Brian Marples

LOW-DOSE HYPERSENSITIVITY: CURRENT STATUS AND POSSIBLE MECHANISMS

PURPOSE To retain cell viability, mammalian cells can increase damage repair in response to excessive radiation-induced injury. The adaptive response to small radiation doses is an example of this induced resistance and has been studied for many years, particularly in human lymphocytes. This review focuses on another manifestation of actively increased resistance that is of potential interest for developing improved radiotherapy, specifically the phenomenon in which cells die from excessive sensitivity to small single doses of ionizing radiation but remain more resistant (per unit dose) to larger single doses. In this paper, we propose possible mechanisms to explain this phenomenon based on our data accumulated over the last decade and a review of the literature. CONCLUSION Typically, most cell lines exhibit hyper-radiosensitivity (HRS) to very low radiation doses (<10 cGy) that is not predicted by back-extrapolating the cell survival response from higher doses. As the dose is increased above about 30 cGy, there is increased radioresistance (IRR) until at doses beyond about 1 Gy, radioresistance is maximal, and the cell survival follows the usual downward-bending curve with increasing dose. The precise operational and activational mechanism of the process is still unclear, but we propose two hypotheses. The greater amount of injury produced by larger doses either (1) is above a putative damage-sensing threshold for triggering faster or more efficient DNA repair or (2) causes changes in DNA structure or organization that facilitates constitutive repair. In both scenarios, this enhanced repair ability is decreased again on a similar time scale to the rate of removal of DNA damage.

Low-Dose Hyper-radiosensitivity: A Consequence of Ineffective Cell Cycle Arrest of Radiation-Damaged G2-Phase Cells

Abstract Marples, B., Wouters, B. G., Collis, S. J., Chalmers, A. J. and Joiner, M. C. Low-Dose Hyper-radiosensitivity: A Consequence of Ineffective Cell Cycle Arrest of Radiation-Damaged G2-Phase Cells. Radiat. Res. 161, 247–255 (2004). This review highlights the phenomenon of low-dose hyper- radiosensitivity (HRS), an effect in which cells die from excessive sensitivity to small single doses of ionizing radiation but become more resistant (per unit dose) to larger single doses. Established and new data pertaining to HRS are discussed with respect to its possible underlying molecular mechanisms. To explain HRS, a three-component model is proposed that consists of damage recognition, signal transduction and damage repair. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell cycle checkpoint that is specific to cells irradiated in the G2phase. This checkpoint exhibits a dose expression profile that is identical to the cell survival pattern that characterizes HRS and is probably the key control element of low-dose radiosensitivity. This premise is strengthened by the recent observation coupling low- dose radiosensitivity of G2-phase cells directly to HRS. The putative role of known damage response factors such as ATM, PARP, H2AX, 53BP1 and HDAC4 is also included within the framework of the HRS model.

Low-dose hyper-radiosensitivity: past, present, and future.

This review article discusses the biology of low-dose hyper-radiosensitivity (HRS) with reference to the molecular regulation of DNA repair and cell cycle control processes. Particular attention is paid to the significance of G2-phase cell cycle checkpoints in overcoming low-dose hyper-radiosensitivity and the impact of HRS on low-dose rate radiobiology. The history of HRS from the original in vivo discovery to the most recent in vitro and clinical data are examined to present a unifying hypothesis concerning the molecular control and regulation of this important low dose radiation response. Finally, preclinical and clinical data are discussed, from a molecular viewpoint, to provide theoretical approaches to exploit HRS biology for clinical gain.

Hypersensitivity of a human tumour cell line to very low radiation doses

Survival of HT29 cells was measured after irradiation with single doses of X-rays (0.05-5 Gy) and neutrons (0.025-1.5 Gy), using a Dynamic Microscopic Imaging Processing Scanner (DMIPS) with which individual cells can be accurately located in tissue culture flasks, their positions recorded, and after an appropriate incubation time the recorded positions revisited to allow the scoring of survivors. The response over the X-ray dose range 2-5 Gy showed a good fit to a Linear-Quadratic (LQ) model. For X-ray doses below 1 Gy, an increased X-ray effectiveness was observed with cell survival below the high-dose LQ prediction. The value of --dose/loge (SF) for each experimental data point, plotted against dose, demonstrated clearly how X-rays are maximally effective at doses approaching zero, becoming less effective as the dose increases and with minimal effectiveness at about 0.6 Gy then becoming more effective again as the dose increases above 1.5 Gy. This phenomenon was not seen with neutrons. Neutron RBE was calculated for each X-ray data point by taking each X-ray survival value and comparing it with the common LQ fit to all the neutron data. Over the X-ray dose range 0.05-0.2 Gy, the RBE is close to 1 indicating that these very low doses of X-rays are of similar effectiveness to neutrons in killing cells. The increase in RBE with increasing dose over the range 0.05-1 Gy, and the slight decrease in RBE above 1 Gy, reflect primarily the changes in X-ray sensitivity over the whole dose range of 0.05-5 Gy. Several arguments suggest that this phenomenon could reflect an induced radioresistance so that in this system low single doses of X-rays are more effective per Gy than higher doses in reducing cell survival because only at higher doses, above a threshold, is there sufficient damage to trigger radioprotective mechanisms.

