M. Folkard

Studies of bystander effects in human fibroblasts using a charged particle microbeam.

PURPOSE To determine the role of cell-to-cell mediated effects in the response of cells to different radiations by using a charged particle microbeam capable of targeting individual cells with counted particles. MATERIALS AND METHODS Primary human fibroblasts were irradiated conventionally with X-rays or alpha-particles and scored for the induction of micronucleated or apoptotic cell formation at various times after irradiation. Cells were also individually irradiated with helium-3 particles from a microbeam and the distribution of damaged cells between hit and non-hit cells scored. RESULTS Conventionally exposed fibroblasts showed a dose-dependent production of micronucleated and apoptotic cells 3 days after irradiation for both X-rays and alpha-particles, with a high RBE value for alpha-particles at low doses. Targeting individual alpha-particles, using a microbeam, to four cells within a population produced more micronucleated and apoptotic cells than expected on the basis of a direct effect only. CONCLUSIONS This study provides direct evidence for the production of transmissible, cell-to-cell effects between hit and non-hit cells individually exposed to charged particles.

New insights on cell death from radiation exposure

Ionising radiation has been an important part of cancer treatment for almost a century, being used in external-beam radiotherapy, brachytherapy, and targeted radionuclide therapy. At the molecular and cellular level, cell killing has been attributed to deposition of energy from the radiation in the DNA within the nucleus, with production of DNA double-strand breaks playing a central part. However, this DNA-centric model has been questioned because cell-death pathways, in which direct relations between cell killing and DNA damage diverge, have been reported. These pathways include membrane-dependent signalling pathways and bystander responses (when cells respond not to direct radiation exposure but to the irradiation of their neighbouring cells). New insights into mechanisms of these responses coupled with technological advances in targeting of cells in experimental systems with microbeams have led to a reassessment of the model of how cells are killed by ionising radiation. This review provides an update on these mechanisms.

Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts

Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment. Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes. In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts. We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles. Individual primary human fibroblasts within a population of 600–800 cells were targeted with between 1 and 15 helium ions (effectively, α-particles). The charged particles were delivered through the centre of the nucleus with an accuracy of ± 2 μm and a detection and counting efficiency of greater than 99%. When scored 3 days later, even though only a single cell had been targeted, typically an additional 80–100 damaged cells were observed in the surviving population of about 5000 cells. The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell. Similar results of a 2–3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish. Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2–3-fold increase in the damage level in an unexposed quadrant of the dish. This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved.

A charged-particle microbeam: I. Development of an experimental system for targeting cells individually with counted particles

Charged-particle microbeams provide a unique opportunity to control precisely, the dose to individual cells and the localization of dose within the cell. The Gray Laboratory is now routinely operating a charged-particle microbeam capable of delivering targeted and counted particles to individual cells, at a dose-rate sufficient to permit a number of single-cell assays of radiation damage to be implemented. By this means, it is possible to study a number of important radiobiological processes in ways that cannot be achieved using conventional methods. This report describes the rationale, development and current capabilities of the Gray Laboratory microbeam.

Role of TGF-β1 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 β1 (anti-TGF-β1), indicating that NO and TGF-β1 are involved in the RIBE. Intracellular NO was detected in the bystander cells, and additional TGF-β1 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-β1 generation in T98G cells. Conversely, treatment of cells with recombinant TGF-β1 could also induce NO and MN in T98G cells. Treatment of T98G cells with anti-TGF-β1 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-β1 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.

DNA double-strand break distributions in X-ray and alpha-particle irradiated V79 cells: Evidence for non-random breakage

Many studies have shown that with increasing LET of ionizing radiation the RBE (relative biological effectiveness) for dsb (double strand breaks) induction remains around 1.0 despite the increase in the RBE for cell killing. This has been attributed to an increase in the complexity of lesions, classified as dsb with current techniques, at multiply damaged sites. This study determines the molecular weight distributions of DNA from Chinese hamster V79 cells irradiated with X-rays or 110 keV/micron alpha-particles. Two running conditions for pulsed-field gel-electrophoresis were chosen to give optimal separation of fragments either in the 225 kbp-5.7 Mbp range or the 0.3 kbp to 225 kbp range. Taking the total fraction of DNA migrating into the gel as a measure of fragmentation, the RBE for dsb induction was less than 1.0 for both molecular weight regions studied. The total yields of dsb were 8.2 x 10(-9) dsb/Gy/bp for X-rays and 7.8 x 10(-9) dsb/Gy/bp for alpha-particles, measured using a random breakage model. Analysis of the RBE of alpha-particles versus molecular weight gave a different response. In the 0.4 Mbp-5.7 Mbp region the RBE was less than 1.0; however, below 0.4 Mbp the RBE increased above 1.0. The frequency distributions of fragment sizes were found to differ from those predicted by a model assuming random breakage along the length of the DNA and the differences were greater for alpha-particles than for X-rays. An excess of fragments induced by a single-hit mechanism was found in the 8-300 kbp region and for X-rays and alpha-particles these corresponded to an extra 0.8 x 10(-9) and 3.4 x 10(-9) dsb/bp/Gy, respectively. Thus for every alpha-particle track that induces a dsb there is a 44% probability of inducing a second break within 300 kbp and for electron tracks the probability is 10%. This study shows that the distribution of damage from a high LET alpha-particle track is significantly different from that observed with low LET X-rays. In particular, it suggests that the fragmentation patterns of irradiated DNA may be related to the higher-order chromatin repeating structures found in intact cells.

