A study of the biological effects of modulated 6 MV radiation fields

The delivery of spatially modulated radiation fields has been shown to impact on in vitro cell survival responses. To study the effect of modulated fields on cell survival, dose response curves were determined for human DU-145 prostate, T98G glioma tumour cells and normal primary AGO-1552 fibroblast cells exposed to modulated and non-modulated field configurations delivered using a 6 MV Linac with multi-leaf collimator. When exposed to uniform fields delivered as a non-modulated or modulated configuration, no significant differences in survival were observed with the exception of DU-145 cells at a dose of 8 Gy (p = 0.024). Survival responses were determined for exposure to non-uniform-modulated beams in DU-145 and T98G and showed no deviation from the survival response observed following uniform non-modulated exposures. The results of these experiments indicate no major deviation in response to modulated fields compared to uniform exposures.

Effect of sub-lethal challenge with Photodynamic Antimicrobial Chemotherapy (PACT) on the antibiotic susceptibility of clinical bacterial isolates

This study aimed to determine the effect of sub-lethal challenge with Photodynamic Antimicrobial Chemotherapy (PACT) on the susceptibility of clinical Staphylococcus aureus and Pseudomonas aeruginosa isolates to both PACT and a range of antibiotics used in the treatment of infection caused by these bacteria. Clinical S. aureus and P. aeruginosa isolates were exposed to sub-lethal PACT with meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP) and methylene blue (MB) over a 72 h period. After exposure, susceptibility of surviving organisms to a range of antibiotics was determined and compared with the susceptibility of an untreated control. Surviving bacteria were also exposed to previously lethal photosensitizer-light combinations, to determine if susceptibility to PACT was affected by sub-lethal exposure. Exposure to sub-lethal PACT did not decrease susceptibility to antibiotics with the minimum inhibitory concentrations for 95% and 100% of P. aeruginosa and S. aureus isolates, respectively, within two doubling dilutions of the MIC of the untreated control. Similarly, habituation with sub-lethal PACT did not reduce susceptibility of P. aeruginosa isolates to PACT levels previously determined as lethal. A reduction in susceptibility to PACT following habituation was apparent for two S. aureus isolates with MB and for 1 S. aureus isolate with IMP. However, for two of these three isolates, the log reduction for habituated cells was still greater than 4 log(10). PACT remains an attractive potential treatment for infection caused by these bacteria. (C) 2010 Elsevier B.V. All rights reserved.

Regulation of FOS by different compartmental stresses induced by low levels of ionizing radiation

We irradiated different cellular compartments and measured changes in expression of the FOS gene at the mRNA and protein levels. [H-3]Thymidine and tritiated water were used to irradiate the nucleus and the whole cell, respectively. I-125-Concanavalin A binding was used to irradiate the cell membrane differentially. Changes in FOS mRNA and protein levels were measured using semi-quantitative RT-PCR and SDS-PAGE Western blotting, respectively, Irradiation of the nucleus or the whole cell at a dose rate of 0.075 Gy/h caused no change in the level of FOS mRNA expression, but modestly (1.5-fold) induced FOS protein after 0.5 h, Irradiation of the nucleus at a dose rate of 0.43 Gy/h induced FOS mRNA by 1.5-fold after 0.5 h, but there was no significant effect after whole-cell irradiation. FOS protein was transiently induced 2.5-fold above control levels 0.5 h after a 0.43-Gy/h exposure of the nucleus or the whole cell. Irradiation of the cell membrane at a dose rate of 1.8 Gy/h for up to 2 h caused no change in the levels of expression of FOS mRNA or protein, but a dose rate of 6.8 Gy/h transiently increased the level of FOS mRNA S-fold after 0.5 h, These data demonstrate the complexity of the cellular response to radiation-induced damage at low doses. The lack of quantitative agreement between the transcript and protein levels for FOS suggests a role for posttranscriptional regulation. (C) 2000 by Radiation Research Society.

