Down-regulation of PERK enhances resistance to ionizing radiation

Although, ionizing radiation (IR) has been implicated to cause stress in endoplasmic reticulum (ER), how ER stress signaling and major ER stress sensors modulate cellular response to IR is unclear. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein which initiates unfolded protein response (UPR) or ER stress signaling when ER homeostasis is disturbed. Here, we report that down-regulation of PERK resulted in increased clonogenic survival, enhanced DNA repair and reduced apoptosis in irradiated cancer cells. Our study demonstrated that PERK has a role in sensitizing cancer cells to IR. 

Gold nanoparticle cellular uptake, toxicity and radiosensitisation in hypoxic conditions

Background and purpose: Gold nanoparticles (GNPs) are novel agents that have been shown to cause radiosensitisation in vitro and in vivo. Tumour hypoxia is associated with radiation resistance and reduced survival in cancer patients. The interaction of GNPs with cells in hypoxia is explored.

Materials and methods: GNP uptake, localization, toxicity and radiosensitisation were assessed in vitro under oxic and hypoxic conditions.

Results: GNP cellular uptake was significantly lower under hypoxic than oxic conditions. A significant reduction in cell proliferation in hypoxic MDA-MB-231 breast cancer cells exposed to GNPs was observed. In these cells significant radiosensitisation occurred in normoxia and moderate hypoxia. However, in near anoxia no significant sensitisation occurred.

Conclusions: GNP uptake occurred in hypoxic conditions, causing radiosensitisation in moderate, but not extreme hypoxia in a breast cancer cell line. These findings may be important for the development of GNPs for cancer therapy.

Combined Analysis of Gamma-H2AX/53BP1 Foci and Caspase Activation in Lymphocyte Subsets Detects Recent and More Remote Radiation Exposures

Analysis of gamma-H2AX foci in blood lymphocytes is a promising approach for rapid dose estimation to support patient triage after a radiation accident but has one major drawback: the rapid decline of foci levels post-exposure cause major uncertainties in situations where the exact timing between exposure and blood sampling is unknown. To address this issue, radiation-induced apoptosis (RIA) in lymphocytes was investigated using fluorogenic inhibitors of caspases (FLICA) as an independent biomarker for radiation exposure, which may complement the gamma-H2AX assay. Ex vivo X-irradiated peripheral blood lymphocytes from 17 volunteers showed dose-and time-dependent increases in radiation-induced apoptosis over the first 3 days after exposure, albeit with considerable interindividual variation. Comparison with gamma-H2AX and 53BP1 foci counts suggested an inverse correlation between numbers of residual foci and radiation-induced apoptosis in lymphocytes at 24 h postirradiation (P = 0.007). In T-helper (CD4), T-cytotoxic (CD8) and B-cells (CD19), some significant differences in radiation induced DSBs or apoptosis were observed, however no correlation between foci and apoptosis in lymphocyte subsets was observed at 24 h postirradiation. While gamma-H2AX and 53BP1 foci were rapidly induced and then repaired after exposure, radiation-induced apoptosis did not become apparent until 24 h after exposure. Data from six volunteers with different ex vivo doses and post-exposure times were used to test the capability of the combined assay. Results show that simultaneous analysis of gamma-H2AX and radiation-induced apoptosis may provide a rapid and more accurate triage tool in situations where the delay between exposure and blood sampling is unknown compared to gamma-H2AX alone. This combined approach may improve the accuracy of dose estimations in cases where blood sampling is performed days after the radiation exposure. 

State of the art in research into the risk of low dose radiation exposure-findings of the fourth MELODI workshop

The fourth workshop of the Multidisciplinary European Low Dose Initiative (MELODI) was organised by STUK-Radiation and Nuclear Safety Authority of Finland. It took place from 12 to 14 September 2012 in Helsinki, Finland. The meeting was attended by 179 scientists and professionals engaged in radiation research and radiation protection. We summarise the major scientific findings of the workshop and the recommendations for updating the MELODI Strategic Research Agenda and Road Map for future low dose research activities.

Increased susceptibility to delayed genetic effects of low dose X-irradiation in DNA repair deficient cells

Purpose: To examine whether the levels of micronuclei induction, as a marker for genomic instability in the progeny of X-irradiated cells, correlates with DNA repair function.

