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The α-emitter Ra-223 induces clustered DNA damage and significantly reduces cell survival
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. (Bo Stenerlöw)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. (Göran Laurell)
Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden. (Sten Nilsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. (Bo Stenerlöw)
(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 [en]
Prostate cancer, ARv7, DNA damage, Ra-223, high-LET
National Category
Radiology, Nuclear Medicine and Medical Imaging
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-378720OAI: oai:DiVA.org:uu-378720DiVA, id: diva2:1294801
Available from: 2019-03-08 Created: 2019-03-08 Last updated: 2019-03-08
In thesis
1. Induction and repair of clustered DNA damage sites after exposure to ionizing radiation
Open this publication in new window or tab >>Induction and repair of clustered DNA damage sites after exposure to ionizing radiation
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
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:nbn:se:uu:diva-378721 (URN)978-91-513-0591-2 (ISBN)
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

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