Mark A. Hill

Low-dose irradiation of nontransformed cells stimulates the selective removal of precancerous cells via intercellular induction of apoptosis

An important stage in tumorigenesis is the ability of a precancerous cell to escape natural anticancer signals imposed on it by neighboring cells and its microenvironment. We have previously characterized a system of intercellular induction of apoptosis whereby nontransformed cells selectively remove transformed cells from coculture via cytokine and reactive oxygen/nitrogen species (ROS/RNS) signaling. We report that irradiation of nontransformed cells with low doses of either high linear energy transfer (LET) alpha-particles or low-LET gamma-rays leads to stimulation of intercellular induction of apoptosis. The use of scavengers and inhibitors confirms the involvement of ROS/RNS signaling and of the importance of transformed cell NADPH oxidase in the selectivity of the system. Doses as low as 2-mGy gamma-rays and 0.29-mGy alpha-particles were sufficient to produce an observable increase in transformed cell apoptosis. This radiation-stimulated effect saturates at very low doses (50 mGy for gamma-rays and 25 mGy for alpha-particles). The use of transforming growth factor-beta (TGF-beta) neutralizing antibody confirms a role for the cytokine in the radiation-induced signaling. The system may represent a natural anticancer mechanism stimulated by extremely low doses of ionizing radiation.

Detector density and small field dosimetry: Integral versus point dose measurement schemes

PURPOSE The Alfonso et al. [Med. Phys. 35, 5179-5186 (2008)] formalism for small field dosimetry proposes a set of correction factors (kQclin,Qmsrfclin,fmsr) which account for differences between the detector response in nonstandard (clinical) and machine-specific-reference fields. In this study, the Monte Carlo method was used to investigate the viability of such small field correction factors for four different detectors irradiated under a variety of conditions. Because kQclin,Qmsrfclin,fmsr values for single detector position measurements are influenced by several factors, a new theoretical formalism for integrated-detector-position [dose area product (DAP)] measurements is also presented and was tested using Monte Carlo simulations. METHODS A BEAMnrc linac model was built and validated for a Varian Clinac iX accelerator. Using the egs++ geometry package, detailed virtual models were built for four different detectors: a PTW 60012 unshielded diode, a PTW 60003 Diamond detector, a PTW 31006 PinPoint (ionization chamber), and a PTW 31018 MicroLion (liquid-filled ionization chamber). The egs_chamber code was used to investigate the variation of kQclin,Qmsrfclin,fmsr with detector type, detector construction, field size, off-axis position, and the azimuthal angle between the detector and beam axis. Simulations were also used to consider the DAP obtained by each detector: virtual detectors and water voxels were scanned through high resolution grids of positions extending far beyond the boundaries of the fields under consideration. RESULTS For each detector, the correction factor (kQclin,Qmsrfclin,fmsr) was shown to depend strongly on detector off-axis position and detector azimuthal angle in addition to field size. In line with previous studies, substantial interdetector variation was also observed. However, it was demonstrated that by considering DAPs rather than single-detector-position dose measurements the high level of interdetector variation could be eliminated. Under small field conditions, mass density was found to be the principal determinant of water equivalence. Additionally, the mass densities of components outside the sensitive volumes were found to influence the detector response. CONCLUSIONS kQclin,Qmsrfclin,fmsr values for existing detector designs depend on a host of variables and their calculation typically relies on the use of time-intensive Monte Carlo methods. Future moves toward density-compensated detector designs or DAP based protocols may simplify the methodology of small field dosimetry.

Effect of Linear Energy Transfer (LET) on the Complexity of α-Particle-Induced Chromosome Aberrations in Human CD34+ Cells

Abstract Anderson, R. M, Stevens, D. L., Sumption, N. D., Townsend, K. M. S., Goodhead, D. T. and Hill, M. A. Effect of Linear Energy Transfer (LET) on the Complexity of α-Particle-Induced Chromosome Aberrations in Human CD34+ Cells. Radiat. Res. 167, 541–550 (2007). The aim of this study was to assess the relative influence of the linear energy transfer (LET) of α particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with α particles of various incident LETs (110–152 keV/μm, with mean LETs through the cell of 119–182 keV/μm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent “an area” of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single α particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the α particle increases, the likelihood of this aberration being classed as complex is greater.

The importance of XRCC2 in RAD51-related DNA damage repair.

