| 1. |
- Abramenkovs, Andris, et al.
(författare)
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Ra-223 induces clustered DNA damage and inhibits cell survival in several prostate cancer cell lines
- 2022
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Ingår i: Translational Oncology. - : Elsevier. - 1944-7124 .- 1936-5233. ; 26
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Tidskriftsartikel (refereegranskat)abstract
- The bone-seeking radiopharmaceutical Xofigo (Radium-223 dichloride) has demonstrated both extended sur-vival and palliative effects in treatment of bone metastases in prostate cancer. The alpha-particle emitter Ra-223, targets regions undergoing active bone remodeling and strongly binds to bone hydroxyapatite (HAp). However, the toxicity mechanism and properties of Ra-223 binding to hydroxyapatite are not fully understood. By exposing 2D and 3D (spheroid) prostate cancer cell models to free and HAp-bound Ra-223 we here studied cell toxicity, apoptosis and formation and repair of DNA double-strand breaks (DSBs). The rapid binding with a high affinity of Ra-223 to bone-like HAp structures was evident (KD= 19.2 x 10-18 M) and almost no dissociation was detected within 24 h. Importantly, there was no significant uptake of Ra-223 in cells. The Ra-223 alpha-particle decay produced track-like distributions of the DNA damage response proteins 53BP1 and gamma H2AX induced high amounts of clustered DSBs in prostate cancer cells and activated DSB repair through non-homologous end-joining (NHEJ). Ra-223 inhibited growth of prostate cancer cells, independent of cell type, and induced high levels of apoptosis. In summary, we suggest the high cell killing efficacy of the Ra-223 was attributed to the clustered DNA damaged sites induced by alpha-particles.
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| 2. |
- Bivehed, Erik, et al.
(författare)
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Visualizing DNA single- and double-strand breaks in the Flash comet assay by DNA polymerase-assisted end-labelling
- 2024
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 52:4
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Tidskriftsartikel (refereegranskat)abstract
- In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays) or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks. Graphical Abstract
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| 3. |
- Haag, Petra, et al.
(författare)
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Caspase-2 is a mediator of apoptotic signaling in response to gemtuzumab ozogamicin in acute myeloid leukemia
- 2022
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Ingår i: Cell Death Discovery. - : Springer Nature. - 2058-7716. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- The antibody conjugate gemtuzumab ozogamicin (GO; Mylotarg((R))) provides targeted therapy of acute myeloid leukemia (AML), with recent approvals for patients with CD33-positive disease at diagnosis or relapse, as monotherapy or combined with chemotherapeutics. While its clinical efficacy is well documented, the molecular routes by which GO induces AML cell death warrant further analyses. We have earlier reported that this process is initiated via mitochondria-mediated caspase activation. Here we provide additional data, focusing on the involvement of caspase-2 in this mechanism. We show that this enzyme plays an important role in triggering apoptotic death of human AML cells after exposure to GO or its active moiety calicheamicin. Accordingly, the caspase-2 inhibitor z-VDVAD-fmk reduced GO-induced caspase-3 activation. This finding was validated with shRNA and siRNA targeting caspase-2, resulting in reduced caspase-3 activation and cleavage of poly [ADP-ribose] polymerase 1 (PARP-1). We previously demonstrated that GO-induced apoptosis included a conformational change of Bax into a pro-apoptotic state. Present data reveal that GO-treatment also induced Bid cleavage, which was partially reduced by caspase-2 specific inhibition while the effect on GO-induced Bax conformational change remained unaltered. In mononuclear cells isolated from AML patients that responded to GO treatment in vitro, processing of caspase-2 was evident, whereas in cells from an AML patient refractory to treatment no such processing was seen. When assessing diagnostic samples from 22 AML patients, who all entered complete remission (CR) following anthracycline-based induction therapy, and comparing patients with long versus those with short CR duration no significant differences in baseline caspase-2 or caspase-3 full-length protein expression levels were found. In summary, we demonstrate that GO triggers caspase-2 cleavage in human AML cells and that the subsequent apoptosis of these cells in part relies on caspase-2. These findings may have future clinical implications.
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| 4. |
- Henthorn, Nicholas T., et al.
(författare)
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Mapping the Future of Particle Radiobiology in Europe : The INSPIRE Project
- 2020
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Ingår i: Frontiers in Physics. - : Frontiers Media SA. - 2296-424X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Particle therapy is a growing cancer treatment modality worldwide. However, there still remains a number of unanswered questions considering differences in the biological response between particles and photons. These questions, and probing of biological mechanisms in general, necessitate experimental investigation. The "Infrastructure in Proton International Research" (INSPIRE) project was created to provide an infrastructure for European research, unify research efforts on the topic of proton and ion therapy across Europe, and to facilitate the sharing of information and resources. This work highlights the radiobiological capabilities of the INSPIRE partners, providing details of physics (available particle types and energies), biology (sample preparation and post-irradiation analysis), and researcher access (the process of applying for beam time). The collection of information reported here is designed to provide researchers both in Europe and worldwide with the tools required to select the optimal center for their research needs. We also highlight areas of redundancy in capabilities and suggest areas for future investment.
