DNA DSB repair pathway choice: an orchestrated handover mechanism

DNA double strand breaks (DSBs) are potential lethal lesions but can also lead to chromosome rearrangements, a step promoting carcinogenesis. DNA non-homologous end-joining (NHEJ) is the major DSB rejoining process and occurs in all cell cycle stages. Homologous recombination (HR) can additionally function to repair irradiation-induced two-ended DSBs in G2 phase. In mammalian cells, HR predominantly uses a sister chromatid as a template for DSB repair; thus HR functions only in late S/G2 phase. Here, we review current insight into the interplay between HR and NHEJ in G2 phase. We argue that NHEJ represents the first choice pathway, repairing approximately 80% of X-ray-induced DSBs with rapid kinetics. However, a subset of DSBs undergoes end resection and repair by HR. 53BP1 restricts resection, thereby promoting NHEJ. During the switch from NHEJ to HR, 53BP1 is repositioned to the periphery of enlarged irradiation-induced foci (IRIF) via a BRCA1-dependent process. K63-linked ubiquitin chains, which also form at IRIF, are also repositioned as well as receptor-associated protein 80 (RAP80), a ubiquitin binding protein. RAP80 repositioning requires POH1, a proteasome component. Thus, the interfacing barriers to HR, 53BP1 and RAP80 are relieved by POH1 and BRCA1, respectively. Removal of RAP80 from the IRIF core is required for loss of the ubiquitin chains and 53BP1, and for efficient replication protein A foci formation. We propose that NHEJ is used preferentially to HR because it is a compact process that does not necessitate extensive chromatin changes in the DSB vicinity.The notion that DNA represents the hereditary component of the cell necessitates that it maintains stability. Yet, pioneering work by Thomas Lindahl revealed that DNA incurs substantial damage, including base and sugar damage, DNA–DNA and DNA-protein cross links, single strand breaks and double strand breaks (DSBs).^1,2 Given such extensive damage, it became evident that cells must have efficient DNA repair mechanisms if the DNA sequence represents the stably inherited determinant of cellular phenotype, a notion strengthened by the finding that DNA repair defective mutants in lower organisms are genetically unstable.³ The evolutionary conservation of DNA repair pathways further supports a critical role in maintaining genetic stability. The study of model organisms has substantially contributed to our understanding of DNA repair mechanisms, particularly DNA DSB repair, which is our focus here. Such studies have shown that mutants in lower organisms deficient in homologous recombination (HR) are exquisitely radiosensitive owing to the important role of HR in repairing DNA DSBs, the major lethal lesion induced by radiation.^4–8 By contrast, mammalian mutants deficient in HR show only modest radiosensitivity. Further, studies examining plasmid rejoining in mammalian cells and DNA integration events revealed a distinct process, initially called illegitimate recombination, which does not require extensive homology.^9,10 The concept of a non-homologous end-joining (NHEJ) pathway for DSB repair was further substantiated by the study of radiosensitive mammalian mutants and consolidated by the identification of NHEJ genes.^8,11 Indeed, mammalian cell lines, mice and patients with marked radiosensitivity have proved to display deficiency in NHEJ rather than HR (excepting ataxia telangiectasia, arguably the most radiosensitive human disorder, which is predominantly proficient in both pathways). HR does function in mammalian cells, however, and can contribute to DSB repair. Having gained a deep understanding of NHEJ and HR in mammalian cells, we can now evaluate the pathway interplay, and why one pathway dominates. This will be the focus of this review.     

Radiosensitizing effect of epothilone?B on human epithelial cancer cells

Background

A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothilone?B on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly.

Methods

Cancer cells were treated with epothilone?B in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a ??H2AX foci assay was used. To determine the effect of epothilone?B on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted.

Results

Epothilone?B induced a concentration- and application-dependent antiproliferative effect on the cells, with IC₅₀?values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothilone?B. The ??H2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. Epothilone?B produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin.

Conclusion

The results of this study showed that epothilone?B displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothilone?B. It was also demonstrated that epothilone?B in fact targets microtubules in a more effective manner than paclitaxel.     

