Radiation cytogenetics has rich history and goes back over 70 years since its inception when McClintock (1931) integrated the radiation-induced mutations of Muller (1927) and Stadler (1928) into chromosome aberrations. Studies on the effects of radiation on chromosomes in the 1930s constituted the bases of later biophysical modeling such as target theory of Lea (1946), dual radiation action theory of Kellerer and Rossi (1972) and threshold energy model of Goodhead (1982). Coupled with the advancement in the molecular biology, cytogenetic technology, radiation sources and computer sciences, the classical theories of breakage-reunion hypothesis of Sax (1938), exchange hypothesis of Revell (1958) and track structure model of Neary (1965) have continued to be a major concern in the more modern understanding of the action of radiation on living cells. In particular, the introduction of human cell culture technology in the 1960s was epoch making in bridging laboratory science and science of human radiation exposures. The expansion of radiation cytogenetics has revealed several controversies as well, which have been a matter of current debate. However, these controversies must have a unified explanation. Indeed, we are now indubitably aware that the DNA double-strand breaks are the integral molecular species of the induction of chromosome aberrations by radiation. When the diverse cytogenetic observations are reviewed in orderly interpretation in the context of their reaction kinetics and double-strand break repair pathways specific to the DNA turnover cycle, the many of the controversies may be understood in a unified model.

View full abstract

Ionizing radiation has abilities both to initiate and promote cancer development. Although initiation can be ascribed to gene mutations and chromosomal aberrations caused by radiation-induced DNA damages, it is still unclear how radiation affects tumor promotion. One of the targets is the microenvironment harboring cancer-initiating cells. Growth factors secreted from surrounding cells in the microenvironment may support cell survival and proliferation. Radiation may also target cancer stem cells that maintain tumors of a heterogeneous cell population. Animal models are useful for investigating these issues of radiation response. Key words of this symposium are radiation, mutation, microenvironment and in vivo carcinogenesis. We want to show hints to the mechanism of radiation carcinogenesis and will discuss future studies on radiation carcinogenesis. (863)

View full abstract

Xeroderma pigmentosum (XP) is a rare inherited recessive disease characterized by severe sun sensitivity that leads to the progressive degeneration of exposed areas of skin, usually causing various forms of cutaneous malignancy. Mutations in seven of eight XP complementation groups, XP-A through -G, confer defects in the nucleotide excision repair (NER) pathway, while cells from XP-V patients have a normal NER pathway but are defective in the replication of damaged DNA. Our group previously identified DNA polymerase η (Pol η) as a product of XP-V responsible gene (POLH gene). Polη catalyzes error-free translesion synthesis (TLS) past cyclobutane pyrimidine dimer (CPD), a major DNA lesion induced by UV irradiation. DNA polymerase ι (Pol ι), encoded by POLI gene, is also a TLS polymerase that can incorporate nucleotides opposite another UV-induced DNA damage, (6-4) photoproduct, in vitro. To investigate physiological role of these polymerases, we have generated Polη and Polι deficient mice and shown that incidences of skin tumors were greatly increased in the Polη-/- Polι+/+ and Polη-/- Polι-/- mice by UV irradiation, and that epithelial and mesenchymal tumors were formed in Polη and Polι deficient mice, respectively. I will present our data on the mutation frequencies and spectra in genomic DNA isolated from UV-irradiated and unirradiated epidermis of wild-type, Polη-/- Polι+/+, Polη+/+ Polι-/-, and Polη-/- Polι-/- mice by using the rpsL transgene serving as a mutational reporter sequence.

View full abstract

Deficiencies in DNA mismatch repair (MMR) result in replication errors within key tumor related genes, and cause hereditary nonpolyposis colorectal cancer (HNPCC) and hematological malignancy. In this study, we examined the possible effects of MMR on radiation tumorigenesis using Mlh1 knockout mice. Mlh1-/- mice spontaneously develop intestinal tumors and lymphomas, and therefore are a good model for HNPCC. Irradiation of 2Gy to 2- or 10-week-old Mlh1-/- mice accelerated both lymphoma and intestinal tumor development. In order to infer the role of radiation, we analyzed mutations of tumor suppressor genes, Ikaros, in lymphomas. Spontaneous lymphomas frequently lacked Ikaros protein expression, which resulted from a frameshift mutation that created a stop codon. Lymphomas induced by radiation harbored a combination of multiple frameshift and point mutations. These results suggest that radiation accelerates replication errors, which is associated with an increased cell proliferation during recovery from massive cell death after irradiation. In addition, pre-existing pre-malignant cells may have chance to expand during recovery. In conclusion, MMR deficiency cooperates with radiation in tumorigenesis via enhancing replication errors. This study was supported by a grant from LRI by JCIA.

