International Journal of Molecular Sciences

Research

12 pages, 1637 KiB

Open AccessArticle

Radiobiological Characterization of Canine Malignant Melanoma Cell Lines with Different Types of Ionizing Radiation and Efficacy Evaluation with Cytotoxic Agents

by Hiroto Yoshikawa, Shigeaki Sunada, Hirokazu Hirakawa, Akira Fujimori, Suad Elmegerhi, Del Leary and Takamitsu A. Kato

Int. J. Mol. Sci. 2019, 20(4), 841; https://doi.org/10.3390/ijms20040841 - 15 Feb 2019

Cited by 10 | Viewed by 6320

Abstract

Canine malignant melanoma (CMM) is a locally and systemically aggressive cancer that shares many biological and clinical characteristics with human mucosal melanoma. Hypofractionated radiation protocols have been used to treat CMM but little is known about its radiation biology. This pilot study is [...] Read more.

Canine malignant melanoma (CMM) is a locally and systemically aggressive cancer that shares many biological and clinical characteristics with human mucosal melanoma. Hypofractionated radiation protocols have been used to treat CMM but little is known about its radiation biology. This pilot study is designed to investigate response of CMM cell lines to various ionizing radiations and cytotoxic agents to better understand this canine cancer. Four CMM cell lines were evaluated by clonogenic survival assay under aerobic and hypoxic conditions and parameters such as alpha beta (α/β) ratio, oxygen enhancement ratio (OER), and relative biological effectiveness (RBE) were calculated after ¹³⁷Cs, 6 megavoltage (MV) photon, or carbon ion irradiation. Six cytotoxic agents (cisplatin, camptothecin, mitomycin C, bleomycin, methtyl methanesulfonate and etoposide) were also assessed for their efficacy. Under aerobic condition with 6 MV photon, the α/β ratio of the four cell lines ranged from 0.3 to >100, indicating a wide variation of cellular sensitivity. The ratio increased under hypoxic condition compared to aerobic condition and this was more dramatic in ¹³⁷Cs and 6 MV photon treatments. OER of carbon was lower than ¹³⁷Cs at D₁₀ in 3 of the 4 cell lines. The RBE values generally increased with the increase of LET. Different cell lines showed sensitivity/resistance to different cytotoxic agents. This study revealed that CMM has a wide range of radiosensitivity and that hypoxia can reduce it, indicating that widely used hypofractionated protocols may not be optimal for all CMM patients. Several cytotoxic agents that have never been clinically assessed can improve treatment outcome. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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10 pages, 1424 KiB

Open AccessCommunication

Relating Linear Energy Transfer to the Formation and Resolution of DNA Repair Foci After Irradiation with Equal Doses of X-ray Photons, Plateau, or Bragg-Peak Protons

by Sebastian Oeck, Klaudia Szymonowicz, Gesa Wiel, Adam Krysztofiak, Jamil Lambert, Benjamin Koska, George Iliakis, Beate Timmermann and Verena Jendrossek

Int. J. Mol. Sci. 2018, 19(12), 3779; https://doi.org/10.3390/ijms19123779 - 28 Nov 2018

Cited by 33 | Viewed by 4547

Abstract

Proton beam therapy is increasingly applied for the treatment of human cancer, as it promises to reduce normal tissue damage. However, little is known about the relationship between linear energy transfer (LET), the type of DNA damage, and cellular repair mechanisms, particularly for [...] Read more.

Proton beam therapy is increasingly applied for the treatment of human cancer, as it promises to reduce normal tissue damage. However, little is known about the relationship between linear energy transfer (LET), the type of DNA damage, and cellular repair mechanisms, particularly for cells irradiated with protons. We irradiated cultured cells delivering equal doses of X-ray photons, Bragg-peak protons, or plateau protons and used this set-up to quantitate initial DNA damage (mainly DNA double strand breaks (DSBs)), and to analyze kinetics of repair by detecting γH2A.X or 53BP1 using immunofluorescence. The results obtained validate the reliability of our set-up in delivering equal radiation doses under all conditions employed. Although the initial numbers of γH2A.X and 53BP1 foci scored were similar under the different irradiation conditions, it was notable that the maximum foci level was reached at 60 min after irradiation with Bragg-peak protons, as compared to 30 min for plateau protons and photons. Interestingly, Bragg-peak protons induced larger and irregularly shaped γH2A.X and 53BP1 foci. Additionally, the resolution of these foci was delayed. These results suggest that Bragg-peak protons induce DNA damage of increased complexity which is difficult to process by the cellular repair apparatus. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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18 pages, 3871 KiB

Open AccessArticle

Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy

by Elizaveta Bobkova, Daniel Depes, Jin-Ho Lee, Lucie Jezkova, Iva Falkova, Eva Pagacova, Olga Kopecna, Mariia Zadneprianetc, Alena Bacikova, Elena Kulikova, Elena Smirnova, Tatiana Bulanova, Alla Boreyko, Evgeny Krasavin, Frederik Wenz, Felix Bestvater, Georg Hildenbrand, Michael Hausmann and Martin Falk

Int. J. Mol. Sci. 2018, 19(12), 3713; https://doi.org/10.3390/ijms19123713 - 22 Nov 2018

Cited by 32 | Viewed by 5364

Abstract

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and [...] Read more.