The combination of hyperthermia or chemotherapy with gimeracil for effective radiosensitization

Purpose

5-chloro-2,4-dihydroxypyridine (gimeracil) is a component of the oral fluoropyrimidine derivative S-1. Gimeracil was originally added to S-1 to yield prolonged 5-fluorouracil (5-FU) concentrations in serum and tumor tissues by inhibiting dihydropyrimidine dehydrogenase, which degrades 5-FU. We previously demonstrated that gimeracil enhances the efficacy of radiotherapy through the suppression of homologous recombination (HR) in DNA double strand repair. The goal of this paper was to examine the effects of gimeracil on the sensitivity of anticancer drugs and hyperthermia in order to obtain effective radiosensitization.

Materials and methods

Various cell lines, including DLD 1 (human colon carcinoma cells) and cells deficient in HR or nonhomologous end-joining (NHEJ), were used in clonogenic assays. The survival of these cells after various treatments (e.g., drug treatment, heat treatment, and radiation) was determined based on their colony-forming ability.

Results

Gimeracil enhanced cell-killing effects of camptothecin (CPT), 5-FU, and hydroxyurea. Gimeracil sensitized effects of CPT or 5-FU to cells deficient in HR or NHEJ to a similar extent as in other cells (DLD1 and a parent cell), indicating that its sensitizing mechanisms may be different from inhibition of HR or NHEJ. Combination of gimeracil and CPT or 5-FU sensitized radiation more effectively than each modality alone. Gimeracil also enhanced heat sensitivity at 42°C or more. The degree of heat sensitization with gimeracil increased as the temperature increased, and the combination of gimeracil and heat-sensitized radiation was more effective than each modality alone.

Conclusion

Gimeracil enhanced sensitivity of CPT, 5-FU, and hyperthermia. Combination of these modalities sensitized radiation more efficiently than each modality alone.     

Increased radiosensitivity of HPV-positive head and neck cancer cell lines due to cell cycle dysregulation and induction of apoptosis

Background and purpose

Human Papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) respond favourably to radiotherapy as compared to HPV-unrelated HNSCC. We investigated DNA damage response in HPV-positive and HPV-negative HNSCC cell lines aiming to identify mechanisms, which illustrate reasons for the increased sensitivity of HPV-positive cancers of the oropharynx.

Methods

Radiation response including clonogenic survival, apoptosis, DNA double-strand break (DSB) repair, and cell cycle redistribution in four HPV-positive (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) and four HPV-negative (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) cell lines was evaluated.

Results

HPV-positive cells were more radiosensitive (mean SF2: 0.198 range: 0.22–0.18) than HPV-negative cells (mean SF2: 0.34, range: 0.45–0.27; p?=?0.010). Irradiated HPV-positive cell lines progressed faster through S-phase showing a more distinct accumulation in G2/M. The abnormal cell cycle checkpoint activation was accompanied by a more pronounced increase of cell death after x-irradiation and a higher number of residual and unreleased DSBs.

Conclusions

The enhanced responsiveness of HPV-related HNSCC to radiotherapy might be caused by a higher cellular radiosensitivity due to cell cycle dysregulation and impaired DNA DSB repair.     

Survivin, a target to modulate the radiosensitivity of Ewing’s sarcoma

Background and purpose

Radiotherapy constitutes an essential element in the multimodal therapy of Ewing’s sarcoma. Compared to other sarcomas, Ewing tumors normally show a good response to radiotherapy. However, there are consistently tumors with a radioresistant phenotype, and the underlying mechanisms are not known in detail. Here we investigated the association between survivin protein expression and the radiosensitivity of Ewing’s sarcoma in vitro.

Material and methods

An siRNA-based knockdown approach was used to investigate the influence of survivin expression on cell proliferation, double-strand break (DSB) induction and repair, apoptosis and colony-forming ability in four Ewing’s sarcoma cell lines with and without irradiation.