View full abstract

Thymus is an organ well studied of cell differentiation and tumorigenesis. Thymus provides a specialized environment that supports proliferation and maturation. However, formation of the thymus and thymocyte development depend on bidirectional signals between the developing T cells and the thymic stroma. Many signaling molecules that are produced by stroma cells, including proteins in Notch signaling, control thymocyte proliferation and differentiation and also elimination of unfavorable cells generated during the processes. Accordingly, the loss of Notch1 signaling results in developmental block whereas the overexpression leads to thymic lymphomas by rendering cells independent of the supply of Notch ligands. Failure to eliminate unregulated proliferating cells is a cause of cancer. Fractionated whole-body γ-irradiation to mice induces thymic lymphomas after the persistence of thymic atrophy, which probably contains prelymphoma cells. However, it is unclear how irradiation contributes to lymphoma development and of hallmarks of the prelymphomas. We have characterized thymocytes in radiation-damaged thymus and changes in signaling. Most those thymuses comprised clonally expanding thymocytes that exhibited hindrance in the cell-cycle progression and high levels of reactive oxygen species. Also, Nrp-1 expression on the cell surface decreased, which may change thymocyte-stromal cell interactions within thymus. We propose that the hallmark of the prelymphoma cells is the coexistence of activation and inhibition of cell cycle accompanying with evasion from stromal inhibitory signals.

View full abstract

The p53 gene encodes a tumor suppressor protein, and it is generally believed that there is a dosage effect for the p53 gene. To examine this gene-dosage effect, we looked at the incidence of cancer and latent period of carcinogenesis in irradiated p53 wild type (+/+), heterozygous (+/-), and knockout mouse (-/-). Using beta rays, we repetitively irradiated the skin of p53 (+/+), (+/-), and (-/-) mice to examine carcinogenesis. Since the lifespan of the p53 (-/-) mouse was short, we were not able to observe any carcinogenesis, but for the (+/+) and (+/-) mice, results showed that (+/+) > (+/-) on the latent period of carcinogenesis, and (+/+) < (+/-) for cancer incidence. When we examined mutation and loss of heterozygosity (LOH) of the p53 gene on obtained tumors, approximately 61% of the (+/-) mouse tumors showed LOH, but there was no mutation. In the (+/+) mouse tumors, mutations occurred in about 78% and LOH was about 33%. These results indicate that there is no dosage effect for the p53 gene protein. However, it is possible that carcinogenesis occurs through different processes based on the status of the p53 gene. Variations in the carcinogenesis process may affect not only the presence and quantity of the gene products for carcinogenesis, but also the status of the genes themselves.

View full abstract

Oxygen radicals are produced through normal cellular metabolism, and the formation of such radicals is further enhanced by exposure to either ionizing radiation or various chemicals. The oxygen radicals attack DNA and its precursor nucleotides, and consequently induce various oxidized forms of bases in DNA within normally growing cells. Among such modified bases, 8-oxoguanine (8-oxoG) and 2-hydroxyadenine (2-OH-A) are highly mutagenic lesions, if not repaired. MUTYH is a DNA glycosylase that excises adenine or 2-OH-A incorporated opposite either 8-oxoG or guanine, respectively, thus considered to prevent G:C to T:A transversions in mammalian cells. The Mutyh-deficient mice showed a marked predisposition to spontaneous tumorigenesis in various tissues when examined at 18 months of age. The incidence of adenoma/carcinoma in the intestine significantly increased in Mutyh-deficient mice, as compared with wild-type mice. This high susceptibility of the intestinal tumor-development was well correlated with the condition observed in MAP (MUTYH-associated polyposis) patient. We performed mutation analysis of the tumor-associated genes amplified from the intestinal tumors developed in four mutant mice that had been treated with KBrO3. Many tumors had G:C to T:A transversions in either Apc or Ctnnb1. No mutations were found in either K-ras (exon 2) or Trp53 (exon 5 - 8). Our findings indicate that the abnormality in the Wnt signaling pathway is causatively associated with oxidative-stress-induced tumorigenesis in the small intestines of the Mutyh-deficient mice.