Radiation-induced intercellular signaling mediated by cytochrome-c via a p53-dependent pathway in hepatoma cells

The tumor suppressor p53 has a crucial role in cellular response to DNA damage caused by ionizing radiation, but it is still unclear whether p53 can modulate radiation-induced bystander effects (RIBE). In the present work, three different hepatoma cell lines, namely HepG2 (wild p53), PLC/PRF/5 (mutation p53) and Hep3B (p53 null), were irradiated with c-rays and then co-cultured with normal Chang liver cell (wild p53) in order to elucidate the mechanisms of RIBE. Results showed that the radiosensitivity of HepG2 cells was higher than that of PLC/PRF/5 and Hep3B cells. Only irradiated HepG2 cells, rather than irradiated PLC/PRF/5 or Hep3B cells, could induce bystander effect of micronuclei (MN) formation in the neighboring Chang liver cells. When HepG2 cells were treated with 20 mu M pifithrin-alpha, an inhibitor of p53 function, or 5 lM cyclosporin A (CsA), an inhibitor of cytochrome- c release from mitochondria, the MN induction in bystander Chang liver cells was diminished. In fact, it was found that after irradiation, cytochrome- c was released from mitochondria into the cytoplasm only in HepG2 cells in a p53- dependent manner, but not in PLC/PRF/5 and Hep3B cells. Interestingly, when 50 lg/ml exogenous cytochrome- c was added into cell co- culture medium, RIBE was significantly triggered by irradiated PLC/PRF/5 and Hep3B cells, which previously failed to provoke a bystander effect. In addition, this exogenous cytochrome- c also partly recovered the RIBE induced by irradiated HepG2 cells even with CsA treatment. Our results provide new evidence that the RIBE can be modulated by the p53 status of irradiated hepatoma cells and that a p53- dependent release of cytochrome- c may be involved in the RIBE. Oncogene (2011) 30, 1947- 1955; doi: 10.1038/onc. 2010.567; published online 6 December 2010

Effect of relativistic plasma on extreme-ultraviolet harmonic emission from intense laser-matter interactions

Experiments were performed in which intense laser pulses (up to 9x10(19) W/cm(2)) were used to irradiate very thin (submicron) mass-limited aluminum foil targets. Such interactions generated high-order harmonic radiation (greater than the 25th order) which was detected at the rear of the target and which was significantly broadened, modulated, and depolarized because of passage through the dense relativistic plasma. The spectral modifications are shown to be due to the laser absorption into hot electrons and the subsequent sharply increasing relativistic electron component within the dense plasma.

Photon Production from the Vacuum Close to the Superradiant Transition: Linking the Dynamical Casimir Effect to the Kibble-Zurek Mechanism

The dynamical Casimir effect (DCE) predicts the generation of photons from the vacuum due to the parametric amplification of the quantum fluctuations of an electromagnetic field. The verification of such an effect is still elusive in optical systems due to the very demanding requirements of its experimental implementation. We show that an ensemble of two-level atoms collectively coupled to the electromagnetic field of a cavity, driven at low frequencies and close to a quantum phase transition, stimulates the production of photons from the vacuum. This paves the way to an effective simulation of the DCE through a mechanism that has recently found experimental demonstration. The spectral properties of the emitted radiation reflect the critical nature of the system and allow us to link the detection of the DCE to the Kibble-Zurek mechanism for the production of defects when crossing a continuous phase transition.

A computational model of cellular response to modulated radiation fields

Purpose:
To develop a model to describe the response of cell populations to spatially modulated radiation exposures of relevance to advanced radiotherapies.

Materials and Methods:
A Monte Carlo model of cellular radiation response was developed. This model incorporated damage from both direct radiation and intercellular communication including bystander signaling. The predictions of this model were compared to previously measured survival curves for a normal human fibroblast line (AGO1522) and prostate tumor cells (DU145) exposed to spatially modulated fields.

Results:
The model was found to be able to accurately reproduce cell survival both in populations which were directly exposed to radiation and those which were outside the primary treatment field. The model predicts that the bystander effect makes a significant contribution to cell killing even in uniformly irradiated cells. The bystander effect contribution varies strongly with dose, falling from a high of 80% at low doses to 25% and 50% at 4 Gy for AGO1522 and DU145 cells, respectively. This was verified using the inducible nitric oxide synthase inhibitor aminoguanidine to inhibit the bystander effect in cells exposed to different doses, which showed significantly larger reductions in cell killing at lower doses.

Conclusions:
The model presented in this work accurately reproduces cell survival following modulated radiation exposures, both in and out of the primary treatment field, by incorporating a bystander component. In addition, the model suggests that the bystander effect is responsible for a significant portion of cell killing in uniformly irradiated cells, 50% and 70% at doses of 2 Gy in AGO1522 and DU145 cells, respectively. This description is a significant departure from accepted radiobiological models and may have a significant impact on optimization of treatment planning approaches if proven to be applicable in vivo.

