Apoptosis is initiated in human keratinocytes exposed to signalling factors from microbeam irradiated cells.

PURPOSE: There is now no doubt that bystander signalling from irradiated cells occurs and causes a variety of responses in cells not targeted by the ionizing track. However, the mechanisms underlying these processes are unknown and the relevance to radiotherapy and risk assessment remains controversial. Previous research by our laboratory has shown bystander effects in a human keratinocyte cell line, HPV-G cells, exposed to medium from gamma irradiated HPV-G cells. The aim of this work was to investigate if similar mechanisms to those identified in medium transfer experiments occurred in these HPV-G cells when they are in the vicinity of microbeam irradiated cells. Demonstration of a commonality of mechanisms would support the idea that the process is not artifactual. MATERIALS AND METHODS: HPV-G cells were plated as two separate populations on mylar dishes. One population was directly irradiated using a charged particle microbeam (1 - 10 protons). The other population was not irradiated. Bystander factor-induced apoptosis was investigated in both populations following treatment by monitoring the levels of reactive oxygen species and mitochondrial membrane potential using fluorescent probes. Expression of the anti-apoptotic protein, bcl-2, and cytochrome c were determined, as well as apoptosis levels. RESULTS: Microbeam irradiation induced increases in reactive oxygen species and decreases in mitochondrial membrane potential at 6 h post-exposure, increased expression of bcl-2 and cytochrome c release at 6.5 h and increased apoptosis at 24 h. CONCLUSION: This study shows that similar bystander signalling pathways leading to apoptosis are induced following microbeam irradiation and following medium transfer. This demonstrates that the mechanisms involved are common across different radiation qualities and conditions and indicates that they may be relevant in vivo.

Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells.

Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells. When conditioned medium from irradiated cells was transferred to unirradiated xrs5 cells, the level of induction was independent of whether the medium came from irradiated wild-type, ssb or dsb repair deficient cells. This result suggests that the activation of a bystander signal is independent of the DNA repair capacity of the irradiated cells. Also, pre-treatment of the irradiated cells with 0.5% DMSO, which suppresses micronuclei induction in CHO but not in xrs5 cells, suppressed bystander effects completely in both conditioned media, suggesting that DMSO is effective for suppression of bystander signal arising independently of DNA damage in irradiated cells. Overall the work presented here adds to the understanding that it is the repair phenotype of the cells receiving bystander signals, which determines overall response rather than that of the cell producing the bystander signal.

Role of TGF-beta1 and nitric oxide in the bystander response of irradiated glioma cells.

The radiation-induced bystander effect (RIBE) increases the probability of cellular response and therefore has important implications for cancer risk assessment following low-dose irradiation and for the likelihood of secondary cancers after radiotherapy. However, our knowledge of bystander signaling factors, especially those having long half-lives, is still limited. The present study found that, when a fraction of cells within a glioblastoma population were individually irradiated with helium ions from a particle microbeam, the yield of micronuclei (MN) in the nontargeted cells was increased, but these bystander MN were eliminated by treating the cells with either aminoguanidine (an inhibitor of inducible nitric oxide (NO) synthase) or anti-transforming growth factor beta1 (anti-TGF-beta1), indicating that NO and TGF-beta1 are involved in the RIBE. Intracellular NO was detected in the bystander cells, and additional TGF-beta1 was detected in the medium from irradiated T98G cells, but it was diminished by aminoguanidine. Consistent with this, an NO donor, diethylamine nitric oxide (DEANO), induced TGF-beta1 generation in T98G cells. Conversely, treatment of cells with recombinant TGF-beta1 could also induce NO and MN in T98G cells. Treatment of T98G cells with anti-TGF-beta1 inhibited the NO production when only 1% of cells were targeted, but not when 100% of cells were targeted. Our results indicate that, downstream of radiation-induced NO, TGF-beta1 can be released from targeted T98G cells and plays a key role as a signaling factor in the RIBE by further inducing free radicals and DNA damage in the nontargeted bystander cells.

Plasma emission spectroscopy of solids irradiated by intense XUV pulses from a free electron laser

The FLASH XUV-free electron laser has been used to irradiate solid samples at intensities of the order 10(16) W cm(-2) at a wavelength of 13.5 nm. The subsequent time integrated XUV emission was observed with a grating spectrometer. The electron temperature inferred from plasma line ratios was in the range 5-8 eV with electron density in the range 10(21)-10(22) cm(-3). These results are consistent with the saturation of absorption through bleaching of the L-edge by intense photo-absorption reported in an earlier publication. (C) 2009 Elsevier B.V. All rights reserved.

Variation of strand break yield for plasmid DNA irradiated with high-Z metal nanoparticles.