The repair of DNA damage by homologous recombination (HR) is a key pathway for the maintenance of genetic stability in mammalian cells, especially during and following DNA replication. The central HR protein is RAD51, which ensures high fidelity DNA repair by facilitating strand exchange between damaged and undamaged homologous DNA segments. Several RAD51-like proteins, including XRCC2, appear to help with this process, but their roles are not well understood. Here we show that XRCC2 is highly conserved and that most substantial truncations of the protein destroy its ability to function. XRCC2 and its partner protein RAD51L3 are found to interact with RAD51 in the 2-hybrid system, and XRCC2 is shown to be important but not essential for the accumulation of RAD51 at the sites of DNA damage. We visualize the localization of XRCC2 protein at the same sites of DNA damage for the first time using specialized irradiation conditions. Our data indicate that an important function of XRCC2 is to enhance the activity of RAD51, so that the loss of XRCC2 results in a severe delay in the early response of RAD51 to DNA damage.

Production and Validation of CR-39-Based Dishes for α-Particle Radiobiological Experiments

Abstract Gaillard, S., Armbruster, V., Hill, M. A., Gharbi, T. and Fromm, M. Production and Validation of CR-39-Based Dishes for α-Particle Radiobiological Experiments. Radiat. Res. 163, 343–350 (2005). The study of radiobiological effects induced in vitro by low fluences of α particles would be significantly enhanced if the precise localization of each particle track in the cell monolayer was known. From this perspective, we developed a new method based on tailor-made UV-radiation-cured CR-39, the production of which is described. Its validation both as a petri dish and as solid-state nuclear track detectors is demonstrated. With respect to the demands on solid-state nuclear track detectors in such experiments, these biologically compatible detectors have a controlled micrometric thickness that allows them to be crossed by the α particles. In this study, we present a method for obtaining 10-μm-thick CR-39, its chemical characterization, and its properties as a solid-state nuclear track detector under the environmental conditions of radiobiological experiments. The experimental studies performed with 3.5 MeV α particles show that their transmitted energy is sufficient enough to cross the entire cellular volume. Under optimal conditions, etched tracks are clearly defined 2 h after etching. Moreover, the UV-radiation-cured CR-39 represents an essentially zero background that is due to the short time between the production and use of the polymer. Under a confocal microscope, this thin solid-state nuclear track detector allows the precise localization of the impact parameter at the subcellular level.

Lethal Effect of Carbon K-Shell Photoionizations in Chinese Hamster V79 Cell Nuclei: Experimental Method and Theoretical Analysis

To test a possible specific effect of carbon K-shell ionizations in DNA, survival curves for Chinese hamster V79 cells were measured for X irradiations at energies below and above the carbon K-shell ionization threshold. Specific values of the X-ray energies (250 and 340 eV) were chosen to ensure isoattenuation of the two kinds of radiation within the cell. An enhancement of lethality by a factor of about 2 was found for X rays at 340 eV compared to below the threshold at 250 eV. This may be attributed to the production of highly efficient carbon K-shell ionizations located on DNA. A model of X-ray lethality (Goodhead et al., Radiat. Prot. Dosim. 52, 217-223, 1994) was extended to allow for a possible lethal effect from clusters of reactive species induced by K-shell photoionizations (K-shell clusters). Within this model, the increase in lethality above the carbon K-shell threshold may be explained by a value of 2% for the lethal efficiency of K-shell clusters overlapping the DNA. An extrapolation to the lethal effect of more complex ion-induced K-shell ionizations indicates that K-shell ionization may be a major process in the biological effectiveness of heavy ions.

Radiosensitization of renal cell carcinoma in vitro through the induction of autophagy

BACKGROUND AND PURPOSE For patients diagnosed with advanced renal cell carcinoma (RCC), there are few therapeutic options. Radiation therapy is predominantly used to treat metastasis and has not proven effective in the adjuvant setting for renal cancer. Furthermore, RCC is resistant to standard cytotoxic chemotherapies. Targeted anti-angiogenics are the standard of care for RCC but are not curative. Newer agents, such as mTOR inhibitors and others that induce autophagy, have shown great promise for treating RCC. Here, we investigate the potential use of the small molecule STF-62247 to modulate radiation. MATERIALS AND METHODS Using RCC cell lines, we evaluate sensitivity to radiation in addition to agents that induce autophagic cell death by clonogenic survival assays. Furthermore, these were also tested under physiological oxygen levels. RESULTS STF-62247 specifically induces autophagic cell death in cells that have lost VHL, an essential mutation in the development of RCC. Treatment with STF-62247 did not alter cell cycle progression but when combined with radiation increased cell killing under oxic and hypoxic/physiological conditions. CONCLUSIONS This study highlights the possibility of combining targeted therapeutics such as STF-62247 or temsirolimus with radiation to reduce the reliance on partial or full nephrectomy and improve patient prognosis.