View full abstract

Scientific evaluation of the biological effects of low dose radiation has been long regarded as a challenging topic in radiation research. However, the application of new technologies in life science and computer science was thought to have potential to clarify the effects. Under such circumstances, a 5-year project was started as a crossover research project in 2004 by the financial support from MEXT. As the project has passed 4.5 years, we hoped to have an occasion to present our results and get discussion and critical comments from the researchers involved in the field of the low dose radiation research. In this symposium, alteration of gene expression in mice exposed to long-term low dose-rate radiation will be presented as one of the achievements of the project. We invite Dr. Ignacia B. Tanaka III to give us a lecture on the pathological aspects of mice exposed to long-term low dose-rate radiation in order to deepen our understanding of the low dose-rate radiation effects. In addition we are happy to have three distinguished scientists from abroad for lectures on the latest advances in the low dose radiation study.

View full abstract

The radiobiological effects of low doses of ionizing radiation are subject to modulations by various parameters including bystander effects, adaptive response, genomic instability and genetic susceptibility of the exposed individuals. Although the inter-relationship between bystander effects and adaptive responses have recently been discussed in the context of low dose effect, the correlation between genomic instability and non-targeted bystander cells are far from clear. Using the Columbia University microbeam, we selectively irradiated an exact fraction of cells in a population of either human fibroblasts or human-hamster hybrid (A_L) cells with a lethal dose of alpha particles. The non-hit, bystander cells were followed over a range of time periods up to 40 days to ascertain both the incidence of mutagenesis at the CD59 locus and chromosomal aberrations using M-FISH. A progressive increase in mutagenesis as well as chromosomal rearrangements characterized by deletions, duplications, insertions, para- and peri- centric inversions, and reciprocal and nonreciprocal translocations involving human chromosome 11 were detected. Furthermore, in the presence of chemical inhibitors of gap junctions and cyclooxygenase-2, the induced genomic instability was significantly suppressed. These data indicate that genomic instability can be induced in both directly irradiated as well as in bystander cells generations after the original radiation exposure, at similar levels, independently of initial cell-cell contact.

View full abstract

There is increasing evidence that cellular responses to low dose / low dose rate ionizing radiation are fundamentally different from responses occurring at high doses. Two low dose responses that appear to be specific to low dose / low dose rate exposures are bystander effects and adaptive responses. Bystander effects refer to those effects occurring in cells that were not directly hit by radiation but were either neighbors of irradiated cells or received soluble secreted signals from irradiated cells. These bystander cells manifest many of the same endpoints as irradiated cells and suggest that the target for irradiation is larger than the target volume actually irradiated. Adaptive responses are induced by low dose/low dose rate irradiation and can make irradiated cells refractory to a second high dose challenge by radiation exposure. Both bystander effects and adaptive responses can be induced by low doses of radiation, have been described both in vivo and in vitro and appear to be strongly influenced by genetic factors. However the biological, cellular and molecular mechanism(s) underlying these effects are unknown. The biological impact of bystander effects is likely detrimental to an organism. However, they can be interpreted as both being detrimental and, by a stretch, beneficial. Mutations and/or micronuclei induction in non-targeted bystander cells are likely to be detrimental because of increased genomic damage out side the irradiated field. In contrast, apoptosis might well be beneficial in that it could eliminate damaged cells that could otherwise accumulate genomic change leading to transformation, as has been suggested in anecdotal literature from clinical radiation oncology. Adaptive responses on the other hand appear to stimulate a cells ability to deal with a subsequent genomic insult and consequently could be considered a beneficial effect of low dose irradiation - provided cells are exposed to a second challenge. This presentation will consider the biological significance of these two phenomena, and their implications for low dose radiation effects.