Results

Survivin protein and mRNA were upregulated in all cell lines tested in a dose-dependent manner. As a result of survivin knockdown, STA-ET-1 cells showed reduced cell proliferation, an increased number of radiation-induced DSBs, and reduced repair. Apoptosis was increased by knockdown alone and increased further in combination with irradiation. Colony formation was significantly reduced by survivin knockdown in combination with irradiation.

Conclusion

Survivin is a radiation-inducible protein in Ewing’s sarcoma and its down-regulation sensitizes cells toward irradiation. Survivin knockdown in combination with radiation inhibits cell proliferation, repair, and colony formation significantly and increases apoptosis more than each single treatment alone. This might open new perspectives in the radiation treatment of Ewing’s sarcoma.     

脑组织对DNA双链断裂的反应

DNA双链断裂是电离辐射引起的最严重DNA损伤形式,脑组织对DNA损伤的反应与其发展程度密切相关,脑组织中增殖细胞主要是通过同源重组进行修复,而分化细胞则通过非同源末端联接修复。DNA双链断裂信号通路相关因子的缺陷会导致一系列的有神经病理表现的人类遗传病。重点讨论电离辐射触发脑细胞DNA双链断裂后,信号转导相关因子和与相关因子导致的神经系统人类遗传疾病。     

Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation

Purpose

Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines.

Methods

Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by ??H2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis.

Results

SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. ??H2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24?h before irradiation to the sarcoma cell cultures. In contrast, ??H2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type.

Conclusion

SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.     

DNA repair pathway choice at various conditions immediately post irradiation

Purpose: To clarify which DNA double-strand break repair pathway, non-homologous end-joining (NHEJ), homologous recombination repair (HRR) or both, plays a key role in potentially lethal damage repair (PLDR).

Methods and materials: Combining published data and our new potentially lethal damage repair (PLDR) data, we explain whether similar to sublethal damage repair (SLDR), PLDR also mainly depends on NHEJ versus HRR. The PLDR data used the same cell lines: wild type, HRR or NHEJ-deficient fibroblast cells, as those SLDR data published by our laboratory previously. The PLDR condition that we used was as commonly described by many other groups: the cells were collected immediately or overnight post ionizing radiation for colony formation after cultured to a plateau phase with a low concentration of serum medium.

Results: Enough data from other groups and our lab showed that wild type or HRR-deficient cells had efficient PLDR, but NHEJ deficient cells did not.

Conclusion: NHEJ contributes more to PLDR than HRR in mammalian cells, which is similar to SLDR. Since both SLDR and PLDR are relevant to clinical tumor status while undergoing radiotherapy, such clarification may benefit radiotherapy in the near future.     

Advances in the biophysical and molecular bases of radiation cytogenetics

Purpose:?For more than 70 years radiation cytogenetics has continued to be a topic of major concern in relation to the action of radiation on living cells. To date, diverse cytogenetic findings have developed into orderly, quantitative interpretations and have stimulated numerous biophysical models. However, it is generally agreed that any one of the models used alone is still unable to explain all aspects of the observed chromosomal effects. In this review, a large number of radiation-induced chromosome aberration findings from the literature are reassessed with special attention given to the reaction kinetics and the relevant molecular processes.

Conclusion:?It is now clear that DNA double-strand breaks (DSB) are an integral component of radiation-induced chromosome aberration. At the nexus of the maintenance of genome integrity, cells are equipped with excellent systems to repair DSB, notably non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). These repair mechanisms are strictly regulated along with the DNA turnover cycle. NHEJ functions in all phases of the cell cycle, whereas HRR has a supplementary role specifically in S/G2 phase, where homologous DNA sequences are available in close proximity. The repair pathways are further regulated by a complex nuclear dynamism, where DSB are sensed and large numbers of repair proteins are recruited and assembled to form a repair complex involving multiple DSB. Considering such DSB repair dynamism, radiation-induced chromosome aberrations could be well understood as DSB-DSB pairwise interactions associated with the NHEJ pathway in all phases of the cell cycle and misrepair of a single DSB associated with the complementary HRR pathway in late S/G2 phase.     