Bystander responses induced by low LET radiation

Radiation-induced bystander responses are observed when cells respond to their neighbours being irradiated. Considerable evidence is now available regarding the importance of these responses in cell and tissue models. Most studies have utilized two approaches where either a media-transferable factor has been assessed or cells have been exposed to low fluences of charged particles, where only a few percent are exposed. The development of microbeams has allowed nontargeted responses such as bystander effects to be more carefully analysed. As well as charged particle microbeams, X-ray microprobes have been developed, and several groups are also developing electron microbeams. Using the Gray Cancer Institute soft X-ray microprobe, it has been possible to follow the response of individual cells to targeted low doses of carbon-characteristic soft X-rays. Studies in human fibroblasts have shown evidence of a significant radiation quality-dependent bystander effect, measured as chromosomal damage in the form of micronuclei which is radiation quality dependent. Other studies show that even under conditions when only a single cell is targeted with soft X-rays, significant bystander-mediated cell killing is observed. The observation of bystander responses with low LET radiation suggests that these may be important in understanding radiation risk from background levels of radiation, where cells observe only single electron track traversals. Also, the indirect evidence for these responses in vivo indicates that they may have a role to play in current radiotherapy approaches and future novel strategies involving modulating nontargeted responses.

DNA damage induction in dry and hydrated DNA by synchrotron radiation

Radiation biophysics has sought to understand at a molecular level, the mechanisms through which ionizing radiations damage DNA, and other molecules within living cells. The complexity of lesions produced in the DNA by ionizing radiations is thought to depend on the amount of energy deposited at the site of each lesion. To study the relationship between the energy deposited and the damage produced, we have developed novel techniques for irradiating dry prasmid DNA, partially re-hydrated DNA and DNA in solution using monochromatic vacuum-UV synchrotron radiation. We have used photons in the energy range 7-150 eV, corresponding to the range of energies typically involved in the efficient production of DNA single-strand (SSB), and double-strand breaks (DSB) by ionizing radiation. The data show that both types of breaks are produced at all energies investigated (with, or without water present). Also, the energy dependence for DSB induction follows a similar trend to SSB induction but at a 20-30-fold reduced incidence, suggesting a common precursor for both types of damage. Preliminary studies where DNA has been irradiated in solution indicate a change in the shape of the dose-effect curve (from linear, to linear-quadratic for double-strand break induction) and a large increase in sensitivity due to the presence of water.

A model for radiation-induced bystander effects, with allowance for spatial position and the effects of cell turnover

Bystander effects, whereby cells that are not directly exposed to ionizing radiation exhibit adverse biological effects, have been observed in a number of experimental systems. A novel stochastic model of the radiation-induced bystander effect is developed that takes account of spatial location, cell killing and repopulation. The ionizing radiation dose- and time-responses of this model are explored, and it is shown to exhibit pronounced downward curvature in the high dose-rate region, similar to that observed in many experimental systems, reviewed in the paper. It is also shown to predict the augmentation of effect after fractionated delivery of dose that has been observed in certain experimental systems. It is shown that the generally intractable solution of the full stochastic system can be considerably simplified by assumption of pairwise conditional dependence that varies exponentially over time. (C) 2004 Elsevier Ltd. All rights reserved.

Calcium fluxes modulate the radiation-induced bystander responses in targeted glioma and fibroblast cells

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. This study investigated the role of changes in calcium levels in bystander responses leading to chromosomal damage in nonirradiated T98G glioma cells and AG01522 fibroblasts that had been either exposed to conditioned medium from irradiated cells or co-cultured with a population where a fraction of cells were individually targeted through the nucleus or cytoplasm with a precise number of microbeam helium-3 particles. After the recipient cells were treated with conditioned medium from T98G or AG01522 cells that had been irradiated through either nucleus or cytoplasm, rapid calcium fluxes were monitored in the nonirradiated recipient cells. Their characteristics were dependent on the source of the conditioned medium but had no dependence on radiation dose. When recipient cells were co-cultured with an irradiated population of either T98G or AG01522 cells, micronuclei were induced in the nonirradiated cells, but this response was eliminated by treating the cells with calcicludine (CaC), a potent blocker of Ca2+ channels. Moreover, both the calcium fluxes and the bystander effect were inhibited when the irradiated T98G cells were treated with aminoguanidine, an inhibitor of nitric oxide synthase (NOS), and when the irradiated AG01522 cells were treated with DMSO, a scavenger of reactive oxygen species (ROS), which indicates that NO and ROS were involved in the bystander responses generated from irradiated T98G and AG01522 cells, respectively. Our findings indicate that calcium signaling may be an early response in radiation-induced bystander effects leading to chromosome damage. (c) 2006 by Radiation Research Society.

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint.

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.