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Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
2014 (engelsk)Inngår i: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 769, s. 1-10Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2014. Vol. 769, s. 1-10
Emneord [en]
DNA repair, DNA-PKcs, Ionizing radiation, DNA-PK deficiency, NU7441
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-237292DOI: 10.1016/j.mrfmmm.2014.06.004ISI: 000343625700001OAI: oai:DiVA.org:uu-237292DiVA, id: diva2:768298
Tilgjengelig fra: 2014-12-03 Laget: 2014-12-01 Sist oppdatert: 2019-03-08bibliografisk kontrollert
Inngår i avhandling
1. Radiation response in human cells: DNA damage formation, repair and signaling
Åpne denne publikasjonen i ny fane eller vindu >>Radiation response in human cells: DNA damage formation, repair and signaling
2015 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.  

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2015. s. 52
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1157
Emneord
DNA damage, DNA repair, DSB, NHEJ, DNA-PK, ionizing radiation, heat-labile sites
HSV kategori
Forskningsprogram
Biomedicinsk strålningsvetenskap
Identifikatorer
urn:nbn:se:uu:diva-265137 (URN)978-91-554-9397-4 (ISBN)
Disputas
2015-12-16, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 14:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2015-11-24 Laget: 2015-10-22 Sist oppdatert: 2018-01-10
2. Induction and repair of clustered DNA damage sites after exposure to ionizing radiation
Åpne denne publikasjonen i ny fane eller vindu >>Induction and repair of clustered DNA damage sites after exposure to ionizing radiation
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 54
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1548
Emneord
NHEJ, DSB repair, clustered DNA damage, DNA repair, DNA-PKcs, HSCS, Ra-223, ionizing radiation
HSV kategori
Forskningsprogram
Medicinsk vetenskap
Identifikatorer
urn:nbn:se:uu:diva-378721 (URN)978-91-513-0591-2 (ISBN)
Disputas
2019-04-29, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 13:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2019-04-04 Laget: 2019-03-08 Sist oppdatert: 2019-05-07

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