Topoisomerase-I-Inhibitor mit potentiell strahlensensibilisierender Wirkung

Aim

In search of new drugs which should be theoretically able to modify radiation effects, the topoisomerase I-inhibitor topotecan was identified. In November 1996, approval for second-line therapy of ovarian carcinoma was given in Germany.

Material and Method

This review article describes mechanisms of action, pharmacokinetic, preclinical and recent clinical data on topotecan with and without concomitant radiation therapy.

Results

Following a 30 min intravenous bolus, median plasma half-lives between 2 and 3 h and a total-body clearance of 0.57 l/min/m² were seen. After oral administration the bioavailability varied between 30 and 35%. Topotecan is able to pass the blood brain barrier. Encouraging clinical results are reported in gastrointestinal tumors, head and neck squamous cell carcinoma, small cell and non-small cell lung cancer, breast cancer, leucaemia and pediatric tumors in addition to ovarian carcinoma. Recent studies indicate that topotecan function as a radiosensitizer and may be applied together with radiation in future trials. In-vivo and in-vitro experiments revealed a good modulation of radiation effects by topotecan.

Conclusions

In experimental and preclinical studies using topotecan both additive and sensitization effects to normal cells and tumor cells were seen by ionizing radiation. Clinical phase II/III-trials may preferentially be indicated in non-small cell lung cancer and primary and metastatic brain tumors.     

Image-Guided Therapy am geschlossenen MRT

Standard radiological methods

Standard imaging modalities for percutaneous minimally invasive therapy are ultrasound, fluoroscopy and computed tomography.

Methodical innovations

Magnetic resonance imaging is becoming increasingly more popular for minimally invasive procedures. The advantages are high soft-tissue contrast, the possibility of free selection of multiple imaging slices, multiple tools for intrainterventional monitoring and the absence of ionizing radiation for the patient and the interventional radiologist.

Achievements

Magnetic resonance imaging is a promising imaging modality for minimally invasive procedures. The most common clinical applications are thermoablative procedures for treatment of hepatic, renal and prostatic malignancies.     

Recombination between homologous chromosomes does not play a dominant role in the formation of radiation-induced chromosomal aberrations

Purpose : In mammalian cells, the relevance of homologous recombination in radiation-induced double-strand break (DSB) repair is not yet well understood. In the present work, the role of recombination between homologous chromosomes and homology-directed repair of DSB were studied, using X-ray-induced chromosomal aberrations as an end-point. Materials and methods : Human-hamster hybrid cells containing one or two copies of human chromosome 8 were used. If recombination between homologous chromosomes plays a dominant role in DSB repair, it is expected that X-irradiation of cells with two copies of chromosome 8 would result in a lower frequency of aberrations involving this chromosome compared with cells with only one copy of chromosome 8. The aberrations involving human chromosome 8 were detected by fluorescence in situ hybridization (FISH). Furthermore, a comparison between the hamster cell line XR-C1 (defective in non-homologous repair), CHO-9 (the wild-type cells) and the cell line XR-C1#8 (in which the defect of XR-C1 is complemented by human chromosome 8) was made to determine, indirectly, the involvement of homology-directed recombination in DSB repair. Results : The observed frequencies of aberrations per human chromosome 8 were not significantly different between cells containing one or two copies of this chromosome. The frequency of chromatid-type aberrations was doubled in XR-C1 cells compared with CHO-9 and XR-C1#8 cells. Conclusions : In hamster cells, recombination between homologous chromosomes appears not to have a major role in the formation of radiation-induced chromosomal aberrations, while nonhomologous repair seems to be important in both the G1 and G2 phases of the cell cycle.     