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET ¹⁵N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10–20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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20 pages, 14587 KiB

Open AccessArticle

Low-Dose Radiotherapy Has No Harmful Effects on Key Cells of Healthy Non-Inflamed Joints

by Lisa Deloch, Michael Rückert, Rainer Fietkau, Benjamin Frey and Udo S. Gaipl

Int. J. Mol. Sci. 2018, 19(10), 3197; https://doi.org/10.3390/ijms19103197 - 16 Oct 2018

Cited by 23 | Viewed by 4914

Abstract

Low-dose radiotherapy (LD-RT) for benign inflammatory and/or bone destructive diseases has been used long. Therefore, mechanistic investigations on cells being present in joints are mostly made in an inflammatory setting. This raises the question whether similar effects of LD-RT are also seen in [...] Read more.

Low-dose radiotherapy (LD-RT) for benign inflammatory and/or bone destructive diseases has been used long. Therefore, mechanistic investigations on cells being present in joints are mostly made in an inflammatory setting. This raises the question whether similar effects of LD-RT are also seen in healthy tissue and thus might cause possible harmful effects. We performed examinations on the functionality and phenotype of key cells within the joint, namely on fibroblast-like synoviocytes (FLS), osteoclasts and osteoblasts, as well as on immune cells. Low doses of ionizing radiation showed only a minor impact on cytokine release by healthy FLS as well as on molecules involved in cartilage and bone destruction and had no significant impact on cell death and migration properties. The bone resorbing abilities of healthy osteoclasts was slightly reduced following LD-RT and a positive impact on bone formation of healthy osteoblasts was observed after in particular exposure to 0.5 Gray (Gy). Cell death rates of bone-marrow cells were only marginally increased and immune cell composition of the bone marrow showed a slight shift from CD8⁺ to CD4⁺ T cell subsets. Taken together, our results indicate that LD-RT with particularly a single dose of 0.5 Gy has no harmful effects on cells of healthy joints. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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13 pages, 2417 KiB

Open AccessArticle

Impact of X-ray Exposure on the Proliferation and Differentiation of Human Pre-Adipocytes

by Kateryna Shreder, Felicitas Rapp, Ioanna Tsoukala, Vanessa Rzeznik, Martin Wabitsch, Pamela Fischer-Posovszky and Claudia Fournier

Int. J. Mol. Sci. 2018, 19(9), 2717; https://doi.org/10.3390/ijms19092717 - 11 Sep 2018

Cited by 6 | Viewed by 6081

Abstract

Radiotherapy is a widely used treatment option for cancer patients as well as for patients with musculoskeletal disorders. Adipocytes, the dominant cell type of adipose tissue, are known to constitute an active part of the tumor microenvironment. Moreover, adipocytes support inflammatory processes and [...] Read more.

Radiotherapy is a widely used treatment option for cancer patients as well as for patients with musculoskeletal disorders. Adipocytes, the dominant cell type of adipose tissue, are known to constitute an active part of the tumor microenvironment. Moreover, adipocytes support inflammatory processes and cartilage degradation in chronic inflammatory diseases, i.e., rheumatoid and osteoarthritis. Since the production of inflammatory factors is linked to their differentiation stages, we set out to explore the radiation response of pre-adipocytes that may influence their inflammatory potential and differentiation capacity. This is the first study investigating the effects of X-ray irradiation on the proliferation and differentiation capacity of human primary pre-adipocytes, in comparison to Simpson–Golabi–Behmel Syndrome (SGBS) pre-adipocytes, an often-used in vitro model of human primary pre-adipocytes. Our results demonstrate a dose-dependent reduction of the proliferation capacity for both cell strains, whereas the potential for differentiation was mostly unaffected by irradiation. The expression of markers of adipogenic development, such as transcription factors (PPARγ, C/EBPα and C/EBPβ), as well as the release of adipokines (visfatin, adiponectin and leptin) were not significantly changed upon irradiation. However, after irradiation with high X-ray doses, an increased lipid accumulation was observed, which suggests a radiation-induced response of adipocytes related to inflammation. Our results indicate that pre-adipocytes are radio-resistant, and it remains to be elucidated whether this holds true for the overall inflammatory response of adipocytes upon irradiation. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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13 pages, 2384 KiB

Open AccessArticle

Heterogeneity of γH2AX Foci Increases in Ex Vivo Biopsies Relative to In Vivo Tumors

by Treewut Rassamegevanon, Steffen Löck, Michael Baumann, Mechthild Krause and Cläre Von Neubeck

Int. J. Mol. Sci. 2018, 19(9), 2616; https://doi.org/10.3390/ijms19092616 - 4 Sep 2018

Cited by 6 | Viewed by 4714

Abstract

The biomarker for DNA double stand breaks, gammaH2AX (γH2AX), holds a high potential as an intrinsic radiosensitivity predictor of tumors in clinical practice. Here, two published γH2AX foci datasets from in and ex vivo exposed human head and neck squamous cell carcinoma (hHNSCC) [...] Read more.

The biomarker for DNA double stand breaks, gammaH2AX (γH2AX), holds a high potential as an intrinsic radiosensitivity predictor of tumors in clinical practice. Here, two published γH2AX foci datasets from in and ex vivo exposed human head and neck squamous cell carcinoma (hHNSCC) xenografts were statistically re-evaluated for the effect of the assay setting (in or ex vivo) on cellular geometry and the degree of heterogeneity in γH2AX foci. Significant differences between the nucleus areas of in- and ex vivo exposed samples were found. However, the number of foci increased linearly with nucleus area in irradiated samples of both settings. Moreover, irradiated tumor cells showed changes of nucleus area distributions towards larger areas compared to unexposed samples, implying cell cycle alteration after radiation exposure. The number of residual γH2AX foci showed a higher degree of intra-tumoral heterogeneity in the ex vivo exposed samples relative to the in vivo exposed samples. In the in vivo setting, the highest intra-tumoral heterogeneity was observed in initial γH2AX foci numbers (foci detected 30 min following irradiation). These results suggest that the tumor microenvironment and the culture condition considerably influence cellular adaptation and DNA damage repair. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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15 pages, 6074 KiB

Open AccessArticle

Radiation Sensitization of Basal Cell and Head and Neck Squamous Cell Carcinoma by the Hedgehog Pathway Inhibitor Vismodegib

by Stephanie Hehlgans, Patrick Booms, Ömer Güllülü, Robert Sader, Claus Rödel, Panagiotis Balermpas, Franz Rödel and Shahram Ghanaati

Int. J. Mol. Sci. 2018, 19(9), 2485; https://doi.org/10.3390/ijms19092485 - 23 Aug 2018

Cited by 31 | Viewed by 6158

Abstract

Vismodegib, an inhibitor of the Hedgehog signaling pathway, is an approved drug for monotherapy in locally advanced or metastatic basal cell carcinoma (BCC). Data on combined modality treatment by vismodegib and radiation therapy, however, are rare. In the present study, we examined the [...] Read more.

Vismodegib, an inhibitor of the Hedgehog signaling pathway, is an approved drug for monotherapy in locally advanced or metastatic basal cell carcinoma (BCC). Data on combined modality treatment by vismodegib and radiation therapy, however, are rare. In the present study, we examined the radiation sensitizing effects of vismodegib by analyzing viability, cell cycle distribution, cell death, DNA damage repair and clonogenic survival in three-dimensional cultures of a BCC and a head and neck squamous cell carcinoma (HNSCC) cell line. We found that vismodegib decreases expression of the Hedgehog target genes glioma-associated oncogene homologue (GLI1) and the inhibitor of apoptosis protein (IAP) Survivin in a cell line- and irradiation-dependent manner, most pronounced in squamous cell carcinoma (SCC) cells. Furthermore, vismodegib significantly reduced proliferation in both cell lines, while additional irradiation only slightly further impacted on viability. Analyses of cell cycle distribution and cell death induction indicated a G1 arrest in BCC and a G2 arrest in HNSCC cells and an increased fraction of cells in SubG1 phase following combined treatment. Moreover, a significant rise in the number of phosphorylated histone-2AX/p53-binding protein 1 (γH2AX/53BP1) foci in vismodegib- and radiation-treated cells was associated with a significant radiosensitization of both cell lines. In summary, these findings indicate that inhibition of the Hedgehog signaling pathway may increase cellular radiation response in BCC and HNSCC cells. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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12 pages, 1833 KiB

Open AccessArticle

Stress-Induced Phosphorylation of Nuclear YB-1 Depends on Nuclear Trafficking of p90 Ribosomal S6 Kinase

by Aadhya Tiwari, Simone Rebholz, Eva Maier, Mozhgan Dehghan Harati, Daniel Zips, Christine Sers, H. Peter Rodemann and Mahmoud Toulany

Int. J. Mol. Sci. 2018, 19(8), 2441; https://doi.org/10.3390/ijms19082441 - 18 Aug 2018

Cited by 21 | Viewed by 6533

Abstract

Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced [...] Read more.

Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimuli. EGF-induced RSK translocation to the nucleus and nuclear YB-1 phosphorylation were completely blocked by the EGF receptor kinase inhibitor erlotinib. Likewise, RSK inhibition blocked RSK nuclear translocation and nuclear YB-1 phosphorylation after irradiation and KRAS(G12V) overexpression. In summary, acute stimulation of YB-1 phosphorylation does not lead to YB-1 translocation from the cytoplasm to the nucleus. Rather, irradiation, EGF treatment, or KRAS(G12V) overexpression induces RSK activation, leading to its translocation to the nucleus, where it activates already-existing nuclear YB-1. Our novel finding illuminates the signaling pathways involved in nuclear YB-1 phosphorylation and provides a rationale for designing appropriate targeting strategies to block YB-1 in oncology as well as in radiation oncology. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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15 pages, 1408 KiB

Open AccessArticle

Application of Fluorescence Lifetime Imaging Microscopy of DNA Binding Dyes to Assess Radiation-Induced Chromatin Compaction Changes

by Elham Abdollahi, Gisela Taucher-Scholz and Burkhard Jakob

Int. J. Mol. Sci. 2018, 19(8), 2399; https://doi.org/10.3390/ijms19082399 - 14 Aug 2018

Cited by 14 | Viewed by 6633

Abstract

In recent years several approaches have been developed to address the chromatin status and its changes in eukaryotic cells under different conditions—but only few are applicable in living cells. Fluorescence lifetime imaging microscopy (FLIM) is a functional tool that can be used for [...] Read more.

In recent years several approaches have been developed to address the chromatin status and its changes in eukaryotic cells under different conditions—but only few are applicable in living cells. Fluorescence lifetime imaging microscopy (FLIM) is a functional tool that can be used for the inspection of the molecular environment of fluorophores in living cells. Here, we present the use of single organic minor groove DNA binder dyes in FLIM for measuring chromatin changes following modulation of chromatin structure in living cells. Treatment with histone deacetylase inhibitors led to an increased fluorescence lifetime indicating global chromatin decompaction, whereas hyperosmolarity decreased the lifetime of the used dyes, thus reflecting the expected compaction. In addition, we demonstrate that time domain FLIM data based on single photon counting should be optimized using pile-up and counting loss correction, which affect the readout even at moderate average detector count rates in inhomogeneous samples. Using these corrections and utilizing Hoechst 34580 as chromatin compaction probe, we measured a pan nuclear increase in the lifetime following irradiation with X-rays in living NIH/3T3 cells thus providing a method to measure radiation-induced chromatin decompaction. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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18 pages, 2530 KiB

Open AccessArticle

Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

by Andreas Hofmann, Matthias Krufczik, Dieter W. Heermann and Michael Hausmann

Int. J. Mol. Sci. 2018, 19(8), 2263; https://doi.org/10.3390/ijms19082263 - 2 Aug 2018

Cited by 28 | Viewed by 4993

Abstract

DNA double strand breaks (DSB) are the most severe damages in chromatin induced by ionizing radiation. In response to such environmentally determined stress situations, cells have developed repair mechanisms. Although many investigations have contributed to a detailed understanding of repair processes, e.g., homologous [...] Read more.

DNA double strand breaks (DSB) are the most severe damages in chromatin induced by ionizing radiation. In response to such environmentally determined stress situations, cells have developed repair mechanisms. Although many investigations have contributed to a detailed understanding of repair processes, e.g., homologous recombination repair or non-homologous end-joining, the question is not sufficiently answered, how a cell decides to apply a certain repair process at a certain damage site, since all different repair pathways could simultaneously occur in the same cell nucleus. One of the first processes after DSB induction is phosphorylation of the histone variant H2AX to γH2AX in the given surroundings of the damaged locus. Since the spatial organization of chromatin is not random, it may be conclusive that the spatial organization of γH2AX foci is also not random, and rather, contributes to accessibility of special repair proteins to the damaged site, and thus, to the following repair pathway at this given site. The aim of this article is to demonstrate a new approach to analyze repair foci by their topology in order to obtain a cell independent method of categorization. During the last decade, novel super-resolution fluorescence light microscopic techniques have enabled new insights into genome structure and spatial organization on the nano-scale in the order of 10 nm. One of these techniques is single molecule localization microscopy (SMLM) with which the spatial coordinates of single fluorescence molecules can precisely be determined and density and distance distributions can be calculated. This method is an appropriate tool to quantify complex changes of chromatin and to describe repair foci on the single molecule level. Based on the pointillist information obtained by SMLM from specifically labeled heterochromatin and γH2AX foci reflecting the chromatin morphology and repair foci topology, we have developed a new analytical methodology of foci or foci cluster characterization, respectively, by means of persistence homology. This method allows, for the first time, a cell independent comparison of two point distributions (here the point distributions of two γH2AX clusters) with each other of a selected ensample and to give a mathematical measure of their similarity. In order to demonstrate the feasibility of this approach, cells were irradiated by low LET (linear energy transfer) radiation with different doses and the heterochromatin and γH2AX foci were fluorescently labeled by antibodies for SMLM. By means of our new analysis method, we were able to show that the topology of clusters of γH2AX foci can be categorized depending on the distance to heterochromatin. This method opens up new possibilities to categorize spatial organization of point patterns by parameterization of topological similarity. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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14 pages, 5321 KiB

Open AccessArticle

Restraining Akt1 Phosphorylation Attenuates the Repair of Radiation-Induced DNA Double-Strand Breaks and Reduces the Survival of Irradiated Cancer Cells

by Klaudia Szymonowicz, Sebastian Oeck, Adam Krysztofiak, Jansje Van der Linden, George Iliakis and Verena Jendrossek

Int. J. Mol. Sci. 2018, 19(8), 2233; https://doi.org/10.3390/ijms19082233 - 31 Jul 2018

Cited by 12 | Viewed by 4209

Abstract

The survival kinase protein kinase B (Akt) participates in the regulation of essential subcellular processes, e.g., proliferation, growth, survival, and apoptosis, and has a documented role in promoting resistance against genotoxic stress including radiotherapy, presumably by influencing the DNA damage response and DNA [...] Read more.

The survival kinase protein kinase B (Akt) participates in the regulation of essential subcellular processes, e.g., proliferation, growth, survival, and apoptosis, and has a documented role in promoting resistance against genotoxic stress including radiotherapy, presumably by influencing the DNA damage response and DNA double-strand break (DSB) repair. However, its exact role in DSB repair requires further elucidation. We used a genetic approach to explore the consequences of impaired phosphorylation of Akt1 at one or both of its key phosphorylation sites, Threonine 308 (T308) or Serine 473 (S473), on DSB repair and radiosensitivity to killing. Therefore, we overexpressed either the respective single or the double phosphorylation-deficient mutants (Akt1-T308A, Akt1-S473A, or Akt1-T308A/S473A) in TRAMPC1 murine prostate cancer cells (TrC1) and measured the DSB repair kinetics and clonogenic cell survival upon irradiation. Only the expression of the Akt1-T308A/S473A induced a significant delay in the kinetics of DSB repair in irradiated TrC1 as determined by the γH2A.X (H2A histone family, member X) assay and the neutral comet assay, respectively. Moreover, Akt1-T308A/S473A-expressing cells were characterized by increased radiosensitivity compared to Akt1-WT (wild type)-expressing cells in long-term colony formation assays. Our data reveal that Akt1’s activation state is important for the cellular radiation response, presumably by modulating the phosphorylation of effector proteins involved in the regulation of DSB repair. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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12 pages, 2530 KiB

Open AccessArticle

DNA Repair Deficient Chinese Hamster Ovary Cells Exhibiting Differential Sensitivity to Charged Particle Radiation under Aerobic and Hypoxic Conditions

by Ian M. Cartwright, Cathy Su, Jeremy S. Haskins, Victoria A. Salinas, Shigeaki Sunada, Hao Yu, Mitsuru Uesaka, Hirokazu Hirakawa, David J. Chen, Akira Fujimori and Takamitsu A. Kato

Int. J. Mol. Sci. 2018, 19(8), 2228; https://doi.org/10.3390/ijms19082228 - 30 Jul 2018

Cited by 17 | Viewed by 4362

Abstract

It has been well established that hypoxia significantly increases both cellular and tumor resistance to ionizing radiation. Hypoxia associated radiation resistance has been known for some time but there has been limited success in sensitizing cells to radiation under hypoxic conditions. These studies [...] Read more.

It has been well established that hypoxia significantly increases both cellular and tumor resistance to ionizing radiation. Hypoxia associated radiation resistance has been known for some time but there has been limited success in sensitizing cells to radiation under hypoxic conditions. These studies show that, when irradiated with low linear energy transfer (LET) gamma-rays, poly (ADP-ribose), polymerase (PARP), Fanconi Anemia (FANC), and mutant Chinese Hamster Ovary (CHO) cells respond similarly to the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair mutant CHO cells. Comparable results were observed in cells exposed to 13 keV/μm carbon ions. However, when irradiated with higher LET spread out Bragg peak (SOBP) carbon ions, we observed a decrease in the oxygen enhancement ratio (OER) in all the DNA of repair mutant cell lines. Interestingly, PARP mutant cells were observed as having the largest decrease in OER. Finally, these studies show a significant increase in the relative biological effectiveness (RBE) of high LET SOBP carbon and iron ions in HR and PARP mutants. There was also an increase in the RBE of NHEJ mutants when irradiated to SOBP carbon and iron ions. However, this increase was lower than in other mutant cell lines. These findings indicate that high LET radiation produces unique types of DNA damage under hypoxic conditions and PARP and HR repair pathways play a role in repairing this damage. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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43 pages, 7577 KiB

Open AccessArticle

The Role of the Nuclear Factor κB Pathway in the Cellular Response to Low and High Linear Energy Transfer Radiation

by Christine E. Hellweg, Luis F. Spitta, Kristina Koch, Arif A. Chishti, Bernd Henschenmacher, Sebastian Diegeler, Bikash Konda, Sebastian Feles, Claudia Schmitz, Thomas Berger and Christa Baumstark-Khan

Int. J. Mol. Sci. 2018, 19(8), 2220; https://doi.org/10.3390/ijms19082220 - 30 Jul 2018

Cited by 11 | Viewed by 6249

Abstract

Astronauts are exposed to considerable doses of space radiation during long-term space missions. As complete shielding of the highly energetic particles is impracticable, the cellular response to space-relevant radiation qualities has to be understood in order to develop countermeasures and to reduce radiation [...] Read more.

Astronauts are exposed to considerable doses of space radiation during long-term space missions. As complete shielding of the highly energetic particles is impracticable, the cellular response to space-relevant radiation qualities has to be understood in order to develop countermeasures and to reduce radiation risk uncertainties. The transcription factor Nuclear Factor κB (NF-κB) plays a fundamental role in the immune response and in the pathogenesis of many diseases. We have previously shown that heavy ions with a linear energy transfer (LET) of 100–300 keV/µm have a nine times higher potential to activate NF-κB compared to low-LET X-rays. Here, chemical inhibitor studies using human embryonic kidney cells (HEK) showed that the DNA damage sensor Ataxia telangiectasia mutated (ATM) and the proteasome were essential for NF-κB activation in response to X-rays and heavy ions. NF-κB’s role in cellular radiation response was determined by stable knock-down of the NF-κB subunit RelA. Transfection of a RelA short-hairpin RNA plasmid resulted in higher sensitivity towards X-rays, but not towards heavy ions. Reverse Transcriptase real-time quantitative PCR (RT-qPCR) showed that after exposure to X-rays and heavy ions, NF-κB predominantly upregulates genes involved in intercellular communication processes. This process is strictly NF-κB dependent as the response is completely absent in RelA knock-down cells. NF-κB’s role in the cellular radiation response depends on the radiation quality. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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13 pages, 1738 KiB

Open AccessArticle

Radioprotective Effects of Dermatan Sulfate in a Preclinical Model of Oral Mucositis—Targeting Inflammation, Hypoxia and Junction Proteins without Stimulating Proliferation

by Sylvia Gruber, Marlene Arnold, Nilsu Cini, Victoria Gernedl, Sabine Hetzendorfer, Lisa-Marie Kowald, Peter Kuess, Julia Mayer, Susanne Morava, Stephanie Pfaffinger, Andreas Rohorzka and Wolfgang Dörr

Int. J. Mol. Sci. 2018, 19(6), 1684; https://doi.org/10.3390/ijms19061684 - 6 Jun 2018

Cited by 3 | Viewed by 3394

Abstract

Oral mucositis is the most frequently occurring early side effect of head-and-neck cancer radiotherapy. Systemic dermatan sulfate (DS) treatment revealed a significant radioprotective potential in a preclinical model of oral mucositis. This study was initiated to elucidate the mechanistic effects of DS in [...] Read more.

Oral mucositis is the most frequently occurring early side effect of head-and-neck cancer radiotherapy. Systemic dermatan sulfate (DS) treatment revealed a significant radioprotective potential in a preclinical model of oral mucositis. This study was initiated to elucidate the mechanistic effects of DS in the same model. Irradiation comprised daily fractionated irradiation (5 × 3 Gy/week) over two weeks, either alone (IR) or in combination with daily dermatan sulfate treatment of 4 mg/kg (IR + DS). Groups of mice (n = 5) were sacrificed every second day over the course of 14 days in both experimental arms, their tongues excised and evaluated. The response to irradiation with and without DS was analyzed on a morphological (cell numbers, epithelial thickness) as well as on a functional (proliferation and expression of inflammation, hypoxia and epithelial junction markers) level. The mucoprotective activity of DS can be attributed to a combination of various effects, comprising increased expression of epithelial junctions, reduced inflammation and reduced hypoxia. No DS-mediated effect on proliferation was observed. DS demonstrated a significant mucositis-ameliorating activity and could provide a promising strategy for mucositis treatment, based on targeting specific, radiation-induced, mucositis-associated signaling without stimulating proliferation. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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16 pages, 2020 KiB