PURPOSE: Poly(ADP-ribose) polymerase (PARP) plays an important role in DNA repair, and PARP inhibitors can enhance the activity of DNA-damaging agents in vitro and in vivo. AG014699 is a potent PARP inhibitor in phase II clinical development. However, the range of therapeutics with which AG014699 could interact via a DNA-repair based mechanism is limited. We aimed to investigate a novel, vascular-based activity of AG014699, underlying in vivo chemosensitization, which could widen its clinical application. EXPERIMENTAL DESIGN: Temozolomide response was analyzed in vitro and in vivo. Vessel dynamics were monitored using "mismatch" following the administration of perfusion markers and real-time analysis of fluorescently labeled albumin uptake in to tumors established in dorsal window chambers. Further mechanistic investigations used ex vivo assays of vascular smooth muscle relaxation, gut motility, and myosin light chain kinase (MLCK) inhibition. RESULTS: AG014699 failed to sensitize SW620 cells to temozolomide in vitro but induced pronounced enhancement in vivo. AG014699 (1 mg/kg) improved tumor perfusion comparably with the control agents nicotinamide (1 g/kg) and AG14361 (forerunner to AG014699; 10 mg/kg). AG014699 and AG14361 relaxed preconstricted vascular smooth muscle more potently than the standard agent, hydralazine, with no impact on gut motility. AG014699 inhibited MLCK at concentrations that relaxed isolated arteries, whereas AG14361 had no effect. CONCLUSION: Increased vessel perfusion elicited by AG014699 could increase tumor drug accumulation and therapeutic response. Vasoactive concentrations of AG014699 do not cause detrimental side effects to gut motility and may increase the range of therapeutics with which AG014699 could be combined with for clinical benefi

Fragmentation and plasmid strand breaks in pure and gold-doped DNA irradiated by beams of fast hydrogen atoms

The results of an investigation into the damage caused to dry plasmid DNA after irradiation by fast (keV) hydrogen atoms are presented. Agarose gel electrophoresis was used to assess single and double strand break yields as a function of dose in dry DNA samples deposited on a mica substrate. Damage levels were observed to increase with beam energy. Strand break yields demonstrated a considerable dependence on sample structure and the method of sample preparation. Additionally, the effect of high-Z nanoparticles on damage levels was investigated by irradiating DNA samples containing controlled amounts of gold nanoparticles. In contrast to previous (photonic) studies, no enhancement of strand break yields was observed with the particles showing a slight radioprotective effect. A model of DNA damage as a function of dose has been constructed in terms of the probability for the creation of single and double strand breaks, per unit ion flux. This model provides quantitative conclusions about the effects of both gold nanoparticles and the different buffers used in performing the assays and, in addition, infers the proportion of multiply damaged fragments.

Observation of the transient charging of a laser-irradiated solid

The proton radiography technique has been used to investigate the incidence of a 3 x10(19) W/cm(2) infrared pulse with a 125 mu m-diameter gold wire. The laser interaction is observed to drive the growth of a radial electric field similar to 10(10) V/m on the surface of the wire which rises and decays over a temporal window of 20 ps. Such studies of the ultrafast charging of a solid irradiated at high-intensity may be of relevance to schemes for laser-driven ion acceleration and the fast-ignitor concept for inertial confinement fusion.

Metals in the Exosphere of the Highly Irradiated Planet WASP-12b

We present near-UV transmission spectroscopy of the highly irradiated transiting exoplanet WASP-12b, obtained with the Cosmic Origins Spectrograph on the Hubble Space Telescope. The spectra cover three distinct wavelength ranges: NUVA (2539-2580 Å), NUVB (2655-2696 Å), and NUVC (2770-2811 Å). Three independent methods all reveal enhanced transit depths attributable to absorption by resonance lines of metals in the exosphere of WASP-12b. Light curves of total counts in the NUVA and NUVC wavelength ranges show a detection at a 2.5s level. We detect extra absorption in the Mg II ??2800 resonance line cores at the 2.8s level. The NUVA, NUVB, and NUVC light curves imply effective radii of 2.69 ± 0.24 R J , 2.18 ± 0.18 R J , and 2.66 ± 0.22 R J respectively, suggesting the planet is surrounded by an absorbing cloud which overfills the Roche lobe. We detect enhanced transit depths at the wavelengths of resonance lines of neutral sodium, tin, and manganese, and at singly ionized ytterbium, scandium, manganese, aluminum, vanadium, and magnesium. We also find the statistically expected number of anomalous transit depths at wavelengths not associated with any known resonance line. Our data are limited by photon noise, but taken as a whole the results are strong evidence for an extended absorbing exosphere surrounding the planet. The NUVA data exhibit an early ingress, contrary to model expectations; we speculate this could be due to the presence of a disk of previously stripped material.