Low dose hyper-radiosensitivity and increased radioresistance in mammalian cells

This manuscript reviews the low-dose survival work using the DMIPS cell analyser that has been carried out at the Gray Laboratory in the U.K. and the British Columbia Cancer Research Centre in Canada. It describes low dose hyper-radiosensitivity (HRS) detected after single doses of X-rays less than approximately 0.3 Gy and the subsequent increased radioresistant response (IRR) seen as the dose increases up to 1 Gy. Work is summarized from studies in V79 cells, normal human and human tumour cell lines and mutant cell lines deficient in DNA repair. The data are considered in light of the hypothesis that hyper-radiosensitivity and increased radioresistance reflect the existence of an inducible protective mechanism, possibly triggered by DNA damage.

An Association between the Radiation-Induced Arrest of G2-Phase Cells and Low-Dose Hyper-Radiosensitivity: A Plausible Underlying Mechanism?

Abstract Marples, B., Wouters, B. G. and Joiner, M. C. An Association between the Radiation-Induced Arrest of G2-Phase Cells and Low-Dose Hyper-Radiosensitivity: A Plausible Underlying Mechanism? Radiat. Res. 160, 38–45 (2003). The survival of asynchronous and highly enriched G1-, S- and G2-phase populations of Chinese hamster V79 cells was measured after irradiation with 60Co γ rays (0.1–10 Gy) using a precise flow cytometry-based clonogenic survival assay. The high-dose survival responses demonstrated a conventional relationship, with G2-phase cells being the most radiosensitive and S-phase cells the most radioresistant. Below 1 Gy, distinct low-dose hyper-radiosensitivity (HRS) responses were observed for the asynchronous and G2-phase enriched cell populations, with no evidence of HRS in the G1- and S-phase populations. Modeling supports the conclusion that HRS in asynchronous V79 populations is explained entirely by the HRS response of G2-phase cells. An association was discovered between the occurrence of HRS and the induction of a novel G2-phase arrest checkpoint that is specific for cells that are in the G2 phase of the cell cycle at the time of irradiation. Human T98G cells and hamster V79 cells, which both exhibit HRS in asynchronous cultures, failed to arrest the entry into mitosis of damaged G2-phase cells at doses less than 30 cGy, as determined by the flow cytometric assessment of the phosphorylation of histone H3, an established indicator of mitosis. In contrast, human U373 cells that do not show HRS induced this G2-phase checkpoint in a dose-independent manner. These data suggest that HRS may be a consequence of radiation-damaged G2-phase cells prematurely entering mitosis.

The elimination of low-dose hypersensitivity in Chinese hamster V79-379A cells by pretreatment with X rays or hydrogen peroxide.

To explain increased radioresistance over the X-ray dose range approximately 0.5-1 Gy an inducible radioprotective mechanism triggered by DNA damage was proposed; hypersensitivity to doses << 0.5 Gy reflected the response prior to the activation of this system (Marples and Joiner, Radiat. Res. 133, 41-51, 1993). To test this hypothesis, cells were pre-exposed to DNA-damaging agents in an attempt to induce the process prematurely. An increase in survival was evident at X-ray doses below 0.3 Gy after a priming treatment of X rays (0.05, 0.2, 1 Gy) given 6 h earlier. The protective effect was found to be transitory, requiring time for development and diminishing after two to three cell cycle times. Cycloheximide administered in the interval between the priming and challenge doses of X rays abolished the protection conferred by pretreatment, indicating the involvement of de novo protein synthesis. Oxidative damage by nontoxic doses of hydrogen peroxide (10(-4) M, but not 10(-6) M) also produced a protective effect against subsequent X irradiation. These experiments indicate survival in the hyper-radiosensitive region (<< 0.5 Gy) can be modified by pretreatment with agents known to affect DNA repair. In addition, the development of increased radioresistance after single doses of X rays was inhibited by cycloheximide treatment. These studies provide evidence to support the explanations proposed previously for the phenomena of increased radioresistance and hyper-radiosensitivity observed at very low X-ray doses.

