Determine the yield of micronucleated cells in primary human fibroblasts exposed to focused soft X-rays.

This project was a small part of a larger collaborative study headed by Dr Aloke Chatterjee, (Lawrence Berkeley National Laboratory) and including Drs Les Braby, John Ford (Texas A&M) and Kathy Held (MGH Boston), which was developing an integrated theoretical and experimental model of the radiation-induced bystander response. Our part of the study has been to determine the effectiveness of soft X-rays at inducing chromosomal damage under conditions of direct and bystander exposure. The aim was to compare this with the effectiveness of the low energy 60 kV electron microbeam available at Texas A&M. Previous studies have been performed with primary human fibroblasts measuring micronuclei formation to determine the relative yields of direct versus bystander mediated micronuclei formation after cells were individually irradiated utilizing our novel focused soft X-ray microprobe, which is capable of producing localized submicron beams of carbon-K (278 eV) X-rays. Only a brief overview is given here as the study has been published in several papers. Our original hypothesis was to study yields of bystander-induced micronucleated cells in both wild-type and mutant fibroblast from mouse embryo fibroblasts. Difficulties with the level of background micronuclei in the MEFs prevented systematic studies of bystander responses in the laboratories involved in the collaboration. We then performed these studies with AG1522 primary human fibroblast cells using a siRNA approach developed by John Ford at Texas A&M to knock down DNA PKcs in the first instance. Our soft X-ray source has been in routine use for carbon-K X-rays and is now available with Aluminium-K (1.49 keV) and titanium-K (4.5 keV), although the dose-rate from titanium is still too low at present for most experiments, where large numbers of cells need to be exposed. A separately funded project developed a new soft X-ray microprobe which will give much greater flexibility for changing energies and giving high dose-rates for exposures (See DE-FG02-01ER63236). However, we performed pilot studies measuring bystander responses with titanium-K. To date we have performed studies with V79 cells measuring cell survival as an endpoint and are starting studies in our human fibroblasts to measure micronuclei yields. A significant bystander response is observed in the V79 cells under conditions where only a single cell within a population was irradiated either with carbon-K or titanium-K X-rays. Typically around 10% cell killing is observed under these conditions. These studies are now being extended to measure micronuclei yields in the AG1522 cells under direct and bystander conditions. Our work has suggested that the yield of micronuclei in fibroblasts exposed to soft X-rays may be reduced in comparison to conventional X-ray exposures (Prise et al., 2003). Although further studies are required to confirm this using a range of scoring times

Pro-inflammatory Signaling in a 3D Organotypic Skin Model after Low LET Irradiation-NF-kappa B, COX-2 Activation, and Impact on Cell Differentiation

Nearly 85% of radiotherapy patients develop acute radiation dermatitis, which is an inflammatory reaction of the skin at the treatment field and in the surrounding area. The aims of this study were to unravel the mechanisms of radiation-induced inflammatory responses after localized irradiation in a human 3D organotypic skin culture model. This could provide possible inflammatory targets for reduction of skin side effects. 3D organotypic skin cultures were set up and locally irradiated with 225 kVp X-rays, using a combination of full exposure and partial shielding (50%) of the cultures. The secretion of pro-inflammatory cytokines, the phenotype, and the differentiation markers expression of the cultures were assessed up to 10 days postirradiation. The pro-inflammatory transcription factor nuclear factor kappa B (NF-kappa B) and cyclooxygenase-2 (COX-2) pathways have been studied. The results showed fast activation of NF-kappa B, most likely triggered by DNA damage in the irradiated cells, followed by upregulation of p38 MAPK and COX-2 in the irradiated and surrounding, non-irradiated, areas of the 3D cultures. The application of the COX-2 inhibitor sc-236 was effective at reducing the COX-2 mRNA levels 4 h postirradiation. The same inhibitor also suppressed the PGE2 secretion significantly 72 h after the treatment. The expression of a pro-inflammatory phenotype and abnormal differentiation markers of the cultures were also reduced. However, the use of an NF-kappa B inhibitor (Bay 11-7085) did not have the predicted positive effect on the cultures phenotype postirradiation. Radiation-induced pro-inflammatory responses have been observed in the 3D skin model. The activated signaling pathways involved NF-kappa B transcription factor and its downstream target COX-2. Further experiments aiming to suppress the inflammatory response via specific inhibitors showed that COX-2 is a suitable target for reduction of the normal skin inflammatory responses at radiotherapy, while NF-kappa B inhibition had detrimental effects on the 3D skin model development.Peer reviewe

