H.C. Newman

A Review of Studies of Ionizing Radiation-Induced Double-Strand Break Clustering

Abstract Prise, K. M., Pinto, M., Newman, H. C. and Michael, B. D. A Review of Studies of Ionizing Radiation-Induced Double-Strand Break Clustering. Radiat. Res. 156, 572–576 (2001). Underpinning current models of the mechanisms of the action of radiation is a central role for DNA damage and in particular double-strand breaks (DSBs). For radiations of different LET, there is a need to know the exact yields and distributions of DSBs in human cells. Most measurements of DSB yields within cells now rely on pulsed-field gel electrophoresis as the technique of choice. Previous measurements of DSB yields have suggested that the yields are remarkably similar for different types of radiation with RBE values ≤1.0. More recent studies in mammalian cells, however, have suggested that both the yield and the spatial distribution of DSBs are influenced by radiation quality. RBE values for DSBs induced by high-LET radiations are greater than 1.0, and the distributions are nonrandom. Underlying this is the interaction of particle tracks with the higher-order chromosomal structures within cell nuclei. Further studies are needed to relate nonrandom distributions of DSBs to their rejoining kinetics. At the molecular level, we need to determine the involvement of clustering of damaged bases with strand breakage, and the relationship between higher-order clustering over sizes of kilobase pairs and above to localized clustering at the DNA level. Overall, these studies will allow us to elucidate whether the nonrandom distributions of breaks produced by high-LET particle tracks have any consequences for their repair and biological effectiveness.

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.

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.

Effect of radiation quality on lesion complexity in cellular DNA

Understanding the critical lesions induced by ionizing radiation in DNA and their relationship to cellular effects is an important challenge in radiation biology. Much evidence has suggested that DNA double-strand breaks (dsb) are important lesions. Establishing a cause and effect relationship between initial levels of DNA dsb, their repair rate or the level of residual unrepaired breaks, and cellular effects has proved difficult in mammalian cells. Several studies have measured yields of DNA dsb after irradiation with radiations of differing linear energy transfer (LET). In general the RBEs for dsb induction (20-100 keV/microns) have been lower than the RBEs measured for cell survival and in many cases are around 1.0. Several studies have shown differences in the rejoining of dsb with less dsb rejoined after high-LET irradiation in comparison with low-LET radiation. These results suggest that there may be differences in the types of lesions induced by different radiations and scored as DNA dsb using current techniques. Track structure modelling studies have suggested that some lesions induced will be clustered at the sites of energy depositions and that uniquely large energy deposition events are produced by high-LET radiations. Assays need to be developed to measure complex lesions in both model DNA and cellular systems. Different levels of complexity need to be considered such as clustering of radicals close to DNA, localized areas of DNA damage (1-20 bp) and lesions which may be induced over larger distances. Studies using new and existing assays of DNA damage, coupled with irradiation at various LETs, are directed at understanding the role of lesion complexity in relation to cellular effects.

Quantification of DNA damage by PFGE : development of an analytical approach to correct for the background distribution

Purpose : To analyse the currently existing methods to infer the extent of cellular DNA damage induced by ionizing radiation when the pulsed field gel electrophoresis (PFGE) technique is used. Results and conclusions : PFGE is currently the method of choice for the measurement of radiation-induced double-strand breaks (dsb). For accurate determination of both the yields and distributions of breaks, separation of a large range of fragment sizes is required. In the conventional analysis of PFGE experiments, the background distribution of fractionated molecules is, normally, simply subtracted from the irradiated measured distribution, for each molecular weight region available. This work shows that this approach may lead to incorrect estimation of the breakage frequencies. An alternative approach based on correcting the fitting functions for the actual nonrandom damage present in the control unirradiated samples has been developed.PURPOSE To analyse the currently existing methods to infer the extent of cellular DNA damage induced by ionizing radiation when the pulsed field gel electrophoresis (PFGE) technique is used. RESULTS AND CONCLUSIONS PFGE is currently the method of choice for the measurement of radiation-induced double-strand breaks (dsb). For accurate determination of both the yields and distributions of breaks, separation of a large range of fragment sizes is required. In the conventional analysis of PFGE experiments, the background distribution of fractionated molecules is, normally, simply subtracted from the irradiated measured distribution, for each molecular weight region available. This work shows that this approach may lead to incorrect estimation of the breakage frequencies. An alternative approach based on correcting the fitting functions for the actual nonrandom damage present in the control unirradiated samples has been developed.