Materials and methods: Two repair deficient cell lines (X-ray repair cross-complementing 1 [XRCC1] deficient cell line [EM9] and X-ray repair cross complementing 5 [XRCC5; Ku80] deficient X-ray sensitive Chinese hamster ovary [CHO] cell line [xrs5]) were used in addition to wild-type CHO cells. These cells were irradiated with low doses of X-rays (up to 1 Gy). Seven days after irradiation, micronuclei formed in binucleated cells were counted. To assess the contribution of the bystander effect micronuclei induction was measured in progeny of non-irradiated cells co-cultured with cells that had been irradiated with 1Gy.

Results: The delayed induction of micronuclei in 1 Gy-irradiated cells was observed in normal CHO and EM9 but not in xrs5. In the clone analysis, progenies of xrs5 under bystander conditions showed significantly higher levels of micronuclei, while CHO and EM9 did not.

Conclusion: Genomic instability induced by X-irradiation is associated with DSB (double-strand break) repair, even at low doses. It is also suggested that bystander signals, which lead to genomic instability, may be enhanced when DSB repair is compromised.

Non-targeted effects of ionising radiation-Implications for low dose risk

Non-DNA targeted effects of ionising radiation, which include genomic instability, and a variety of bystander effects including abscopal effects and bystander mediated adaptive response, have raised concerns about the magnitude of low-dose radiation risk. Genomic instability, bystander effects and adaptive responses are powered by fundamental, but not clearly understood systems that maintain tissue homeostasis. Despite excellent research in this field by various groups, there are still gaps in our understandfng of the likely mechanisms associated with non-DNA targeted effects, particularly with respect to systemic (human health) consequences at low and intermediate doses of ionising radiation. Other outstanding questions include links between the different non-targeted responses and the variations. in response observed between individuals and cell lines, possibly a function of genetic background. Furthermore, it is still not known what the initial target and early interactions in cells are that give rise to non-targeted responses in neighbouring or descendant cells. This paper provides a commentary on the current state of the field as a result of the non-targeted effects of ionising radiation (NOTE) Integrated Project funded by the European Union. Here we critically examine the evidence for non-targeted effects, discuss apparently contradictory results and consider implications for low-dose radiation health effects. (C) 2012 Elsevier B.V. All rights reserved.

Conventional in vivo irradiation procedures are insufficient to accurately determine tumor responses to non-uniform radiation fields

Purpose: To determine differences in overall tumor responses measured by volumetric assessment and bioluminescence imaging (BLI) following exposure to uniform and non-uniform radiation fields in an ectopic prostate tumor model.

Materials and methods: Bioluminescent human prostate tumor xenografts were established by subcutaneous implantation into male mice. Tumors were irradiated with uniform or non-uniform field configurations using conventional in vivo irradiation procedures performed using a 225 kVp generator with custom lead shielding. Tumor responses were measured using Vernier calipers and by BLI using an in vivo imaging system. Survival was defined as the time to quadroupling of pre-treatment tumor volume. 

Results: The correlation between BLI and tumor volume measurements was found to be different for un-irradiated (R = 0.61), uniformly irradiated (R = 0.34) and partially irradiated (R = 0.30) tumors. Uniformly irradiated tumors resulted in an average tumor growth delay of 60 days with median survival of 75 days, compared to partially irradiated tumors which showed an average growth delay of 24 days and median survival of 38 days. 

Conclusions: Correlation between BLI and tumor volume measurements is lower for partially irradiated tumors than those exposed to uniform dose distributions. The response of partially irradiated tumors suggests non-uniformity in response beyond physical dose distribution within the target volume. Dosimetric uncertainty associated with conventional in vivo irradiation procedures prohibits their ability to accurately determine tumor response to non-uniform radiation fields and stresses the need for image guided small animal radiation research platforms.

