ATR-dependent radiation-induced gammaH2AX foci in bystander primary human astrocytes and glioma cells.

Radiotherapy is an important treatment for patients suffering from high-grade malignant gliomas. Non-targeted (bystander) effects may influence these cells' response to radiation and the investigation of these effects may therefore provide new insights into mechanisms of radiosensitivity and responses to radiotherapy as well as define new targets for therapeutic approaches. Normal primary human astrocytes (NHA) and T98G glioma cells were irradiated with helium ions using the Gray Cancer Institute microbeam facility targeting individual cells. Irradiated NHA and T98G glioma cells generated signals that induced gammaH2AX foci in neighbouring non-targeted bystander cells up to 48 h after irradiation. gammaH2AX bystander foci were also observed in co-cultures targeting either NHA or T98G cells and in medium transfer experiments. Dimethyl sulphoxide, Filipin and anti-transforming growth factor (TGF)-beta 1 could suppress gammaH2AX foci in bystander cells, confirming that reactive oxygen species (ROS) and membrane-mediated signals are involved in the bystander signalling pathways. Also, TGF-beta 1 induced gammaH2AX in an ROS-dependent manner similar to bystander foci. ROS and membrane signalling-dependent differences in bystander foci induction between T98G glioma cells and normal human astrocytes have been observed. Inhibition of ataxia telangiectasia mutated (ATM) protein and DNA-PK could not suppress the induction of bystander gammaH2AX foci whereas the mutation of ATM- and rad3-related (ATR) abrogated bystander foci induction. Furthermore, ATR-dependent bystander foci induction was restricted to S-phase cells. These observations may provide additional therapeutic targets for the exploitation of the bystander effect.

Dissociation of H2+ from a short, intense, infrared laser pulse: proton emission spectra and pulse calibration

The proton energy spectrum from photodissociation of the hydrogen molecular ion by short intense pulses of infrared light is calculated. The time-dependent Schrödinger equation is discretized and integrated. For few-cycle pulses one can resolve vibrational structure, arising from the experimental preparation of the molecular ion. We calculate the corresponding energy spectrum and analyse the dependence on the pulse time delay, pulse length and intensity of the laser for ? ~ 790 nm. We conclude that the proton spectrum is a sensitive probe of both the vibrational populations and phases, and allows us to distinguish between adiabatic and nonadiabatic dissociation. Furthermore, the sensitivity of the proton spectrum from H2+ is a practical means of calibrating the pulse. Our results are compared with recent measurements of the proton spectrum for 65 fs pulses using a Ti:Sapphire laser (? ~ 790 nm) including molecular orientation and focal-volume averaging. Integrating over the laser focal volume, for the intensity I ~ 3 × 1015 W cm-2, we find our results are in excellent agreement with these experiments.

Dynamic control of laser-produced proton beams

The emission characteristics of intense laser driven protons are controlled using ultrastrong (of the order of 10(9) V/m) electrostatic fields varying on a few ps time scale. The field structures are achieved by exploiting the high potential of the target (reaching multi-MV during the laser interaction). Suitably shaped targets result in a reduction in the proton beam divergence, and hence an increase in proton flux while preserving the high beam quality. The peak focusing power and its temporal variation are shown to depend on the target characteristics, allowing for the collimation of the inherently highly divergent beam and the design of achromatic electrostatic lenses.

The radiation-inducible pE9 promoter driving inducible nitric oxide synthase radiosensitizes hypoxic tumour cells to radiation

Driving high-level transgene expression in a tumour-specific manner remains a key requirement in the development of cancer gene therapy. We have previously demonstrated the strong anticancer effects of generating abnormally high levels of intracellular NO• following the overexpression of the inducible nitric oxide synthase (iNOS) gene. Much of this work has focused on utilizing exogenously activated promoters, which have been primarily induced using X-ray radiation. Here we further examine the potential of the pE9 promoter, comprising a combination of nine CArG radio-responsive elements, to drive the iNOS transgene. Effects of X-ray irradiation on promoter activity were compared in vitro under normoxic conditions and various degrees of hypoxia. The pE9 promoter generated high-level transgene expression, comparable with that achieved using the constitutively driven cytomegalovirus promoter. Furthermore, the radio-resistance of radiation-induced fibrosarcoma-1 (RIF-1) mouse sarcoma cells exposed to 0.1 and 0.01% O2 was effectively eliminated following transfection with the pE9/iNOS construct. Significant inhibition of tumour growth was also observed in vivo following direct intratumoural injection of the pE9/iNOS construct compared to empty vector alone (P<0.001) or to a single radiation dose of 10?Gy (P<0.01). The combination of both therapies resulted in a significant 4.25 day growth delay compared to the gene therapy treatment alone (P<0.001). In summary, we have demonstrated the potential of the pE9/iNOS construct for reducing radio-resistance conferred by tumour cell hypoxia in vitro and in vivo, with greater tumour growth delay observed following the treatment with the gene therapy construct as compared with radiotherapy alone.