View full abstract

The Janus Program at Argonne National Laboratory involved a large-scale mouse study of 49,000 animals exposed to a variety of doses of neutrons and gamma-rays administered at a range of dose rates. The database for these tissues is available at www.janus.northwestern.edu and tissues from the study are also available. In this work, a retrospective statistical analysis of data obtained from the Janus Program on cancerous and non-cancerous diseases of irradiated B6CF1 mice was performed in order to assess the effects of low-dose chronic radiation, with 2-40 cGy of Janus reactor fission neutrons or 100-600 cGy of gamma rays, given in 60 fractions. We specifically assessed: (1) the difference of individual tissue toxicities due to each radiation dose; (2) the effect of gender on a tissue toxicity within a given dose; (3) the effect of neutron and gamma ray irradiations at doses with presumably similar relative biological effectiveness (RBE); we did this by comparing risk for individual toxicities and toxicity incidence rate ratios for organ systems; (4) the incidence rate ratio of organ system failure due to each radiation dose; and (5) the relationship between multiple organ systems failure, radiation type, and radiation dose. The results from our per-toxicity analyses support previous findings that radiation increases the overall incidence of both neoplastic and non-neoplastic tissue toxicities, however the data also suggest that the effects of radiation are tissue-type specific. Additionally, our data also showed that gender has a role in the response to radiation. When analyzing the toxicities grouped as affecting whole organ systems, the data provided useful information supporting the theoretical model of linear no-threshold effect for neutron irradiation, and the concept of adaptive for gamma ray and neutron irradiation.

View full abstract

Late effects of low-dose and low-dose-rates of ionizing radiation are potential hazards to radiation workers and to the general public, thereby becoming a serious concern in the recent years. Using a total of 4,000 mice, we studied the late biological effects of chronic exposure to low-dose-rate radiation on life span and neoplasia. Two thousand male and 2000 female 8-week-old specific pathogen free (SPF) B6C3F1 mice were randomly divided into 4 groups, one non-irradiated (control) and three irradiated. The irradiated groups were exposed to ¹³⁷Cs gamma rays at dose-rates of 21, 1.1 and 0.05 mGy day^-1 for approximately 400 days with total doses equivalent to 8000, 400 and 20 mGy, respectively. All mice were kept under SPF conditions until natural death and pathological examination was performed to determine the cause of death. Statistical analyses showed that life spans of both sexes of mice irradiated with 21 mGy day^-1 (P < 0.0001) and of females irradiated with 1.1 mGy day^-1 (P < 0.05) were significantly shorter than those of the control group. Our results show no evidence of lengthened life span in mice chronically exposed to very low-dose-rates of gamma rays. Neoplasms accounted for >86.7% of all deaths. Compared to non-irradiated controls, incidences of lethal neoplasms were significantly increased for myeloid leukaemia and hemangiosarcomas in males, soft tissue neoplasms and malignant granulosa cell tumors in females exposed to 21 mGy day^-1. The number of multiple primary neoplasms per mouse was significantly increased in mice irradiated at 21 mGy day^-1. Our results suggest that life shortening in mice continuously exposed to low- dose-rate gamma rays is due to early death from a variety of neoplasms and not from increased incidence of specific lethal neoplasms. This study was performed under contract with the Aomori Prefectural Government, Japan.

View full abstract

Biological responses to low dose radiation are not well understood. Above all, responses to low dose-rate irradiation such as 20 to 500 mGy per year have special meaning because the dose level corresponds to the dose limit of radiation workers or the exposure level of astronauts in space. Here, we have challenged to obtain molecular clues to understand biological responses to long-term low dose-rate radiation. C57BL/6J mice were irradiated continuously for 401 or 485 days at the dose rates of 38 or 32 nGy/min, 767 or 650 nGy/min, or 15.3 or 12.5 µGy/min. The total doses were about 20 mGy, 400 mGy or 8 Gy. Immediately after irradiation, liver and kidney were examined for alteration in gene expression. Microarray analysis of liver RNA showed that the levels of some mRNAs changes at the three dose ranges used. RT-PCR analysis confirmed the changes. Proteome analysis showed an increase of one mitochondrial protein at middle and high doses. Since mRNA level of the protein remained unchanged as judged by microarray analysis, the increase would be post-translational effect. Microarray analysis of kidney RNA also showed alterations in about 50 kinds of mRNA. Among them, 4 mRNAs which are involved in oxidative phosphorylation in mitochondria were found to be elevated by irradiation. These results suggest a presence of some kinds of effects at gene expression level. However, physiological meanings of these molecular changes are yet to be proved.