Mechanistic Modelling of DNA Repair and Cellular Survival Following Radiation-Induced DNA Damage

Characterising and predicting the effects of ionising radiation on cells remains challenging, with the lack of robust models of the underlying mechanism of radiation responses providing a significant limitation to the development of personalised radiotherapy. In this paper we present a mechanistic model of cellular response to radiation that incorporates the kinetics of different DNA repair processes, the spatial distribution of double strand breaks and the resulting probability and severity of misrepair. This model enables predictions to be made of a range of key biological endpoints (DNA repair kinetics, chromosome aberration and mutation formation, survival) across a range of cell types based on a set of 11 mechanistic fitting parameters that are common across all cells. Applying this model to cellular survival showed its capacity to stratify the radiosensitivity of cells based on aspects of their phenotype and experimental conditions such as cell cycle phase and plating delay (correlation between modelled and observed Mean Inactivation Doses R(2) > 0.9). By explicitly incorporating underlying mechanistic factors, this model can integrate knowledge from a wide range of biological studies to provide robust predictions and may act as a foundation for future calculations of individualised radiosensitivity

Commentary: Opportunities for research in molecular radiotherapy

Cancer has been treated with radiopharmaceuticals for 80 years. A recent National Cancer Research Institute report from the Clinical and Translational Radiotherapy Research Working Group reviews the current status of molecular radiotherapy and has highlighted the barriers to and opportunities for increased research activities. The report recommends a number of actions to promote this field, which in the dawning age of personalized medicine and theragnostics is of increasing importance, particularly with the clinical introduction of a range of new commercial radiotherapeutics at costs in line with those seen for conventional chemotherapeutics. These recommendations recognize the importance of a multidisciplinary approach to the development of molecular radiotherapy and the particular need for investment in radiopharmacies and personalized dosimetry. There are many areas to be investigated including adaptive treatment planning, the use of radiosensitizers and translational radiation biology. Progress in these areas will result in significant patient benefit and more cost-effective use of increasingly expensive therapeutic radiopharmaceuticals. A concerted effort from the community, from funding bodies and from health service providers is now needed to address the scientific and logistical changes necessary to realize the potential offered by this currently underused treatment modality

Mechanistic Modelling of Radiation Responses

Radiobiological modelling has been a key part of radiation biology and therapy for many decades, and many aspects of clinical practice are guided by tools such as the linear-quadratic model. However, most of the models in regular clinical use are abstract and empirical, and do not provide significant scope for mechanistic interpretation or making predictions in novel cell lines or therapies. In this review, we will discuss the key areas of ongoing mechanistic research in radiation biology, including physical, chemical, and biological steps, and review a range of mechanistic modelling approaches which are being applied in each area, highlighting the possible opportunities and challenges presented by these techniques

Biological Mechanisms of Gold Nanoparticle Radiosenstitisation

There has been growing interest in the use of nanomaterials for a range of biomedical applications over the last number of years. In particular, gold nanoparticles (GNPs) possess a number of unique properties that make them ideal candidates as radiosensitizers on the basis of their strong photoelectric absorption coefficient and ease of synthesis. However, despite promising preclinical evidence in vitro supported by a limited amount of in vivo experiments, along with advances in mechanistic understanding, GNPs have not yet translated into the clinic. This may be due to disparity between predicted levels of radiosensitization based on physical action, observed biological response and an incomplete mechanistic understanding, alongside current experimental limitations. This paper provides a review of the current state of the field, highlighting the potential underlying biological mechanisms in GNP radiosensitization and examining the barriers to clinical translation

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

A Constrained Coding Approach to Error-Free Half-Duplex Relay Networks

We show that the broadcast capacity of an infinite-depth tree-structured network of error-free half-duplex-constrained relays can be achieved using constrained coding at the source and symbol forwarding at the relays.Comment: To appear in IEEE Transactions on Information Theory, 201