Michael C. Joiner

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.

Hypersensitivity to very-low single radiation doses: its relationship to the adaptive response and induced radioresistance.

There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.

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.

Renal damage in the mouse: the response to very small doses per fraction

Experiments were undertaken to study the effect on the mouse kidney of repeated X-ray doses in the range 0.2 to 1.6 Gy per fraction and neutron doses in the range 0.05 to 0.25 Gy per fraction. A top-up design of experiment was used, so that additional graded doses of d(4)-Be neutrons (EN = 2.3 MeV) were given to bring the subthreshold damage produced by these treatments into the measurable range. This approach avoided the necessity to use a large number of fractions to study low doses per fraction. Renal damage was assessed using three methods: 51Cr-EDTA clearance, urine output, and hematocrit at 16-50 weeks postirradiation. The dose-response curves obtained were resolved best at 29 weeks. However, the results were also examined by fitting second-order polynomials to the data for response versus time postirradiation and using interpolated values from these functions at 29 weeks to construct dose-response curves. This method reduced slightly the variation in the dose-response data, but the interrelationship between the dose-response curves remained the same. The data were used to test the linear-quadratic (LQ) description of the underlying X-ray dose-fractionation relationship. The model fits well down to X-ray doses per fraction of approximately 1 Gy, but lower X-ray doses were more effective per gray than predicted by LQ, as seen previously in skin [M. C. Joiner et al., Int. J. Radiat. Biol. 49, 565-580 (1986)]. This increased X-ray effectiveness and deviation from LQ are reflected directly in a decrease in the RBE of d(4)-Be neutrons relative to X-rays at low doses, since the underlying response to these neutrons is linear in this low-dose region. The RBE decreases from 9.9 to 4.7 as the X-ray dose per fraction is reduced below 0.8 Gy to 0.2 Gy, reflecting an increase in X-ray effectiveness by a factor of 2.1. A model is discussed which attempts to explain this behavior at low doses per fraction.

A therapeutic benefit from combining normobaric carbogen or oxygen with nicotinamide in fractionated X-ray treatments.

The ability of normobaric oxygen and carbogen (95% O2 + 5% CO2) combined with nicotinamide to enhance the radiosensitivity of two rodent adenocarcinomas and of mouse skin and kidneys, using a 10 fraction radiation schedule, was compared with the effect of radiation in air with and without the drug. Tumour response was assayed using local control and regrowth delay, and compared with acute skin reactions, decreased renal 51Cr-EDTA clearance and reduction in haematocrit. Nicotinamide increased the radiation sensitivity of CaNT tumours under all three different oxygen concentrations tested (21, 95 and 100% oxygen). The effect was statistically significant for oxygen and carbogen but not for air; the combination of nicotinamide with carbogen gave the greatest increase in tumour radiosensitivity. Relative to treatments in air without the drug, the enhancement ratios (ER) at the TCD50 level were 1.17, 1.65 and 1.83 for CaNT tumours irradiated in air, oxygen or carbogen and injected with nicotinamide 1 h before each fraction. The ER in CaRH tumours irradiated in carbogen plus the drug was 1.83, which was greater, but statistically not significantly different, to that seen with carbogen alone (ER = 1.68). In skin, relative to air without the drug, the increase in radiosensitivity by nicotinamide was greater in oxygen and carbogen than in air (1.29, 1.36 and 1.08, respectively). The ERs for both assays of renal damage were similar and lower than those in skin: less than or equal to 1.07, less than or equal to 1.13 and less than or equal to 1.16 for irradiations done in air, oxygen and carbogen plus nicotinamide, relative to air alone. A comparison of these results in the tumours and normal tissues showed that a significant therapeutic benefit was obtained with normobaric oxygen and carbogen combined with nicotinamide. This benefit is greater than observed with other radiosensitizers tested so far. Toxic side effects of the treatment are unlikely in a clinical situation, since prolonged administration of nicotinamide is well tolerated in man. The combination of normobaric carbogen with nicotinamide could be an effective method of enhancing tumour radiosensitivity in clinical radiotherapy where hypoxia limits the outcome of treatment.

Relationship between Radiation-Induced Low-Dose Hypersensitivity and the Bystander Effect

Abstract Mothersill, C., Seymour, C. B. and Joiner, M. C. Relationship between Radiation-Induced Low-Dose Hypersensitivity and the Bystander Effect. Radiat. Res. 157, 526–532 (2002). Recent advances in our knowledge of the biological effects of low doses of ionizing radiation have shown two unexpected phenomena: a “bystander effect” that can be demonstrated at low doses as a transferable factor(s) causing radiobiological effects in unexposed cells, and low-dose hyper-radiosensitivity and increased radioresistance that can be demonstrated collectively as a change in the dose–effect relationship, occurring around 0.5–1 Gy of low-LET radiation. In both cases, the effect of very low doses is greater than would be predicted by conventional DNA strand break/repair-based radiobiology. This paper addresses the question of whether the two phenomena have similar or exclusive mechanisms. Cells of 13 cell lines were tested using established protocols for expression of both hyper-radiosensitivity/increased radioresistance and a bystander response. Both were measured using clonogenicity as an end point. The results showed considerable variation in the expression of both phenomena and suggested that cell lines with a large bystander effect do not show hyper-radiosensitivity. The reverse was also true. This inverse relationship was not clearly related to the TP53 status or malignancy of the cell line. There was an indication that cell lines that have a radiation dose–response curve with a wide shoulder show hyper-radiosensitivity/increased radioresistance and no bystander effect. The results may suggest new approaches to understanding the factors that control cell death or the sectoring of survival at low radiation doses.

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.

Hypersensitive Response of Normal Human Lung Epithelial Cells at Low Radiation Doses

The effect of very low single X-ray doses (0.05-4 Gy) was investigated in a human lung epithelial cell line (L132). Cell survival measurements were made using a Dynamic Microscopic Imaging Processing Scanner (DMIPS), which allowed single cells to be located accurately, their positions recorded and these positions revisited after an appropriate incubation period at 37 degrees C; surviving cells were identified by their ability to produce a colony > or = 50 cells. The survival data at doses > or = 2 Gy were well-fitted by a linear-quadratic (LQ) model. For doses < 1 Gy, increased X-ray effectiveness was observed with cell survival below the prediction from the fit of the LQ model to the higher dose data, extrapolated into the low dose region. This is the first evidence for the existence of a hypersensitive survival response to very low doses in normal human cells. The transition between the low dose hypersensitive region and greater resistance at higher doses, could result from induced radioresistance which requires a threshold of radiation-induced damage before being triggered.

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.