Effects of cell cycle phase on low-dose hyper-radiosensitivity

Purpose : To examine the low-dose radiation response of human glioma cell lines separated into different cell-cycle phases and to determine if low-dose hyper-radiosensitivity (HRS) differs in populations defined by cell-cycle position. To assess whether predictions of the outcome of multiple low-dose regimens should take account of cell-cycle effects. Materials and methods : The clonogenic survival of G1, G2 and S phase cells was measured after exposure to single doses of X-rays in two human glioma cell lines. One cell line (T98G) showed marked HRS when asynchronous cells were irradiated, while the other (U373) did not. Separation of populations and high-resolution cell counting was achieved using a fluorescence activated cell sorter. Sorted cell populations were irradiated with 240 kVp X-rays to doses between 0.05 and 5Gy. The resulting cell-survival versus dose data were comparatively fitted using the linear-quadratic and induced-repair models in order to assess the degree of HRS. Results : In both cell lines the low-dose response was altered when different populations were irradiated. In T98G cells, all populations showed HRS, but this was most marked in G2 phase cells. In U373 cells, no HRS was found in G1 or S phase cells, but HRS was demonstrable in G2 phase cells. Conclusions : HRS was expressed by the whole cell population of T98G cells but the size of the effect varied with cell-cycle phase and was most marked in the G2 population. In U373 cells, the effect could only be demonstrated in G2 cells. This implies that HRS is primarily a response of G2 phase cells and that this response dominates that seen in asynchronous populations. Actively proliferating cell populations may therefore demonstrate a greater increase in radiosensitivity to very low radiation doses compared with quiescent populations.

Role of Apoptosis in Low-Dose Hyper-radiosensitivity

Abstract Krueger, S. A., Joiner, M. C., Weinfeld, M., Piasentin, E. and Marples, B. Role of Apoptosis in Low-Dose Hyper-radiosensitivity. Radiat. Res. 167, 260–267 (2007). Little is known about the mode of cell killing associated with low-dose hyper-radiosensitivity, the radiation response that describes the enhanced sensitivity of cells to small doses of ionizing radiation. Using a technique that measures the activation of caspase 3, we have established a relationship between apoptosis detected 24 h after low-dose radiation exposure and low-dose hyper-radiosensitivity in four mammalian cell lines (T98G, U373, MR4 and 3.7 cells) and two normal human lymphoblastoid cell lines. The existence of low-dose hyper-radiosensitivity in clonogenic survival experiments was found to be associated with an elevated level of apoptosis after low-dose exposures, corroborating earlier observations (Enns et al., Mol. Cancer Res. 2, 557–566, 2004). We also show that enriching populations of MR4 and V79 cells with G1-phase cells, to minimize the numbers of G2-phase cells, abolished the enhanced low-dose apoptosis. These cell-cycle enrichment experiments strengthen the reported association between low-dose hyper-sensitivity and the radioresponse of G2-phase cells. These data are consistent with our current hypothesis to explain low-dose hyper-radiosensitivity, namely that the enhanced sensitivity of cells to low doses of ionizing radiation reflects the failure of ATM-dependent repair processes to fully arrest the progression of damaged G2-phase cells harboring unrepaired DNA breaks entering mitosis.

Development of synthetic promoters for radiation-mediated gene therapy.

Exposure of cells to ionising radiation results in the activation of specific transcriptional control (CArG) elements within the early growth response 1 (Egr1) gene promoter, leading to increased gene expression. As part of a study investigating the potential use of these elements in radiation-controlled gene therapy vectors, we have incorporated their sequences into a synthetic gene promoter and assayed for the ability to induce expression of a downstream reporter gene following irradiation. In vector-transfected MCF-7 breast adenocarcinoma cells, the synthetic promoter was more effective than the wild-type Egr1 counterpart in up-regulating expression of the reporter gene after exposure to a single 5 Gy dose, and equally effective as the wild-type in U87-MG glioma cells. The level of gene expression achieved using the synthetic promoter was dependent on the inducing radiation dose for both U87-MG and MCF-7 cells, being maximal at 3 Gy and decreasing at 5 and 10 Gy. Furthermore, induction could be repeated by additional radiation treatments. The latter indicates that up-regulation should be additive during fractionated radiotherapy schedules. To demonstrate the potential clinical benefit of such an approach, the synthetic promoters were also shown to drive expression of the herpes simplex virus thymidine kinase gene, leading to enhanced cell killing in the presence of the prodrug ganciclovir (GCV) when compared with cells treated with radiation alone. Our results demonstrate that the synthetic promoter is responsive to low doses of ionising radiation and therefore isolated CArG elements function as radiation-mediated transcriptional enhancers outside their normal sequence context. The continued development and optimisation of such radiation-responsive synthetic promoters is expected to make a valuable contribution to the development of future radiation-responsive vectors for cancer gene therapy.