THz irradiation inhibits cell division by affecting actin dynamics.

Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.

G2 Premature Chromosome Condensation/Chromosome Aberration Assay: Drug-Induced Premature Chromosome Condensation (PCC) Protocols and Cytogenetic Approaches in Mitotic Chromosome and Interphase Chromatin for Radiation Biology.

Chromosome analysis is a fundamental technique for a wide range of cytogenetic studies. Chromosome aberrations are easily introduced by many kinds of clastogenic agents such as ionizing irradiation, UV, or alkylating agents, and damaged chromosomes may be prone to cancer. Chromosomes are conventionally prepared from mitotic cells arrested by the colcemid block method. However, obtaining of mitotic chromosomes is sometimes hampered under several circumstances, for example after high-dose (over several Gys of γ-rays) ionizing irradiation exposure accident. As a result, cytogenetic analysis will be often difficult or even impossible in such cases. Premature chromosome condensation (PCC) is an alternative technique that has proved to be a unique and useful way in chromosome analysis. Previously, PCC has been achieved following cell fusion mediated either by fusogenic viruses (for example Sendai virus) or by polyethylene glycol (PEG) (cell-fusion PCC), but the cell-fusion PCC has several drawbacks. The novel drug-induced PCC use of specific inhibitors for serine/threonine protein phosphatase was introduced about 20 years ago. This method is much simple and easy even than the conventional mitotic chromosome preparation using colcemid block protocol and the obtained PCC index (equivalent to mitotic index for metaphase chromosome) is much higher. Furthermore, this method allows the interphase chromatin to be condensed and visualized like mitotic chromosomes, and thus has been opening the way for chromosome analysis not only in metaphase chromosomes but also in interphase chromatin. The drug-induced PCC has therefore proven the usefulness in cytogenetics and other many cell biology fields. Since the first version of drug-induced PCC protocol has been published in 2009 (Gotoh, Methods in molecular biology. Humana Press, New York, 2009), many newer applications of drug-induced PCC in radiation biology and chromosome science fields in a wide range of species from animal to plant have been reported (Gotoh et al., Biomed Res 16:63-68, 1995; Lamadrid Boada et al., Mutat Res 757:45-51, 2013; Ravi et al., Biochimie 95:124-33, 2013; Ono et al., J Cell Biol 200:429-41, 2013; Vagnarelli, Exp Cell Res 318:1435-41, 2012; Roukos et al., Nat Protoc 9:2476-92, 2014; Miura and Blakely, Cytometry A 79:1016-22, 2013; Zabka et al., J Plant Physiol 174:62-70, 2015; Samaniego et al., Planta 215:195-204, 2002; Rybaczek et al., Folia Histochem Cytobiol 40:51-9, 2002; Gotoh and Durante J Cell Physiol 209:297-304, 2006). Therefore as a new edition, I will write in this chapter the drug-induced PCC technique with newer findings, in particular focused drug-induced PCC protocols in radiation biology with referring updated articles published recently.

Cell-Type Specific Responses to DNA Replication Stress in Early C. elegans Embryos.

To better understand how the cellular response to DNA replication stress is regulated during embryonic development, we and others have established the early C. elegans embryo as a model system to study this important problem. As is the case in most eukaryotic cell types, the replication stress response is controlled by the ATR kinase in early worm embryos. In this report we use RNAi to systematically characterize ATR pathway components for roles in promoting cell cycle delay during a replication stress response, and we find that these genetic requirements vary, depending on the source of stress. We also examine how individual cell types within the embryo respond to replication stress, and we find that the strength of the response, as defined by duration of cell cycle delay, varies dramatically within blastomeres of the early embryo. Our studies shed light on how the replication stress response is managed in the context of embryonic development and show that this pathway is subject to developmental regulation.

Low concentrations of caffeine induce asymmetric cell division as observed in vitro by means of the CBMN-assay and iFISH.