Comments on the Recently Reported Low Biological Effectiveness of Ultrasoft X Rays

In a recent paper, C. K. Hill et al. (1) reported the results of experiments carried out at the University of Wisconsin in which Chinese hamster V79 and mouse C3H 10T1⁄2 cells were irradiated with synchrotronproduced 273 eV and 860 eV ultrasoft X rays. X rays at these energies are heavily attenuated through a single mammalian cell. Therefore, they chose two energies that would be equally attenuated, enabling a direct comparison between the biological effectiveness of the two energies. The two main results of the cell survival experiments carried out by the Wisconsin group were: 1. The biological effectiveness for these two energies, for both cell lines, was similar. 2. After the mean nuclear dose was calculated, taking into account attenuation through the cells, there were no significant differences in cell survival between these two ultrasoft X-ray energies and 250 kVp X rays. Based on this second result, the Wisconsin group (1) concluded that, in general, ultrasoft X rays are no more biologically effective per unit dose than are standard orthovoltage X rays and g rays, and hence they refuted the suggestion (2, 3) that the majority of critical damage that follows exposure to standard X rays and g rays results from low-energy, secondary electrons. Their second result is inconsistent with previously published experimental data from several other groups, and it therefore warrants close scrutiny to understand the origin of this difference, especially in view of the recent increase in the use of ultrasoft X rays for radiobiology experiments. The first result mentioned above is not inconsistent with other published experimental data or with the main interpretations that have been suggested for a high biological effectiveness of ultrasoft X rays. We first briefly discuss Result 1, and then we consider in more detail some potential reasons for the difference of Result 2.

Relative sensitivities of repair-deficient mammalian cells for clonogenic survival after α-particle irradiation

Abstract Hill, M. A., Herdman, M. T., Stevens, D. L., Jones, N. J., Thacker, J. and Goodhead, D. T. Relative Sensitivities of Repair-Deficient Mammalian Cells for Clonogenic Survival after α-Particle Irradiation. Radiat. Res. 162, 667–676 (2004). The clonogenic survival of cells of the radiation-sensitive hamster cell lines irs1, irs2, irs3 and xrs5, representing different DNA repair pathways, was compared to that of their parent lines after α-particle irradiation. Measurements of nuclear area were made to calculate the probability of surviving a single α-particle traversal, the average number of lethal lesions per track and per unit dose, along with the “intrinsic radiosensitivity” of these cells, allowing for the potential of multiple lethal lesions per traversal. For all cell lines studied, α particles were found to be more biologically effective per unit absorbed dose than X rays at inducing cell inactivation. The repair-deficient cells showed an enhanced sensitivity to α particles compared to their parent line, but the degree of enhancement was less than for X rays. The reduction in additional sensitivity for α-particle irradiation was shown not to be due predominantly to differences in cell geometry limiting the probability of a cell nucleus being traversed. The results suggest that both the nonhomologous end-joining pathway and to a lesser extent the homologous recombination repair pathway play a role in successful repair of α-particle-induced damage, although a large proportion of damage is not repaired by either pathway.

Do the various radiations present in BNCT act synergistically? Cell survival experiments in mixed alpha-particle and gamma-ray fields

In many radiotherapy situations patients are exposed to mixed field radiation. In particular in BNCT, as with all neutron beam exposures, a significant fraction of the dose is contributed by low LET gamma ray photons. The components of such a mixed field may show a synergistic interaction and produce a greater cell kill effect than would be anticipated from the independent action of the different radiation types. Such a synergy would have important implications for treatment planning and in the interpretation of clinical results. An irradiation setup has been created at the Medical Research Council in Harwell to allow simultaneous irradiation of cells by cobalt-60 gamma rays and plutonium-238 alpha-particles. The setup allows for variation of dose and dose rates for both sources along with variation of the alpha particle energy. A series of cell survival assays for this mixed field have been carried out using V79-4 cells and compared to exposures to the individual components of the field under identical conditions. In the experimental setup described no significant synergistic effect was observed.