was more than 100-fold increase in cell killing when Ra-223 was attached to the bone-like hydroxyapatite structure, compared to when the radioactivity was distributed in the cell growth media. However, independent of the exposure condition, the high cell killing efficacy of the Ra-223 was attributed to the clustered DNA damaged sites induced by the released α-particles.

Keywords
Prostate cancer, ARv7, DNA damage, Ra-223, high-LET
National Category
Radiology, Nuclear Medicine and Medical Imaging
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-378720 (URN)
Available from: 2019-03-08 Created: 2019-03-08 Last updated: 2019-03-08
4. Removal of heat-sensitive clustered damaged DNA sites is independent of double-strand break repair
Open this publication in new window or tab >>Removal of heat-sensitive clustered damaged DNA sites is independent of double-strand break repair
2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 12, article id e0209594Article in journal (Refereed) Published
Abstract [en]

DNA double-strand breaks (DSBs) are the most deleterious lesions that can arise in cells after ionizing radiation or radiometric drug treatment. In addition to prompt DSBs, DSBs may also be produced during repair, evolving from a clustered DNA damaged site, which is composed of two or more distinct lesions that are located within two helical turns. A specific type of cluster damage is the heat-sensitive clustered site (HSCS), which transforms into DSBs upon treatment at elevated temperatures. The actual lesions or mechanisms that mediate the HSCS transformation into DSBs are unknown. However, there are two possibilities; either these lesions are transformed into DSBs due to DNA lesion instability, e.g., transfer of HSCS into single-strand breaks (SSBs), or they are formed due to local DNA structure instability, e.g., DNA melting, where two SSBs on opposite strands meet and transform into a DSB. The importance of these processes in living cells is not understood, but they significantly affect estimates of DSB repair capacity. In this study, we show that HSCS removal in human cells is not affected by defects in DSB repair or inhibition of DSB repair. Under conditions where rejoining of prompt DSBs was almost completely inhibited, heat-sensitive DSBs were successfully rejoined, without resulting in increased DSB levels, indicating that HSCS do not transfer into DSB in cells under physiological conditions. Furthermore, analysis by atomic force microscopy suggests that prolonged heating of chromosomal DNA can induce structural changes that facilitate transformation of HSCS into DSB. In conclusion, the HSCS do not generate additional DSBs at physiological temperatures in human cells, and the repair of HSCS is independent of DSB repair.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-374120 (URN)10.1371/journal.pone.0209594 (DOI)000454621900032 ()30592737 (PubMedID)
Funder
Swedish Cancer Society, CAN2014/661Swedish Cancer Society, CAN2016/649Swedish Radiation Safety Authority, SSM2017-2374Swedish Radiation Safety Authority, SSM2018-2181
Available from: 2019-01-23 Created: 2019-01-23 Last updated: 2019-03-08Bibliographically approved

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