Recombination between homologous chromosomes does not play a dominant role in the formation of radiation-induced chromosomal aberrations

PURPOSE: In mammalian cells, the relevance of homologous recombination in radiation-induced double-strand break (DSB) repair is not yet well understood. In the present work, the role of recombination between homologous chromosomes and homology-directed repair of DSB were studied, using X-ray-induced chromosomal aberrations as an end-point. MATERIALS AND METHODS: Human-hamster hybrid cells containing one or two copies of human chromosome 8 were used. If recombination between homologous chromosomes plays a dominant role in DSB repair, it is expected that X-irradiation of cells with two copies of chromosome 8 would result in a lower frequency of aberrations involving this chromosome compared with cells with only one copy of chromosome 8. The aberrations involving human chromosome 8 were detected by fluorescence in situ hybridization (FISH). Furthermore, a comparison between the hamster cell line XR-C1 (defective in non-homologous repair), CHO-9 (the wild-type cells) and the cell line XR-C1#8 (in which the defect of XR-C1 is complemented by human chromosome 8) was made to determine, indirectly, the involvement of homology-directed recombination in DSB repair. RESULTS: The observed frequencies of aberrations per human chromosome 8 were not significantly different between cells containing one or two copies of this chromosome. The frequency of chromatid-type aberrations was doubled in XR-C1 cells compared with CHO-9 and XR-C1#8 cells. CONCLUSIONS: In hamster cells, recombination between homologous chromosomes appears not to have a major role in the formation of radiation-induced chromosomal aberrations, while nonhomologous repair seems to be important in both the G and G2 phases of the cell cycle.     

Nachweis einer strahleninduzierten Produktion von Tumor-Nekrose-Faktor alpha im Ewing-Sarkom RM 82 in vitro und in vivo

Aim

The expression of cytokines plays an important role in the transmission of the effects of ionizing radiation to tumor cells and normal tissue. Tumor necrosis factor alpha (TNF α), a pleiotropic monokine, is of special interest because of its cytotoxic effect on tumor cells and the induction of hemorrhagic necrosis in tumors. We examined the influence of ionizing radiation on TNF α production in a human Ewing’s sarcoma cell line in vitro and in vivo.

Methods

The protein and mRNA levels of the Ewing’s sarcoma cell line RM 82 were examined in vitro with ?Enhanced Amplified Sensitivity Immunoassay” (EASIA) and semiquantitative RT-PCR before and after treatment with single doses of 2 to 40 Gy, 1 to 72 hours after irradiation. After successful transplantation to nude mice, the time and dose correlation of TNF α mRNA production was examined in vivo.

Results

In vitro, RM 82 had a basal protein level of TNF α of 20.1±4.3 pg/ml/10⁶ cells. We observed a time- and dose-dependent increase of TNF α expression with a maximum of 125 pg/ml/10⁶ (5.9fold) 24 hours after irradiation with 20 Gy. At the mRNA level, the maximal up-regulation occurred 6 to 12 hours after 10 Gy. In vivo, the xenograft tumor maintained the capacity of TNF α expression. Time-and dose-dependency in mRNA production showed a maximum increase 6 hours after treatment with 10 Gy.

Conclusions

The presented experiments show in vitro a dose- and time-dependent up-regulation of TNF α in the Ewing’s sarcoma cell line RM 82 on protein and mRNA level. For the first time this phenomenon was also observed in vivo in a human xenograft tumor. This tumor model could be used for further experiments to examine the role of TNF α as a biologic radiation response modifier in human tumors.     