The dual role of caffeine as a chromosomal damage inducer and G2/M-checkpoint abrogator is well known but it is observed mainly at relatively high concentrations. At low concentrations, caffeine enhances the cytogenetic effects of several carcinogens and its intake during pregnancy has been recently reported to cause adverse birth outcomes. Interestingly, a threshold below which this association is not apparent was not identified. Since chromosomal abnormalities and aneuploidy are the major genetic etiologies of spontaneous abortions and adverse birth outcomes, we re-evaluate here the effects of caffeine at the cytogenetic level and propose a model for the mechanisms involved. Our hypothesis is that low caffeine concentrations affect DNA replication and cause chromosomal aberrations and asymmetric cell divisions not easily detected at metaphase since damaged cells are delayed during their G2/M-phase transition and the low caffeine concentrations cannot abrogate the G2-checkpoint. To test this hypothesis, caffeine-induced chromatid breaks and micronuclei in peripheral blood lymphocytes (PBLs) were evaluated in vitro after low caffeine concentration exposures, followed by a short treatment with 4mM of caffeine to abrogate the G2-checkpoint. The results show a statistically significant increase in chromatid breaks at caffeine concentrations ≥1mM. When caffeine was applied for G2/M-checkpoint abrogation, a statistically significant increase in chromatid breaks, compared to an active checkpoint, was only observed at 4mM of caffeine. The potential of low concentrations to induce asymmetric cell divisions was tested by applying a methodology combining the cytochalasin-B mediated cytokinesis-block micronucleus assay (CBMN) with interphase FISH (iFISH), using selected centromeric probes. Interestingly, low caffeine concentrations induce a dose dependent aneuploidy through asymmetric cell divisions, which are caused by misalignment of chromosomes through a mechanism unrelated to the formation of chromatid breaks. The cytogenetic approach used, combining CBMN with iFISH, is proposed as a valuable tool to test chemically induced asymmetric cell divisions.

Nuclear localization of mouse Ku70 in interphase cells and focus formation of mouse Ku70 at DNA damage sites immediately after irradiation.

To elucidate the mechanisms of DNA repair pathway is critical for developing next-generation radiotherapies and chemotherapeutic drugs for cancer. Ionizing radiation and many chemotherapeutic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). The classical nonhomologous DNA-end joining (NHEJ) (C-NHEJ) pathway repairs a predominant fraction of DSBs in mammalian cells. The C-NHEJ pathway appears to start with the binding of Ku (heterodimer of Ku70 and Ku80) to DNA break ends. Therefore, recruitment of Ku to DSB sites might play a critical role in regulating NHEJ activity. Indeed, human Ku70 and Ku80 localize in the nuclei and accumulate at microirradiated DSB sites. However, the localization and regulation mechanisms of Ku70 and Ku80 homologues in animal models, such as mice and other species, have not been elucidated in detail, particularly in cells immediately after microirradiation. Here, we show that EYFP-tagged mouse Ku70 localizes in the interphase nuclei of mouse fibroblasts and epithelial cells. Furthermore, our findings indicate that EYFP-mouse Ku70 accumulates with its heterodimeric partner Ku80 immediately at laser-microirradiated DSB sites. We also confirmed that the structure of Ku70 nuclear localization signal (NLS) is highly conserved among various rodent species, such as the mouse, rat, degu and ground squirrel, supporting the idea that NLS is important for the regulation of rodent Ku70 function. Collectively, these results suggest that the mechanisms of regulating the localization and accumulation of Ku70 at DSBs might be well conserved between the mouse and human species.

Computational model of dose response for low-LET-induced complex chromosomal aberrations.

Experiments with full-colour mFISH chromosome painting have revealed high yield of radiation-induced complex chromosomal aberrations (CAs). The ratio of complex to simple aberrations is dependent on cell type and linear energy transfer. Theoretical analysis has demonstrated that the mechanism of CA formation as a result of interaction between lesions at a surface of chromosome territories does not explain high complexes-to-simples ratio in human lymphocytes. The possible origin of high yields of γ-induced complex CAs was investigated in the present work by computer simulation. CAs were studied on the basis of chromosome structure and dynamics modelling and the hypothesis of CA formation on nuclear centres. The spatial organisation of all chromosomes in a human interphase nucleus was predicted by simulation of mitosis-to-interphase chromosome structure transition. Two scenarios of CA formation were analysed, 'static' (existing in a nucleus prior to irradiation) centres and 'dynamic' (formed in response to irradiation) centres. The modelling results reveal that under certain conditions, both scenarios explain quantitatively the dose-response relationships for both simple and complex γ-induced interchromosomal exchanges observed by mFISH chromosome painting in the first post-irradiation mitosis in human lymphocytes.

Micronucleus frequencies and clonogenic cell survival in TK6 cells exposed to changing dose rates under controlled temperature conditions.

PURPOSE: In most exposure scenarios the dose rate of exposure is not constant. Despite this, very little information exists on the possible biological effects of exposing cells to radiation under the conditions of a changing dose rate. The current study highlights interesting effects following exposure under these conditions. MATERIALS AND METHODS: We constructed a new exposure facility that allows exposing cells inside an incubator and used it to irradiate human lymphoblastoid TK6 cells both after a moderate (0.48 Gy) and a high (1.1 Gy) dose, where the change in dose rate was, respectively, ≈ 17-fold (2.2-37 mGy/min) and ≈ 39-fold (2.7-106 mGy/min). Clonogenic survival and micronuclei (MN) induction were the chosen endpoints. RESULTS: The obtained results confirm the outcome of our first study that TK6 cells exposed to a decreasing dose rate express more MN than cells exposed to an increasing or constant dose rate. The effect was not seen after the moderate dose of 0.48 Gy or detectable at the level of clonogenic cell survival. CONCLUSIONS: We speculate that the high level of MN is probably related to a delayed elimination of damaged cells by interphase death, as opposed to mechanisms relating to DNA damage and repair.

Relative proximity of chromosome territories influences chromosome exchange partners in radiation-induced chromosome rearrangements in primary human bronchial epithelial cells.

It is well established that chromosomes exist in discrete territories (CTs) in interphase and are positioned in a cell-type specific probabilistic manner. The relative localisation of individual CTs within cell nuclei remains poorly understood, yet many cancers are associated with specific chromosome rearrangements and there is good evidence that relative territorial position influences their frequency of exchange. To examine this further, we characterised the complexity of radiation-induced chromosome exchanges in normal human bronchial epithelial (NHBE) cells by M-FISH analysis of PCC spreads and correlated the exchanges induced with their preferred interphase position, as determined by 1/2-colour 2D-FISH analysis, at the time of irradiation. We found that the frequency and complexity of aberrations induced were reduced in ellipsoid NHBE cells in comparison to previous observations in spherical cells, consistent with aberration complexity being dependent upon the number and proximity of damaged CTs, i.e. lesion proximity. To ask if particular chromosome neighbourhoods could be identified we analysed all radiation-induced pair-wise exchanges using SCHIP (statistics for chromosome interphase positioning) and found that exchanges between chromosomes (1;13), (9;17), (9;18), (12;18) and (16;21) all occurred more often than expected assuming randomness. All of these pairs were also found to be either sharing similar preferred positions in interphase and/or sharing neighbouring territory boundaries. We also analysed a human small cell lung cancer cell line, DMS53, by M-FISH observing the genome to be highly rearranged, yet possessing rearrangements also involving chromosomes (1;13) and (9;17). Our findings show evidence for the occurrence of non-random exchanges that may reflect the territorial organisation of chromosomes in interphase at time of damage and highlight the importance of cellular geometry for the induction of aberrations of varying complexity after exposure to both low and high-LET radiation.

[Effect of of distamycin A on histone H1 methylation, extraction and formation of UV-inducible crosslinks with DNA in the interphase rat liver nucleus].

Incubation in vitro of rat liver nuclei in the presence of S-adenosyl[methyl-(3)H]methionine ([(3)H] SAM) leads to incorporation of the radioactive label not only into core-histones H3 and H4, but also into linker histone H1. Addition of distamycine A to the incubation medium stimulates label incorporation into histone H1 ~ in 6 times and into histone H3 ~ in 2 times. The presence of distamycine facilitates histone H1 extraction by polyglutamic acid (poly(Glu)) and decreases of UV-induced DNA-histone cross-links formation. These effects give evidence of weakening of H1-chromatin interaction by distamycin to be results of histone H1 position change relative to nucleosome and(or) disturbance of histones H1-H3 interactions so as these histones are exposed to additional methylation.

A mouse PRMT1 null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation.

Protein arginine methyltransferase 1 (PRMT1) is the major enzyme that generates monomethylarginine and asymmetrical dimethylarginine. We report here a conditional null allele of PRMT1 in mice and that the loss of PRMT1 expression leads to embryonic lethality. Using the Cre/lox-conditional system, we show that the loss of PRMT1 in mouse embryonic fibroblasts (MEFs) leads to the loss of arginine methylation of substrates harboring a glycine-arginine rich motif, including Sam68 and MRE11. The loss of PRMT1 in MEFs leads to spontaneous DNA damage, cell cycle progression delay, checkpoint defects, aneuploidy, and polyploidy. We show using a 4-hydroxytamoxifen-inducible Cre that the loss of PRMT1 in MEFs leads to a higher incidence of chromosome losses, gains, structural rearrangements, and polyploidy, as documented by spectral karyotyping. Using PRMT1 small interfering RNA in U2OS cells, we further show that PRMT1-deficient cells are hypersensitive to the DNA damaging agent etoposide and exhibit a defect in the recruitment of the homologous recombination RAD51 recombinase to DNA damage foci. Taken together, these data show that PRMT1 is required for genome integrity and cell proliferation. Our findings also suggest that arginine methylation by PRMT1 is a key posttranslational modification in the DNA damage response pathway in proliferating mammalian cells.

[The reentrant binomial model of nuclear anomalies growth in rhabdomyosarcoma RA-23 cell populations under increasing doze of rare ionizing radiation].

Distributions of nuclear morphology anomalies in transplantable rabdomiosarcoma RA-23 cell populations were investigated under effect of ionizing radiation from 0 to 45 Gy. Internuclear bridges, nuclear protrusions and dumbbell-shaped nuclei were accepted for morphological anomalies. Empirical distributions of the number of anomalies per 100 nuclei were used. The adequate model of reentrant binomial distribution has been found. The sum of binomial random variables with binomial number of summands has such distribution. Averages of these random variables were named, accordingly, internal and external average reentrant components. Their maximum likelihood estimations were received. Statistical properties of these estimations were investigated by means of statistical modeling. It has been received that at equally significant correlation between the radiation dose and the average of nuclear anomalies in cell populations after two-three cellular cycles from the moment of irradiation in vivo the irradiation doze significantly correlates with internal average reentrant component, and in remote descendants of cell transplants irradiated in vitro - with external one.

Interphase chromosome positioning affects the spectrum of radiation-induced chromosomal aberrations.

In interphase, chromosomes occupy defined nuclear volumes known as chromosome territories. To probe the biological consequences of the described nonrandom spatial positioning of chromosome territories in human lymphocytes, we performed an extensive FISH-based analysis of ionizing radiation-induced interchanges involving chromosomes 1, 4, 18 and 19. Since the probability of exchange formation depends strongly on the spatial distance between the damage sites in the genome, a preferential formation of exchanges between proximally positioned chromosomes is expected. Here we show that the spectrum of interchanges deviates significantly from one expected based on random chromosome positioning. Moreover, the observed exchange interactions between specific chromosome pairs as well as the interactions between homologous chromosomes are consistent with the proposed gene density-related radial distribution of chromosome territories. The differences between expected and observed exchange frequencies are more pronounced after exposure to densely ionizing neutrons than after exposure to sparsely ionizing X rays. These experiments demonstrate that the spatial positioning of interphase chromosomes affects the spectrum of chromosome rearrangements.