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| 5. |
- Pour Khavari, Ali, 1985-
(författare)
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Role of oxidative stress response in radiosensitivity
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The quality of the ionizing radiation (IR) can be described in terms of its nature, photons or particles, and their corresponding energies. The energy is classified in terms of High or Low linear energy transfer that will produce a different distribution of DNA damage and other molecules in the cell either by direct action or indirect action. Indirect action leads to the production of reactive oxygen species (ROS) modifying nucleotides in DNA or free dNTPs. 8-oxo-dGTP is formed through ROS endogenously when there is an imbalance between the antioxidants defence systems and the production of ROS levels in favour of ROS, leading to an oxidative stress condition. Organisms, organs, and cell types show different degrees of radiosensitivity, and this thesis aimed to investigate the underlying mechanisms of IR induced oxidative stress and its relation with radiosensitivity.In previous studies, we identified proteins involved in radiation response with a focus on low dose radiation response. Cell models were established in which the expression of some protein/s was downregulated by knocking down/out using CRISPR/Cas9 or shRNA technology. The knockdown or knockout cells were exposed to different doses at low dose rates (LDR) or high dose rate (HDR) to investigate the role of these genes/proteins for survival (radiosensitivity), mutation induction, stress response, differentiation, etc. and they were subjected to further studies in this thesis.Publication I, cell lines with hMTH1, and MYH knockdown were established and exposed to 0.5 and 1 Gy administered at different dose rates. We found that LDR induces significantly increased levels of extracellular 8-oxo-dG compared to HDR. We also found that hMTH1 and MYH play together an important role in the protection of cells against ROS-induced mutagenicity.Publication II, the role of NRF2 was investigated for the radiosensitivity of glioblastoma cancer stem cells (CSCs). The neutrosphere cells from the U87MG cell line were irradiated with three different radiation qualities. The results show that cells exposed to LDR produce significantly higher levels of extracellular 8-oxo-dG compared to HDR and carbon ion irradiated cells. Lower proliferation, self-renewal, and neurosphere formation were observed in both LDR and HDR irradiated NRF2-knockdown cells as compared with the wild type. The results show that NRF2 plays an important role in the radiosensitivity of neurosphere cells isolated from the U87MG cell line.Publication III, we examined the relation between 8-oxo-dG levels and the outcome of radiotherapy and chemotherapy in gastrointestinal cancer patients. The results showed that patients with improved treatment outcomes (responders), had lower levels of the stress marker extracellular 8-oxo-dG before the start of the treatment and the levels were increased 2 weeks after completing the treatment.Publication IV, mice were whole-body irradiated with different doses administered at LDR and HDR. Three hours or three weeks after exposure, the immune cell populations in the spleens were phenotyped. The effects of dose, dose rate, and time after exposure and interaction between them were investigated to check which of the factors had the main effect on the change of immune cell populations. The results indicate that there was a pro-inflammatory short-term effect at high doses for both HDR and LDR. The results also indicate a pro-inflammatory effect of low doses of radiation three weeks after exposure.
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| 6. |
- Spiegelberg, Diana, 1982-, et al.
(författare)
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The HSP90 inhibitor Onalespib exerts synergistic anti-cancer effects when combined with radiotherapy : an in vitro and in vivo approach
- 2020
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Oncogenic client-proteins of the chaperone Heat shock protein 90 (HSP90) insure unlimited tumor growth and are involved in resistance to chemo- and radiotherapy. The HSP90 inhibitor Onalespib initiates the degradation of oncoproteins, and might also act as a radiosensitizer. The aim of this study was therefore to evaluate the efficacy of Onalespib in combination with external beam radiotherapy in an in vitro and in vivo approach. Onalespib downregulated client proteins, lead to increased apoptosis and caused DNA-double-strands. Monotherapy and combination with radiotherapy reduced colony formation, proliferation and migration assessed in radiosensitive HCT116 and radioresistant A431 cells. In vivo, a minimal treatment regimen for 3 consecutive days of Onalespib (3 x 10 mg/kg) doubled survival, whereas Onalespib with radiotherapy (3 x 2 Gy) caused a substantial delay in tumor growth and prolonged the survival by a factor of 3 compared to the HCT116 xenografted control group. Our results demonstrate that Onalespib exerts synergistic anti-cancer effects when combined with radiotherapy, most prominent in the radiosensitive cell models. We speculate that the depletion and downregulation of client proteins involved in signalling, migration and DNA repair mechanisms is the cause. Thus, individually, or in combination with radiotherapy Onalespib inhibits tumor growth and has the potential to improve radiotherapy outcomes, prolonging the overall survival of cancer patients.
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