BRCA1, FANCD2 and Chk1 are potential molecular targets for the modulation of a radiation-induced DNA damage response in bystander cells

Radiotherapy is an important treatment option for many human cancers. Current research is investigating the use of molecular targeted drugs in order to improve responses to radiotherapy in various cancers. The cellular response to irradiation is driven by both direct DNA damage in the targeted cell and intercellular signalling leading to a broad range of bystander effects. This study aims to elucidate radiation-induced DNA damage response signalling in bystander cells and to identify potential molecular targets to modulate the radiation induced bystander response in a therapeutic setting. Stalled replication forks in T98G bystander cells were visualised via bromodeoxyuridine (BrdU) nuclear foci detection at sites of single stranded DNA. γH2AX co-localised with these BrdU foci. BRCA1 and FANCD2 foci formed in T98G bystander cells. Using ATR mutant F02-98 hTERT and ATM deficient GM05849 fibroblasts it could be shown that ATR but not ATM was required for the recruitment of FANCD2 to sites of replication associated DNA damage in bystander cells whereas BRCA1 bystander foci were ATM-dependent. Phospho-Chk1 foci formation was observed in T98G bystander cells. Clonogenic survival assays showed moderate radiosensitisation of directly irradiated cells by the Chk1 inhibitor UCN-01 but increased radioresistance of bystander cells. This study identifies BRCA1, FANCD2 and Chk1 as potential targets for the modulation of radiation response in bystander cells. It adds to our understanding of the key molecular events propagating out-of-field effects of radiation and provides a rationale for the development of novel molecular targeted drugs for radiotherapy optimisation.

Radioprotection of targeted and bystander cells by methylproamine

INTRODUCTION: Radioprotective agents are of interest for application in radiotherapy for cancer and in public health medicine in the context of accidental radiation exposure. Methylproamine is the lead compound of a class of radioprotectors which act as DNA binding anti-oxidants, enabling the repair of transient radiation-induced oxidative DNA lesions. This study tested methylproamine for the radioprotection of both directly targeted and bystander cells.

METHODS: T98G glioma cells were treated with 15 μM methylproamine and exposed to (137)Cs γ-ray/X-ray irradiation and He(2+) microbeam irradiation. Radioprotection of directly targeted cells and bystander cells was measured by clonogenic survival or γH2AX assay.

RESULTS: Radioprotection of directly targeted T98G cells by methylproamine was observed for (137)Cs γ-rays and X-rays but not for He(2+) charged particle irradiation. The effect of methylproamine on the bystander cell population was tested for both X-ray irradiation and He(2+) ion microbeam irradiation. The X-ray bystander experiments were carried out by medium transfer from irradiated to non-irradiated cultures and three experimental designs were tested. Radioprotection was only observed when recipient cells were pretreated with the drug prior to exposure to the conditioned medium. In microbeam bystander experiments targeted and nontargeted cells were co-cultured with continuous methylproamine treatment during irradiation and postradiation incubation; radioprotection of bystander cells was observed.

DISCUSSION AND CONCLUSION: Methylproamine protected targeted cells from DNA damage caused by γ-ray or X-ray radiation but not He(2+) ion radiation. Protection of bystander cells was independent of the type of radiation which the donor population received.

The role of mitochondrial function in gold nanoparticle mediated radiosensitisation

Gold nanoparticles (GNPs), have been demonstrated as effective preclinical radiosensitising agents in a range of cell models and radiation sources. These studies have also highlighted difficulty in predicted cellular radiobiological responses mediated by GNPs, based on physical assumptions alone, and therefore suggest a significant underlying biological component of response. This study aimed to determine the role of mitochondrial function in GNP radiosensitisation. Using assays of DNA damage and mitochondrial function through levels of oxidation and loss of membrane potential, we demonstrate a potential role of mitochondria as a central biological mechanism of GNP mediated radiosensitisation.

High dose bystander effects in spatially fractionated radiation therapy

Traditional radiotherapy of bulky tumors has certain limitations. Spatially fractionated radiation therapy (GRID) and intensity modulated radiotherapy (IMRT) are examples of advanced modulated beam therapies that help in significant reductions in normal tissue damage. GRID refers to the delivery of a single high dose of radiation to a large treatment area that is divided into several smaller fields, while IMRT allows improved dose conformity to the tumor target compared to conventional three-dimensional conformal radiotherapy. In this review, we consider spatially fractionated radiotherapy approaches focusing on GRID and IMRT, and present complementary evidence from different studies which support the role of radiation induced signaling effects in the overall radiobiological rationale for these treatments.

Small animal image-guided radiotherapy: status, considerations and potential for translational impact.

Radiation biology is being transformed by the implementation of small animal image-guided precision radiotherapy into pre-clinical research programmes worldwide. We report on the current status and developments of the small animal radiotherapy field, suggest criteria for the design and execution of effective studies and contend that this powerful emerging technology, used in combination with relevant small animal models, holds much promise for translational impact in radiation oncology.