Pairing of heterochromatin in response to cellular stress.

We previously reported that exposure of human cells to DNA-damaging agents (X-rays and mitomycin C (MMC)) induces pairing of the homologous paracentromeric heterochromatin of chromosome 9 (9q12-13). Here, we show that UV irradiation and also heat shock treatment of human cells lead to similar effects. Since the various agents induce very different types and frequencies of damage to cellular constituents, the data suggest a general stress response as the underlying mechanism. Moreover, local UV irradiation experiments revealed that pairing of heterochromatin is an event that can be triggered without induction of DNA damage in the heterochromatic sequences. The repair deficient xeroderma pigmentosum cells (group F) previously shown to fail pairing after MMC displayed elevated pairing after heat shock treatment but not after UV exposure. Taken together, the present results indicate that pairing of heterochromatin following exposure to DNA-damaging agents is initiated by a general stress response and that the sensing of stress or the maintenance of the paired status of the heterochromatin might be dependent on DNA repair.

Exploration of "over kill effect" of high-LET Ar- and Fe-ions by evaluating the fraction of non-hit cell and interphase death.

The reason why RBE for cell killing fell to less than unity (1.0) with very high-LET heavy-ions ((40)Ar: 1,640 keV/microm; (56)Fe: 780, 1,200, 2,000 keV/microm) was explored by evaluating the fraction of non-hit cell (time-lapse observation) and cells undergoing interphase death (calculation based on our previous data). CHO cells were exposed to 4 Gy (30% survival dose) of Ar (1,640 keV/microm) or Fe-ions (2,000 keV/microm). About 20% of all cells were judged to be non-hit, and about 10% cells survived radiation damage. About 70% cells died after dividing at least once (reproductive death) or without dividing (interphase death). RBE for reproductive (RBE[R]) and interphase (RBE[I]) death showed a similar LET dependence with maximum around 200 keV/microm. In this LET region, at 30% survival level, about 10% non-survivors underwent interphase death. The corresponding value for very high-LET Fe-ions (2,000 keV/microm) was not particularly high (approximately 15%), whereas that for X-rays was less than 3%. However, reproductive death (67%) predominated over interphase death (33%) even in regard to rather severely damaged cells (1% survival level) after exposure to Fe-ions (2,000 keV/microm). These indicate that interphase death is a type of cell death characteristic for the cells exposed to high-LET radiation and is not caused by "cellular over kill effect". Both NHF37 (non-hit fraction at 37% survival) and inactivation cross-section for reproductive death (sigma[R]) began to increase when LET exceeded 100 keV/microm. The exclusion of non-hit fraction in the calculation of surviving fraction partially prevented the fall of RBE[R] when LET exceeded 200 keV/microm. On the other hand, the mean number of lethal damage per unit dose (NLD/Gy) showed the same LET-dependent pattern as RBE[R]. These suggest that the increase in non-hit fraction and sigma[R] with an increasing LET is caused by enhanced clustering of ionization and DNA damage which lowers the energy efficiency for producing damage and RBE.

SCHIP: statistics for chromosome interphase positioning based on interchange data.

MOTIVATION: The position of chromosomes in the interphase nucleus is believed to be associated with a number of biological processes. Here, we present a web-based application that helps analyze the relative position of chromosomes during interphase in human cells, based on observed radiogenic chromosome aberrations. The inputs of the program are a table of yields of pairwise chromosome interchanges and a proposed chromosome geometric cluster. Each can either be uploaded or selected from provided datasets. The main outputs are P-values for the proposed chromosome clusters. SCHIP is designed to be used by a number of scientific communities interested in nuclear architecture, including cancer and cell biologists, radiation biologists and mathematical/computational biologists.

Human Rad51C deficiency destabilizes XRCC3, impairs recombination, and radiosensitizes S/G2-phase cells.