Open AccessArticle

Detrimental Effects of Helium Ion Irradiation on Cognitive Performance and Cortical Levels of MAP-2 in B6D2F1 Mice

by Jacob Raber, Eileen Ruth S. Torres, Tunde Akinyeke, Joanne Lee, Sydney J. Weber Boutros, Mitchell S. Turker and Amy Kronenberg

Int. J. Mol. Sci. 2018, 19(4), 1247; https://doi.org/10.3390/ijms19041247 - 20 Apr 2018

Cited by 18 | Viewed by 4640

Abstract

The space radiation environment includes helium (⁴He) ions that may impact brain function. As little is known about the effects of exposures to ⁴He ions on the brain, we assessed the behavioral and cognitive performance of C57BL/6J × DBA2/J F1 [...] Read more.

The space radiation environment includes helium (⁴He) ions that may impact brain function. As little is known about the effects of exposures to ⁴He ions on the brain, we assessed the behavioral and cognitive performance of C57BL/6J × DBA2/J F1 (B6D2F1) mice three months following irradiation with ⁴He ions (250 MeV/n; linear energy transfer (LET) = 1.6 keV/μm; 0, 21, 42 or 168 cGy). Sham-irradiated mice and mice irradiated with 21 or 168 cGy showed novel object recognition, but mice irradiated with 42 cGy did not. In the passive avoidance test, mice received a slight foot shock in a dark compartment, and latency to re-enter that compartment was assessed 24 h later. Sham-irradiated mice and mice irradiated with 21 or 42 cGy showed a higher latency on Day 2 than Day 1, but the latency to enter the dark compartment in mice irradiated with 168 cGy was comparable on both days. ⁴He ion irradiation, at 42 and 168 cGy, reduced the levels of the dendritic marker microtubule-associated protein-2 (MAP-2) in the cortex. There was an effect of radiation on apolipoprotein E (apoE) levels in the hippocampus and cortex, with higher apoE levels in mice irradiated at 42 cGy than 168 cGy and a trend towards higher apoE levels in mice irradiated at 21 than 168 cGy. In addition, in the hippocampus, there was a trend towards a negative correlation between MAP-2 and apoE levels. While reduced levels of MAP-2 in the cortex might have contributed to the altered performance in the passive avoidance test, it does not seem sufficient to do so. The higher hippocampal and cortical apoE levels in mice irradiated at 42 than 168 cGy might have served as a compensatory protective response preserving their passive avoidance memory. Thus, there were no alterations in behavioral performance in the open filed or depressive-like behavior in the forced swim test, while cognitive impairments were seen in the object recognition and passive avoidance tests, but not in the contextual or cued fear conditioning tests. Taken together, the results indicate that some aspects of cognitive performance are altered in male mice exposed to ⁴He ions, but that the response is task-dependent. Furthermore, the sensitive doses can vary within each task in a non-linear fashion. This highlights the importance of assessing the cognitive and behavioral effects of charged particle exposure with a variety of assays and at multiple doses, given the possibility that lower doses may be more damaging due to the absence of induced compensatory mechanisms at higher doses. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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15 pages, 7117 KiB

Open AccessArticle

Radiation-Induced Gene Expression Changes in High and Low Grade Breast Cancer Cell Types

by Valentina Bravatà, Claudia Cava, Luigi Minafra, Francesco Paolo Cammarata, Giorgio Russo, Maria Carla Gilardi, Isabella Castiglioni and Giusi Irma Forte

Int. J. Mol. Sci. 2018, 19(4), 1084; https://doi.org/10.3390/ijms19041084 - 4 Apr 2018

Cited by 27 | Viewed by 5246

Abstract

Background: There is extensive scientific evidence that radiation therapy (RT) is a crucial treatment, either alone or in combination with other treatment modalities, for many types of cancer, including breast cancer (BC). BC is a heterogeneous disease at both clinical and molecular levels, [...] Read more.