Hyperthermic radiosensitization: mode of action and clinical relevance

PURPOSE: To provide an update on the recent knowledge about the molecular mechanisms of thermal radiosensitization and its possible relevance to thermoradiotherapy. SUMMARY: Hyperthermia is probably the most potent cellular radiosensitizer known to date. Heat interacts with radiation and potentiates the cellular action of radiation by interfering with the cells' capability to deal with radiation-induced DNA damage. For ionizing irradiation, heat inhibits the repair of all types of DNA damage. Genetic and biochemical data suggest that the main pathways for DNA double-strand break (DSB) rejoining, non-homologous end-joining and homologous recombination, are not the likely primary targets for heat-induced radiosensitization. Rather, heat is suggested to affect primarily the religation step of base excision repair. Subsequently additional DSB arise during the DNA repair process in irradiated and heated cells and these additional DSB are all repaired with slow kinetics, the repair of which is highly error prone. Both mis- and non-rejoined DSB lead to an elevated number of lethal chromosome aberrations, finally causing additional cell killing. Heat-induced inhibition of DNA repair is considered not to result from altered signalling or enzyme inactivation but rather from alterations in higher-order chromatin structure. Although, the detailed mechanisms are not yet known, a substantial body of indirect and correlative data suggests that heat-induced protein aggregation at the level of attachment of looped DNA to the nuclear matrix impairs the accessibility of the damaged DNA for the repair machinery or impairs the processivity of the repair machinery itself. CONCLUSION: Since recent phase III clinical trials have shown significant benefit of adding hyperthermia to radiotherapy regimens for a number of malignancies, it will become more important again to determine the molecular effects underlying this success. Such information could eventually also improve treatment quality in terms of patient selection, improved sequencing of the heat and radiation treatments, the number of heat treatments, and multimodality treatments (i.e. thermochemoradiotherapy).     

Genetische Prädisposition und Strahlenempfindlichkeit bei normalen Geweben

Background

After radiotherapy there are always some patients, who develop strong acute and late reactions in normal tissues. In these patients frequently a genetic predisposition is observed. There are found DNA-repair deficiencies and changes in the regulation of the cell cycle which are responsible for the increased radiosensitivity with enhanced cell killing.

Methods

The micronucleus test and the comet assay appear to be appropriate tests in order to measure this increased radiosensitivity. Both tests are characterized by being relatively quick and simple and can be performed with small cell numbers. It is possible to study blood lymphocytes and fibroblasts with these tests.

Results

Both tests can predict the radiosensitivity of normal tissues especially if they are applied in combination.

Conclusions

Epidemiological studies with patients after radiotherapy show evidence that the increased radiosensitivity also causes an enhanced induction of secondary tumors by ionizing radiation. This is supported by corresponding animal models.     

Purple sweet potato pigments protect murine thymocytes from 60Co γ-ray-induced mitochondria-mediated apoptosis

Purpose:?Ionising radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumourigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H₂O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies.

Conclusions:?In the past 50 years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation.     

Modeling the absorbed dose to the common carotid arteries following radioiodine treatment of benign thyroid disease

Introduction

External fractionated radiotherapy of cancer increases the risk of cardio- and cerebrovascular events, but less attention has been paid to the potential side effects on the arteries following internal radiotherapy with radioactive iodine (RAI), i.e. 131-iodine. About 279 per million citizens in the western countries are treated each year with RAI for benign thyroid disorders (about 140,000 a year in the EU), stressing that it is of clinical importance to be aware of even rare radiation-induced side effects. In order to induce or accelerate atherosclerosis, the dose to the carotid arteries has to exceed 2 Gy which is the known lower limit of ionizing radiation to affect the endothelial cells and thereby to induce atherosclerosis.

Objective

To estimate the radiation dose to the carotid arteries following RAI therapy of benign thyroid disorders.

Methods

Assuming that the lobes of the thyroid gland are ellipsoid, that the carotid artery runs through a part of the lobes, that there is a homogeneous distribution of RAI in the lobes, and that the 24 h RAI uptake in the thyroid is 35 % of the ¹³¹I orally administrated, we used integrated modules for bioassay analysis and Monte Carlo simulations to calculate the dose in Gy/GBq of administrated RAI.

Results

The average radiation dose along the arteries is 4–55 Gy/GBq of the ¹³¹I orally administrated with a maximum dose of approximately 25–85 Gy/GBq. The maximum absorbed dose rate to the artery is 4.2 Gy/day per GBq ¹³¹I orally administrated.

Conclusion

The calculated radiation dose to the carotid arteries after RAI therapy of benign thyroid disorder clearly exceeds the 2 Gy known to affect the endothelial cells and properly induce atherosclerosis. This simulation indicates a relation between the deposited dose in the arteries following RAI treatment and an increased risk of atherosclerosis and subsequent cerebrovascular events such as stroke.     

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells

Background and purpose

Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation.

Materials and methods

A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p.

Results

A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo.

Conclusion

miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells.