Biological Cell Irradiation At Ultrahigh Dose Rate Employing Laser Driven Protons

The ultrashort duration of laser-driven multi-MeV ion bursts offers the possibility of radiobiological studies at extremely high dose rates. Employing the TARANIS Terawatt laser at Queen's University, the effect of proton irradiation at MeV-range energies on live cells has been investigated at dose rates exceeding 10⁹Gy/s as a single exposure. A clonogenic assay showed consistent lethal effects on V-79 live cells, which, even at these dose rates, appear to be in line with previously published results employing conventional sources. A Relative Biological Effectiveness (RBE) of 1.4±0.2 at 10% survival is estimated from a comparison with a 225 kVp X-ray source. 

Relative biological effectiveness (RBE) and out-of-field cell survival responses to passive scattering and pencil beam scanning proton beam deliveries

The relative biological effectiveness (RBE) of passive scattered (PS) and pencil beam scanned (PBS) proton beam delivery techniques for uniform beam configurations was determined by clonogenic survival. The radiobiological impact of modulated beam configurations on cell survival occurring in- or out-of-field for both delivery techniques was determined with intercellular communication intact or physically inhibited. Cell survival responses were compared to those observed using a 6 MV photon beam produced with a linear accelerator. DU-145 cells showed no significant difference in survival response to proton beams delivered by PS and PBS or 6 MV photons taking into account a RBE of 1.1 for protons at the centre of the spread out Bragg peak. Significant out-of-field effects similar to those observed for 6 MV photons were observed for both PS and PBS proton deliveries with cell survival decreasing to 50-60% survival for scattered doses of 0.05 and 0.03 Gy for passive scattered and pencil beam scanned beams respectively. The observed out-of-field responses were shown to be dependent on intercellular communication between the in-and out-of-field cell populations. These data demonstrate, for the first time, a similar RBE between passive and actively scanned proton beams and confirm that out-of-field effects may be important determinants of cell survival following exposure to modulated photon and proton fields

Development of a novel experimental model to investigate radiobiological implications of respiratory motion in advanced radiotherapy

Respiratory motion introduces complex spatio-temporal variations in the dosimetry of radiotherapy. There is a paucity of literature investigating the radiobiological consequences of intrafraction motion and concerns regarding the impact of movement when applied to cancer cell lines in vitro exist. We have addressed this by developing a novel model which accurately replicates respiratory motion under experimental conditions to allow clinically relevant irradiation of cell lines. A bespoke phantom and motor driven moving platform was adapted to accommodate flasks containing medium and cells in order to replicate respiratory motion using varying frequencies and amplitude settings. To study this effect on cell survival in vitro, dose response curves were determined for human lung cancer cell lines H1299 and H460 exposed to a uniform 6 MV radiation field under moving or stationary conditions. Cell survival curves showed no significant difference between irradiation at different dose points for these cell lines in the presence or absence of motion. These data indicate that motion of unshielded cells in vitro does not affect cell survival in the presence of uniform irradiation. This model provides a novel research platform to investigate the radiobiological consequences of respiratory motion in radiotherapy.

In-vitro investigation of out-of-field cell survival following the delivery of conformal, intensity-modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT) plans

The aim of this work is to determine the out-of-field survival of cells irradiated with either the primary field or scattered radiation in the presence and absence of intercellular communication following delivery of conformal, IMRT and VMAT treatment plans. Single beam, conformal, IMRT and VMAT plans were created to deliver 3 Gy to half the area of a T80 flask containing either DU-145 or AGO-1522 cells allowing intercellular communication between the in-and out-of-field cell populations. The same plans were delivered to a similar custom made phantom used to hold two T25 culture flasks, one flask in-field and one out-of-field to allow comparison of cell survival responses when intercellular communication is physically inhibited. Plans were created for the delivery of 8 Gy to the more radio-resistant DU-145 cells only in the presence and absence of intercellular communication. Cell survival was determined by clonogenic assay. In both cell lines, the out-of-field survival was not statistically different between delivery techniques for either cell line or dose. There was however, a statistically significant difference between survival out-of-field when intercellular communication was intact (single T80 culture flask) or inhibited (multiple T25 culture flasks) to in-field for all plans. No statistically significant difference was observed in-field with or without cellular communication to out-of-field for all plans. These data demonstrate out-of-field effects as important determinants of cell survival following exposure to modulated irradiation fields when cellular communication between differentially irradiated cell populations is present. This data is further evidence that refinement of existing radiobiological models to include indirect cell killing effects is required.

Physical basis and biological mechanisms of gold nanoparticle radiosensitization

The unique properties of nanomaterials, in particular gold nanoparticles (GNPs) have applications for a wide range of biomedical applications. GNPs have been proposed as novel radiosensitizing agents due to their strong photoelectric absorption coefficient. Experimental evidence supporting the application of GNPs as radiosensitizing agents has been provided from extensive in vitro investigation and a relatively limited number of in vivo studies. Whilst these studies provide experimental evidence for the use of GNPs in combination with ionising radiation, there is an apparent disparity between the observed experimental findings and the level of radiosensitization predicted by mass energy absorption and GNP concentration. This review summarises experimental findings and attempts to highlight potential underlying biological mechanisms of response in GNP radiosensitization.

KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1α survival pathways

KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1a (HIF-1a). HIF-1a is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1a. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1a in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1a levels in KNK437-treated cells. This suggested that the absence of HIF-1a in hypoxic cells was not due to the enhanced protein degradation. HIF-1a is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1a mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1a levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

DNA damage responses following exposure to modulated radiation fields

During the delivery of advanced radiotherapy treatment techniques modulated beams are utilised to increase dose conformity across the target volume. Recent investigations have highlighted differential cellular responses to modulated radiation fields particularly in areas outside the primary treatment field that cannot be accounted for by scattered dose alone. In the present study, we determined the DNA damage response within the normal human fibroblast AG0-1522B and the prostate cancer cell line DU-145 utilising the DNA damage assay. Cells plated in slide flasks were exposed to 1 Gy uniform or modulated radiation fields. Modulated fields were delivered by shielding 25%, 50% or 75% of the flask during irradiation. The average number of 53BP1 or ?H2AX foci was measured in 2 mm intervals across the slide area. Following 30 minutes after modulated radiation field exposure an increase in the average number of foci out-of-field was observed when compared to non-irradiated controls. In-field, a non-uniform response was observed with a significant decrease in the average number of foci compared to uniformly irradiated cells. Following 24 hrs after exposure there is evidence for two populations of responding cells to bystander signals in-and out-of-field. There was no significant difference in DNA damage response between 25%, 50% or 75% modulated fields. The response was dependent on cellular secreted intercellular signalling as physical inhibition of intercellular communication abrogated the observed response. Elevated residual DNA damage observed within out-of-field regions decreased following addition of an inducible nitric oxide synthase inhibitor (Aminoguanidine). These data show, for the first time, differential DNA damage responses in-and out-of-field following modulated radiation field delivery. This study provides further evidence for a role of intercellular communication in mediating cellular radiobiological response to modulated radiation fields and may inform the refinement of existing radiobiological models for the optimization of advanced radiotherapy treatment plans. © 2012 Trainor et al.

A Kinetic-Based Model of Radiation-Induced Intercellular Signalling

It is now widely accepted that intercellular communication can cause significant variations in cellular responses to genotoxic stress. The radiation-induced bystander effect is a prime example of this effect, where cells shielded from radiation exposure see a significant reduction in survival when cultured with irradiated cells. However, there is a lack of robust, quantitative models of this effect which are widely applicable. In this work, we present a novel mathematical model of radiation-induced intercellular signalling which incorporates signal production and response kinetics together with the effects of direct irradiation, and test it against published data sets, including modulated field exposures. This model suggests that these so-called "bystander" effects play a significant role in determining cellular survival, even in directly irradiated populations, meaning that the inclusion of intercellular communication may be essential to produce robust models of radio-biological outcomes in clinically relevant in vivo situations.

Antiproton induced DNA damage: proton like in flight, carbon-ion like near rest

Biological validation of new radiotherapy modalities is essential to understand their therapeutic potential. Antiprotons have been proposed for cancer therapy due to enhanced dose deposition provided by antiproton-nucleon annihilation. We assessed cellular DNA damage and relative biological effectiveness (RBE) of a clinically relevant antiproton beam. Despite a modest LET (,19 keV/mm), antiproton spread out Bragg peak (SOBP) irradiation caused significant residual c-H2AX foci compared to X-ray, proton and antiproton plateau irradiation. RBE of ,1.48 in the SOBP and ,1 in the plateau were measured and used for a qualitative effective dose curve comparison with proton and carbon-ions. Foci in the antiproton SOBP were larger and more structured compared to X-rays, protons and carbon-ions. This is likely due to overlapping particle tracks near the annihilation vertex, creating spatially correlated DNA lesions. No biological effects were observed at 28–42 mm away from the primary beam suggesting minimal risk from long-range secondary particles.

An in vitro study of the radiobiological effects of flattening filter free radiotherapy treatments

Flattening filter free (FFF) linear accelerators allow for an increase in instantaneous dose-rate of the x-ray pulses by a factor of 2-6 over the conventional flattened output. As a result, radiobiological investigations are being carried out to determine the effect of these higher dose-rates on cell response. The studies reported thus far have presented conflicting results, highlighting the need for further investigation. To determine the radiobiological impact of the increased dose-rates from FFF exposures a Varian Truebeam medical linear accelerator was used to irradiate two human cancer cell lines in vitro, DU-145 prostate and H460 non-small cell lung, with both flattened and FFF 6 MV beams. The fluence profile of the FFF beam was modified using a custom-designed Nylon compensator to produce a similar dose profile to the flattened beam (6X) at the cell surface but at a higher instantaneous dose-rate. For both cell lines there appeared to be no significant change in cell survival. Curve fitting coefficients for DU145 cells irradiated with constant average dose-rates were 6X: alpha = 0.09 +/- 0.03, beta = 0.03 +/- 0.01 and 6FFF: alpha = 0.14 +/- 0.13, beta = 0.03 +/- 0.02 with a significance of p = 0.75. For H460 cells irradiated with the same instantaneous dose-rate but different average dose-rate the fit coefficients were 6FFF (low dose-rate): alpha = 0.21 +/- 0.11, 0.07 +/- 0.02 and 6FFF (high dose-rate): alpha = 0.21 +/- 0.16, 0.07 +/- 0.03, with p = 0.79. The results indicate that collective damage behaviour does not occur at the instantaneous dose-rates investigated here and that the use of either modality should result in the same clinical outcome, however this will require further validation in vivo.

DNA double strand break repair: a radiation perspective

SIGNIFICANCE:
Ionizing radiation (IR) can induce a wide range of unique deoxyribonucleic acid (DNA) lesions due to the spatiotemporal correlation of the ionization produced. Of these, DNA double strand breaks (DSBs) play a key role. Complex mechanisms and sophisticated pathways are available within cells to restore the integrity and sequence of the damaged DNA molecules.
RECENT ADVANCES:
Here we review the main aspects of the DNA DSB repair mechanisms with emphasis on the molecular pathways, radiation-induced lesions, and their significance for cellular processes.
CRITICAL ISSUES:
Although the main characteristics and proteins involved in the two DNA DSB repair processes present in eukaryotic cells (homologous recombination and nonhomologous end-joining) are reasonably well established, there are still uncertainties regarding the primary sensing event and their dependency on the complexity, location, and time of the damage. Interactions and overlaps between the different pathways play a critical role in defining the repair efficiency and determining the cellular functional behavior due to unrepaired/miss-repaired DNA lesions. The repair pathways involved in repairing lesions induced by soluble factors released from directly irradiated cells may also differ from the established response mechanisms.
FUTURE DIRECTIONS:
An improved understanding of the molecular pathways involved in sensing and repairing damaged DNA molecules and the role of DSBs is crucial for the development of novel classes of drugs to treat human diseases and to exploit characteristics of IR and alterations in tumor cells for successful radiotherapy applications.

Bystander Signalling: Exploring Clinical Relevance Through New Approaches and New Models

Classical radiation biology research has centred on nuclear DNA as the main target of radiation-induced damage. Over the past two decades, this has been challenged by a significant amount of scientific evidence clearly showing radiation-induced cell signalling effects to have important roles in mediating overall radiobiological response. These effects, generally termed radiation-induced bystander effects (RIBEs) have challenged the traditional DNA targeted theory in radiation biology and highlighted an important role for cells not directly traversed by radiation. The multiplicity of experimental systems and exposure conditions in which RIBEs have been observed has hindered precise definitions of these effects. However, RIBEs have recently been classified for different relevant human radiation exposure scenarios in an attempt to clarify their role in vivo. Despite significant research efforts in this area, there is little direct evidence for their role in clinically relevant exposure scenarios. In this review, we explore the clinical relevance of RIBEs from classical experimental approaches through to novel models that have been used to further determine their potential implications in the clinic. 

Identification of RBCK1 as a novel regulator of FKBPL: implications for tumor growth and response to tamoxifen

FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identifed a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-b-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-a (ERa). Furthermore, FKBPL and RBCK1 associate with ERa at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.

Radiobiology at ultra-high dose rates employing laser-driven ions

The potential that laser based particle accelerators offer to solve sizing and cost issues arising with conventional proton therapy has generated great interest in the understanding and development of laser ion acceleration, and in investigating the radiobiological effects induced by laser accelerated ions. Laser-driven ions are produced in bursts of ultra-short duration resulting in ultra-high dose rates, and an investigation at Queen's University Belfast was carried out to investigate this virtually unexplored regime of cell rdaiobiology. This employed the TARANIS terawatt laser producing protons in the MeV range for proton irradiation, with dose rates exceeding 10 Gys on a single exposure. A clonogenic assay was implemented to analyse the biological effect of proton irradiation on V79 cells, which, when compared to data obtained with the same cell line irradiated with conventionally accelerated protons, was found to show no significant difference. A Relative Biological effectiveness of 1.4±0.2 at 10 % Survival Fraction was estimated from a comparison with a 225 kVp X-ray source. © 2013 SPIE.