Cytoplasmic Irradiation Induces Mitochondrial-Dependent 53BP1 Protein Relocalization in Irradiated and Bystander Cells

The accepted paradigm for radiation effects is that direct DNA damage via energy deposition is required to trigger the downstream biological consequences. The radiation-induced bystander effect is the ability of directly irradiated cells to interact with their nonirradiated neighbors, which can then show responses similar to those of the targeted cells. p53 binding protein 1 (53BP1) forms foci at DNA double-strand break sites and is an important sensor of DNA damage. This study used an ionizing radiation microbeam approach that allowed us to irradiate specifically the nucleus or cytoplasm of a cell and quantify response in irradiated and bystander cells by studying ionizing radiation-induced foci (IRIF) formation of 53BP1 protein. Our results show that targeting only the cytoplasm of a cell is capable of eliciting 53BP1 foci in both hit and bystander cells, independently of the dose or the number of cells targeted. Therefore, direct DNA damage is not required to trigger 53BP1 IRIF. The use of common reactive oxygen species and reactive nitrogen species (RNS) inhibitors prevent the formation of 53BP1 foci in hit and bystander cells. Treatment with filipin to disrupt membrane-dependent signaling does not prevent the cytoplasmic irradiation-induced 53BP1 foci in the irradiated cells, but it does prevent signaling to bystander cells. Active mitochondrial function is required for these responses because pseudo-rho(0) cells, which lack mitochondrial DNA, could not produce a bystander signal, although they could respond to a signal from normal rho+ cells.

Low-Dose Binary Behavior of Bystander Cell Killing after Microbeam Irradiation of a Single Cell with Focused CK X Rays

Abstract Schettino, G., Folkard, M., Michael, B. D. and Prise, K. M. Low-Dose Binary Behavior of Bystander Cell Killing after Microbeam Irradiation of a Single Cell with Focused CK X Rays. Radiat. Res. 163, 332–336 (2005). Although conclusive evidence has been obtained for the presence of radiation-induced bystander effects, the mechanisms that trigger and regulate these processes are still largely unknown. The bystander effect may play a critical role in determining the biological effectiveness of low-dose exposures, but questions on how to incorporate it into current models and extrapolate the risks of radiation-induced carcinogenesis are still open. The Gray Cancer Institute soft X-ray microbeam has been used to investigate the dose–response relationship of the bystander effect below 0.5 Gy. The survival response of V79 cells was assessed after the irradiation of a single cell within a population with a submicrometer-size beam of carbon K X rays (278 eV). Above 0.3 Gy, the measured bystander cell killing was in agreement with previously published data; however, a significant increase in the scatter of the data was observed in the low-dose region (<0.3 Gy). The data distribution observed indicates a binary behavior for triggering of the bystander response. According to our hypothesis, the probability of triggering a bystander response increases approximately linearly with the dose delivered to the single selected cell, reaching 100% above about 0.3 Gy. The magnitude of the bystander effect, when triggered, is approximately constant with the dose and results in an overall ∼10% reduction in survival in our system. This suggests that the event that triggers the emission of the bystander signal by the hit cell is an all-or-nothing process. Extrapolation of the data indicates that when a single fast electron traverses a V79 cell, there is a probability of ∼0.3% that the cell will emit the bystander signal. The data presented in this paper have also been analyzed statistically to test the possibility that complex DNA double-strand breaks may be the initial critical event.

Low-Dose Studies of Bystander Cell Killing with Targeted Soft X Rays

Abstract Schettino, G., Folkard, M., Prise, K. M., Vojnovic, B., Held, K. D. and Michael, B. D. Low-Dose Studies of Bystander Cell Killing with Targeted Soft X Rays. Radiat. Res. 160, 505–511 (2003). The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic CK X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 μm at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a CK X-ray beam was measured in the 0–2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (<200 mGy) and then reached a plateau (>200 mGy). In the low-dose region (<200 mGy), the response after irradiation of a single cell was not significantly different from that when all cells were exposed to radiation. Damaged cells were distributed uniformly over the area of the dish scanned (∼25 mm2). However, critical analysis of the distance of the damaged, unirradiated cells from other damaged cells revealed the presence of clusters of damaged cells produced under bystander conditions.

Inactivation of V79 cells by low-energy protons, deuterons and helium-3 ions

Previous work by ourselves and by others has demonstrated that protons with a linear energy transfer (LET) about 30 keVmum(-1)are more effective at killing cells than doubly charged particles of the same LET. In this work we show that by using deuterons, which have about twice the range of protons with the same LET, it is possible to extend measurements of the RBE of singly charged particles to higher LET (up to 50 keVmum(-1). We report the design and use of a new arrangement for irradiating V79 mammalian cells. Cell survival measurements have been made using protons in the energy range 1.0-3.7 MeV, deuterons in the energy range 0.9-3.4MeV and 3He2+ ions in the energy range 3.4-6.9 MeV. This corresponds to volume-averaged LET (within the cell nucleus) between 10 and 28 keVmum(-1) for protons, 18-50 keVmum(-1) for deuterons, and 59-106 keVmum(-1) for helium ions. Our results show no difference in the effectiveness of protons and deuterons matched for LET. However, for LET above about 30 keVmum(-1) singly charged particles are more effective at inactivating cells than doubly-charged particles of the same LET and that this difference can be understood in terms of the radial dose distribution around the primary ion track.