The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) are expressed in mitotically growing cells and are thought to play mediating roles in homologous recombination, although their precise functions remain unclear. Among the five paralogs, Rad51C was found to be a central component present in two complexes, Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2. We have shown previously that the human Rad51C protein exhibits three biochemical activities, including DNA binding, ATPase, and DNA duplex separation. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA-cross-linking agent mitomycin C and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G(2)/M phases of the cell cycle but not in G(1) phase. Together, these results provide direct cellular evidence for the function of human Rad51C in homologous recombinational repair.

Estimation of radiation-induced interphase cell death in cultures of human tumor material and in cell lines.

A short-term assay method able to estimate the radiation response of human cancer tissue samples would be of great advantage to the individualization of radiotherapy in cancer patients. However, the effect of radiation on [3H]thymidine incorporation by proliferating cells reflects a composite of cell cycle arrest and induced cell death pathways. Here we consider whether it is feasible to correct for cell cycle effects based on comparison of the effects of radiation and the mitotic inhibitor paclitaxel on [3H]thymidine incorporation. Sixty-two short-term (7-day) cultures of human tumor tissue from 61 patients with melanoma, gynecological cancer, brain cancer, and head and neck cancer, as well as 18 5-day cultures of low passage human tumor cell lines, were irradiated at doses from 2 to 9 Gy, or exposed to paclitaxel (200 nM). [3H]Thymidine incorporation was measured at the end of the incubation. Cell cycle times could be estimated from the paclitaxel data and were 2.7 to 18.6 days for melanomas, 2.5 to >40 days for carcinomas, 3.9 to 39 days for brain tumors, and 1.1 to 3.8 days for cell lines. The effects of radiation on [3H]thymidine incorporation varied widely (0-97% and 0-99% inhibition for 2 and 9 Gy, respectively), and in 23 of the clinical samples, but in none of the cell lines, radiation caused significantly greater inhibition of [3H]thymidine incorporation than paclitaxel (p < 0.05). We argue that that these differences reflect radiation-induced cell loss from G1 phase and/or S phase. Responses of short-term cultures of clinical tumor material to radiation, with appropriate correction for cell cycle effects, might have the potential to provide information on radiation-induced cell death in individual patients.

Chromosome aberrations induced by high-LET radiations.

Measurements of chromosome aberrations in peripheral blood lymphocytes are currently the most sensitive and reliable indicator of radiation exposure that can be used for biological dosimetry. This technique has been implemented recently to study radiation exposures incurred by astronauts during space flight, where a significant proportion of the dose is delivered by high-LET particle exposure. Traditional methods for the assessing of cytogenetic damage in mitotic cells collected at one time point after exposure may not be suitable for measuring high-LET radiation effects due to the drastic cell cycle perturbations and interphase cell death induced by this type of exposure. In this manuscript we review the recent advances in methodology used to study high-LET induced cytogenetic effects and evaluate the use of chemically-induced Premature Chromosome Condensation (PCC) as an alternative to metaphase analysis. Published data on the cytogenetic effects of in vitro exposures of high-LET radiation is reviewed, along with biodosimetry results from astronauts after short or long space missions.

Biological effectiveness of accelerated particles for the induction of chromosome damage measured in metaphase and interphase human lymphocytes.

Chromosome aberrations were investigated in human lymphocytes after in vitro exposure to 1H-, 3He-, 12C-, 40Ar-, 28Si-, 56Fe-, or 197Au-ion beams, with LET ranging from approximately 0.4-1393 keV/microm in the dose range of 0.075-3 Gy. Dose-response curves for chromosome exchanges, measured at the first mitosis postirradiation using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosomal damage with respect to low- or high-dose-rate gamma rays. Estimates of RBEmax values for mitotic spreads, which ranged from near 0.7 to 11.1 for total exchanges, increased with LET, reaching a maximum at about 150 keV/microm, and decreased with further increase in LET. RBEs for complex aberrations are undefined due to the lack of an initial slope for gamma rays. Additionally, the effect of mitotic delay on RBE values was investigated by measuring chromosome aberrations in interphase after chemically induced premature chromosome condensation (PCC), and values were up to threefold higher than for metaphase analysis.