View full abstract

For many years, it was thought that genetic changes caused by radiation were only due to DNA damage derived from the incident free radical species produced at the time of exposure. However, it was discovered that the number of surviving cells exhibiting increased delayed genetic effects following ionizing radiation is greater than would be predicted if only the cells exposed at the time of radiation were involved. In other words, the genetic effects of ionizing radiation were found to be trans-generational. These phenomena have been called non-targeted or bystander effects of radiation, but the mechanisms governing these phenomena are not well understood. The aim of this symposium is focused to discuss what is the mediator of this trans-genetical effects and how metabolic oxidative stress produced by steady-state energy production system contribute to this phenomena.

View full abstract

Metabolic oxidative stress and dysfunctional mitochondria have been suggested to play a role in radiation-induced genomic instability but their precise role is unclear. We used hamster fibroblasts (GM10115), genomically unstable (CS-9, LS-12) and stable (114, 118) clones derived from GM10115 following 10 Gy X-ray exposure as a model system. Genomically unstable clones demonstrated increased steady-state levels of reactive oxygen species (ROS) particularly hydrogen peroxide (H2O2) relative to the wild-type and stable clones. In contrast, the genomically stable clones demonstrated increased antioxidant capacity particularly H2O2 scavenging enzymes such as glutathione peroxidase and catalase. Modulating the levels of H2O2 by overexpression of catalase in unstable clones or inhibition of catalase in stable clones, could significantly reduce or increase genomic instability respectively. Furthermore, the unstable clones had depolarized mitochondrial membrane potential and increased mitochondrial content. They also demonstrated increased oxygen consumption, increased mitochondrial electron transport chain complex II activity and improper assembly of complex II suggesting alterations in the electron transfer processes. Intervention in the mitochondrial electron transfer processes using complex II inhibitors was able to reduce H2O2 levels as well as genomic instability in the unstable clones. This work provides clear evidence for mitochondrial oxidative phosphorylation related proteins as targets for radiation injury and augments our understanding of the radiation-induced mutator phenotype. This work suggests that H2O2 scavenging enzymes and complex II inhibitors can reduce radiation-induced genomic instability which is potentially significant for preventing normal tissue injury in patients undergoing radiotherapy.

View full abstract

Manganese superoxide dismutase (MnSOD) is a nuclear encoded primary antioxidant enzyme localized in mitochondria. Because expression of MnSOD plays a major role in maintaining cellular redox status and reactive oxygen species are known to play a role in signal transduction and carcinogenesis, we investigated the role of MnSOD in the development of cancer using a two-stage [7,12-dimethylbenz(a)-anthracene plus 12-O- tetradecanoylphorbol-13-acetate (TPA)] skin carcinogenesis model. Fe-male transgenic mice expressing the human MnSOD gene in the skin and their nontransgenic counterparts were used in this study. Pathological examination demonstrated significant reduction of papilloma formation in transgenic mice. Quantitative analysis of 4-hydroxy-2-nonenal-modified proteins showed greater accumulation of oxidative damage products in nontransgenic compared with transgenic mice, and this oxidative damage was demonstrated to be present in both mitochondria and nucleus. TPA increased activator protein-1 (AP-1) binding activity within 6 h in non-transgenic mice, but increased AP-1 binding activity was delayed in the transgenic mice. Electrophoretic mobility shift assay, transcription of the target genes, and Western analysis studies indicated that the increased AP-1 binding activity was attributable to induction of the Jun but not the Fos protein families. Overexpression of MnSOD selectively inhibited the TPA-induced activation of protein kinase C and prevented subsequent activation of c-Jun NH2-terminal kinase in response to TPA. Overall, these results indicate that MnSOD regulates both cellular redox status and selectively modulates PKC signaling, thereby delaying AP-1 activation and inhibiting tumor promotion, resulting in reduction of tumors in MnSOD transgenic mice.