Background: There is extensive scientific evidence that radiation therapy (RT) is a crucial treatment, either alone or in combination with other treatment modalities, for many types of cancer, including breast cancer (BC). BC is a heterogeneous disease at both clinical and molecular levels, presenting distinct subtypes linked to the hormone receptor (HR) status and associated with different clinical outcomes. The aim of this study was to assess the molecular changes induced by high doses of ionizing radiation (IR) on immortalized and primary BC cell lines grouped according to Human epidermal growth factor receptor (HER2), estrogen, and progesterone receptors, to study how HR status influences the radiation response. Our genomic approach using in vitro and ex-vivo models (e.g., primary cells) is a necessary first step for a translational study to describe the common driven radio-resistance features associated with HR status. This information will eventually allow clinicians to prescribe more personalized total doses or associated targeted therapies for specific tumor subtypes, thus enhancing cancer radio-sensitivity. Methods: Nontumorigenic (MCF10A) and BC (MCF7 and MDA-MB-231) immortalized cell lines, as well as healthy (HMEC) and BC (BCpc7 and BCpcEMT) primary cultures, were divided into low grade, high grade, and healthy groups according to their HR status. At 24 h post-treatment, the gene expression profiles induced by two doses of IR treatment with 9 and 23 Gy were analyzed by cDNA microarray technology to select and compare the differential gene and pathway expressions among the experimental groups. Results: We present a descriptive report of the substantial alterations in gene expression levels and pathways after IR treatment in both immortalized and primary cell cultures. Overall, the IR-induced gene expression profiles and pathways appear to be cell-line dependent. The data suggest that some specific gene and pathway signatures seem to be linked to HR status. Conclusions: Genomic biomarkers and gene-signatures of specific tumor subtypes, selected according to their HR status and molecular features, could facilitate personalized biological-driven RT treatment planning alone and in combination with targeted therapies. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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16 pages, 9228 KiB

Open AccessArticle

Live Dynamics of 53BP1 Foci Following Simultaneous Induction of Clustered and Dispersed DNA Damage in U2OS Cells

by Alice Sollazzo, Beata Brzozowska, Lei Cheng, Lovisa Lundholm, Harry Scherthan and Andrzej Wojcik

Int. J. Mol. Sci. 2018, 19(2), 519; https://doi.org/10.3390/ijms19020519 - 8 Feb 2018

Cited by 38 | Viewed by 6250

Abstract

Cells react differently to clustered and dispersed DNA double strand breaks (DSB). Little is known about the initial reaction to simultaneous induction of DSBs with different complexities. Here, we used live cell microscopy to analyse the behaviour of 53BP1-GFP (green fluorescence protein) foci [...] Read more.

Cells react differently to clustered and dispersed DNA double strand breaks (DSB). Little is known about the initial reaction to simultaneous induction of DSBs with different complexities. Here, we used live cell microscopy to analyse the behaviour of 53BP1-GFP (green fluorescence protein) foci formation at DSBs induced in U2OS cells by alpha particles, X-rays or mixed beams over a 75 min period post irradiation. X-ray-induced foci rapidly increased and declined over the observation interval. After an initial increase, mixed beam-induced foci remained at a constant level over the observation interval, similarly as alpha-induced foci. The average areas of radiation-induced foci were similar for mixed beams and X-rays, being significantly smaller than those induced by alpha particles. Pixel intensities were highest for mixed beam-induced foci and showed the lowest level of variability over time as compared to foci induced by alphas and X-rays alone. Finally, mixed beam-exposed foci showed the lowest level of mobility as compared to alpha and X-ray exposure. The results suggest paralysation of chromatin around foci containing clustered DNA damage. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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17 pages, 3821 KiB

Open AccessArticle

Histone Deacetylase Inhibitor Induced Radiation Sensitization Effects on Human Cancer Cells after Photon and Hadron Radiation Exposure

by Ariungerel Gerelchuluun, Junko Maeda, Eri Manabe, Colleen A. Brents, Takeji Sakae, Akira Fujimori, David J. Chen, Koji Tsuboi and Takamitsu A. Kato

Int. J. Mol. Sci. 2018, 19(2), 496; https://doi.org/10.3390/ijms19020496 - 7 Feb 2018

Cited by 30 | Viewed by 5230

Abstract

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor, which has been widely utilized throughout the cancer research field. SAHA-induced radiosensitization in normal human fibroblasts AG1522 and lung carcinoma cells A549 were evaluated with a combination of γ-rays, proton, and carbon ion exposure. [...] Read more.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor, which has been widely utilized throughout the cancer research field. SAHA-induced radiosensitization in normal human fibroblasts AG1522 and lung carcinoma cells A549 were evaluated with a combination of γ-rays, proton, and carbon ion exposure. Growth delay was observed in both cell lines during SAHA treatment; 2 μM SAHA treatment decreased clonogenicity and induced cell cycle block in G1 phase but 0.2 μM SAHA treatment did not show either of them. Low LET (Linear Energy Transfer) irradiated A549 cells showed radiosensitization effects on cell killing in cycling and G1 phase with 0.2 or 2 μM SAHA pretreatment. In contrast, minimal sensitization was observed in normal human cells after low and high LET radiation exposure. The potentially lethal damage repair was not affected by SAHA treatment. SAHA treatment reduced the rate of γ-H2AX foci disappearance and suppressed RAD51 and RPA (Replication Protein A) focus formation. Suppression of DNA double strand break repair by SAHA did not result in the differences of SAHA-induced radiosensitization between human cancer cells and normal cells. In conclusion, our results suggest SAHA treatment will sensitize cancer cells to low and high LET radiation with minimum effects to normal cells. Full article

(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)

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