View full abstract

Although radiation induced non-targeted response has been well documented in a variety of in vitro and in vivo biological systems, the mechanism is not known. Since mitochondria are the main source of energy production as well as generators of free radicals in cells, the role of mitochondrial function in mediating the non-targeted response in mammalian cells was examined. A microbeam was used to lethally irradiate either mitochondrial DNA depleted (ρo) or wild type (ρ+) human skin fibroblasts with 20 alpha particles each in a mixed, confluent culture, and the bystander response was determined in the non-irradiated fraction. ρ0 cells, when compared with ρ+ cells, showed a higher bystander HPRT- mutagenic response in confluent monolayer when 10% of the same population was lethally irradiated. However, using mixed cultures of ρ0 and ρ+ cells and targeting only one population of cells with a lethal dose of alpha particles, a decreased bystander mutagenesis was uniformly found with both cell types indicating that mitochondrial deficient cells cannot effectively communicate the bystander signals to wild type cells; or alternatively, signals from one cell type can modulate expression of the bystander response in another cell type. These data suggest that mitochondrial signaling pathways are involved in mediating the non-targeted response and a better understanding of the process will allow us to formulate a more accurate model in assessing the health effects of low doses of ionizing radiation.

View full abstract

Reactive oxygen species, produced in energy production, or by exposed stresses such as radiation, is thought to be involved in the critical cause of life-style related disease, aging and cancer. We have reported that irradiated cells are accumulated genetic instability such as unstable telomere, or hypoxically-cultured cells are prevented genetic instability, and we predict that oxidative stresses are closely involved in genetic instability. On the other hand, there is clear difference in transformation sensitivity between human and rodent cells. Therefore this study was carried out to the intervention of mitochondrial function in cell transformation. We cultured human, mouse, and hamster cells obtained from carcass of embryos under 0.5, 2, and 20% oxygen. As a result, mitochondria number or membrane potential was not change in human cells, and they never immortalized or transformed under all the oxygen conditions. Meanwhile, mouse cells spontaneously overcame replicative senescence easily and transformed. Moreover, intracellular oxidative levels of hypoxic-cultured mouse cells were significantly higher, and the tendency to immortalization was increased. This suggests that the response to the oxygen stress via mitochondria involve in cell immortalization and transformation. Additionally, there is a critical difference in responsibility to oxygen stress, between human and rodent cells, and increase of intracellular oxidative stress result in cell immortalization and malignant transformation. We will present the relation of cell transformation and mitochondrial function.

View full abstract

Delayed effects of radiation, such as bystander efffects or genomic instability, are very interesting topics in the field of radiation biology. Although several researchers have found that levels of ROS around mitochondria in unstable cells were higher than those of normal ones, it is rather hard to explain how they reach to gene damage in nuclei. Recently we newly found novel slow-releasing long-lived radicals (SRLLRs) in 4 Gy γ-irradiated Syrian hamster embryo (SHE) cells by ESR spectroscopy without any spin-trapping agents. Levels of SRLLRs increased 6, 21, 20 and 18% with an increase at intervals of 1, 5, 12 and 20 h after irradiation, respectively. Post-treatment of ascorbate for 2 h suppressed the increase in the levels of SRLLRs. Addition of myxothiazol, as an inhibitor of electron transportation in mitochondria, suppressed the increase in the levels of SRLLRs in irradiated cells, and direct addition of hydrogen peroxide to the culture medium increased the levels of them. These results indicate that SRLLRs are likely to be produced by hydrogen peroxide from dysfunctional mitochondria. Because SRLLRs have a long lifetime over 20 h and reduced by L-ascorbic acid treatment, they might be responsible of delayed effects of radiation. Very recently we have succeded to detect the increase of SRLLRs (+18%) in normal CHO cells by the transfer of irradiated cultured medium co-irradiated with CHO cells (1 Gy) in culture flusks. This is the first obsevation of medium mediated bystander effects detected by the ESR measurements of SRLLRs. Because the cells exposed to the irradiated meidum showed two times larger mutation frequency, the SRLLRs produced by the bystander effect are related to the mutation induction. Although we have little idea for the bystander factors, future works should be done to investigate the mechanisms of induction of SRLLRs by the bystander effects.

View full abstract

Low levels of endogenous reactive oxygen species (ROS) originating from NADPH oxidase have been implicated in various signaling pathways induced by growth factors and mediated by cytokines. However, the main source of ROS is known to be the mitochondria, and increased levels of ROS from the mitochondria have been observed in many cancer cells. Thus far, the mechanism of ROS production in cancer cell proliferation in the mitochondria is not well-understood. We recently identified a novel protein, ROS modulator 1 (Romo1), and reported that increased expression of Romo1 triggered ROS-production in the mitochondria. The experiments conducted in the present study showed that Romo1-derived ROS were indispensable for the proliferation of both normal and cancer cells. We also show that Romo1 expression directly contribute to invasive activity of cancer cells and a higher level of Romo1 expression was detected in 39 out of 79 tumor tissuess, compared with the corresponding normal tissues. Interestingly, Romo1 expression showed a strong negative correlation for 12-year survival in tumor patients. The results of this study suggest that Romo1-induced ROS may play an important role in redox signaling in cancer cells.

View full abstract

Radiobiological research with microbeam started at the Gray Cancer Institute and Columbia University. Dr. Hatsumi Nagasawa proposed a phrase "Bystander Effects", and the research thereafter spread out all over the world. We have five working facilities in Japan and abound in variety of beams such as Heavy-ions, Lighter-ions and X-rays. Number of microbeam facilities in the world expands and the scientific interests also spread out to cellular effects by nuclear direct hit, cytoplasm irradiation, effects on non-irradiated cells, survey of bystander factors, gene expression, and so on. We plan this symposium to introduce history and trends in microbeam research in the world as a satellite symposium of the 8th International Workshop on Microbeam Probes of Cellular Radiation Response that will be held in 13-15 November at NIRS cosponsored by the Microbeam Biology Research Union.

View full abstract

From the early 1990s there have been radical changes in our understanding of radiobiological effects and in methods of investigating them. Following earlier indications, it has now become clear that there is a range of non-(DNA)-targeted effects. They play a significant role in radiation responsiveness in relation to that of the classical, DNA-targeted effects. Recognition of these effects has coincided with and stimulated the development of a number of low-dose and targeted microirradiation techniques that have enabled these more newly recognized effects to be investigated in considerable detail. The key phenomena are bystander effects, genomic instability and adaptive responses and they are clearly interrelated and share some common pathways. Cell-to-cell communication is a dominant feature of these responses and the ability of new methods to target specifically, either with microbeams or with targeted radioisotopes, is helping to provide new insights into the mechanisms. Individual cells and subcellular targets can be selected within populations and the responses can often be registered by time-lapse imaging. Much of the emphasis has been to apply these methods to simple in vitro systems and the present challenge is to explore the role of non-targeted effects in 3-D and model tissue systems. The microbeam approach is of particular value in studies related to radiation risk. This is because it is possible to determine the actions of single particle tracks and thereby mimic in vitro the exposure conditions that generally apply at protection levels.

View full abstract

The development of microbeam facilities in many countries over the past fifteen years has provided a unique tool to radiobiologists in addressing many fundamental issues of interest including inter- and intra-cell signaling, DNA damage and repair kinetics, as well as non-targeted responses at both the cellular, tissue and organism levels. The long accepted paradigm that nucleus is the quintessential target for radiation damages have now been overturned. Using a microbeam, there is evidence that targeted cytoplasmic irradiation results in mutations in the nuclei of hit cells. These initial extranuclear observations provides the impetus for numerous non-targeted responses made using microbeams involving multiple endpoints in a variety of cell systems including three dimensional tissues. Studies on microbeam-induced DNA damage and repair foci clearly illustrate the kinetics as well as the complexity of the repair process. Non-targeted responses in whole organisms as well as the induction of genomic instability in the progeny of non-hit human epithelial cells have reinforcing the relevance of the new radiobiology to human health.

View full abstract

Soon after the construction of microbeam irradiation facilities in UK and USA, a project of microbeam irradiation using heavy ions started in JAEA, Takasaki in Japan. Since then, several proposals for constructing microbeam irradiation systems have been proposed, and presently four systems are working and two, under development or being planned. One of the characteristics in microbeam research status in Japan is that various types of radiation are available, including soft X-rays, X-rays, light ions and heavy ions. This indicates a potentiality of explosive development in microbeam radiobiology in Japan. Present status and characteristics of the microbeam irradiation facilities and several highlights of recent researches obtained in these facilities will be presented.

View full abstract

Accumulating evidences indicate that the biological effects of low dose radiation are different from those of high dose radiation. To evaluate the biological effects of low dose radiation certainly, experimental or epidemiological results in low dose region are really important. However, there are many issues, such as the population, the dispersion in the effect among individuals, the difference in experimental procedure, dosimetry, and so on. In addition, non-targeted effects such as bystander response have been reported without dose dependence and their biological mechanism has not yet fully elucidated. On the other hand, the study using animal model has found that the animals show radiation resistance in consequence of the defense response activated by low dose radiation. At this symposium, we would like to introduce recent studies of the biological effects of low dose radiation from various viewpoints and to discuss dose response, dose region to be evaluated and their biological meanings.

View full abstract

The risks of exposure to low dose ionizing radiation are estimated by extrapolating from data obtained after exposure to high dose radiation, using a linear no-threshold model. However, the validity of using this model is controversial because evidence accumulated over the past two decade has indicated that living organisms, including humans, respond differently to low dose/low dose-rate radiation than they do to high dose/high dose-rate radiation. Briefly, there are accumulated findings which cannot be explained by the classical "target theory" of radiation biology. The radioadaptive response, radiation-induced bystander responses, low-dose radio-hypersensitivity, and genomic instability are specifically observed in response to low dose/low dose-rate radiation. However, the mechanisms of these phenomena are not fully known. To elucidate the mechanisms of radiation-induced bystander responses, we analyzed the formation of γH2A.X and pNBS1 foci after irradiation of a targeted cell with 460 MeV argon microbeams. We found that the foci of γH2A.X and pNBS1 were formed in the unirradiated cells in the colony including the targeted cell 6 h after the irradiation and that this formation of these foci was almost completely suppressed by the addition of DMSO or Lindane. Also we found that the foci of γH2A.X and pNBS1 were formed in the unirradiated cells in the colonies not including the targeted cell 6 h after irradiation and that this formation of these foci was almost completely suppressed by the addition of aminoguanidine or c-PTIO. Our findings strongly suggested that ROS and NO may be actually initiators/mediators for evoking charged particles-induced bystander responses.

View full abstract

Phosphorylated ataxia telangiectasia mutated forms foci (p-ATM foci) shows the initial DNA damage, such as DNA double strand breaks, by radiation. In this study, we analyzed the dose-response relationship for the number of p-ATM foci in MRC-5 cells that irradiated of X-rays with doses ranging for ~200 mGy and indicated that the number of p-ATM foci showed a supralinear dose-response relationship. However, this dose-response relationship was not observed in cells treated with lindane that is an inhibitor of bystander effects. Moreover, the number of p-ATM foci obtained by subtracting the number of p-ATM foci in lindane-treated cells from the number of p-ATM foci in untreated cells was proportional to the dose at low doses ~10 mGy. Thus, the increase in the number of p-ATM foci in the range of ~10 mGy was largely due to bystander effects. It is well known that the radioadaptive response is important biological effect of low dose radiation. We investigated whether the bystander effect related to the induction of the radioadaptive response. Conditioning irradiation of the MRC-5 cells with ~10 mGy resulted in a significant protective effect against the occurrence of p-ATM foci induced by a challenging dose of 200 mGy. However, this phenomenon was inhibited by lindane. This result indicated that the radioadaptive response was induced in dose region where bystander effect has occurred. The findings of our present study provide a direct evidence of relationship between the bystander effect and the radioadaptive response.

View full abstract

To determine whether the linear non-threshold model for stochastic effects of ionizing radiation is applicable to very low dose radiation at a low dose rate, we irradiated immature male germ cells of fruit fly Drosophila melanogaster, with several doses of gamma rays from 60Co, at a dose rate of 22.4 mGy/h. Thereafter, we performed the sex-linked recessive lethal mutation assay by mating the irradiated males with non-irradiated females. The mutation frequency in the group subjected to 500 µGy irradiation was found to be significantly lower than that in the control group (p<0.01), whereas in the group subjected to 10 Gy, it was significantly higher than the control (p<0.03). Several genetic studies suggest that most spontaneous mutations are related to mobile elements, while high dose rate irradiation is known to cause deletions due to restoration of double strand break. Our observation showed that a part of the mutations that should have happened at a spontaneous mutation were not generated apparently. Molecular experiments using microarray indicated that Hsp70, gene for a heat shock protein, was up-regulated after 500 µGy irradiation together with grim, a positive regulator of apoptosis and diver2, a kind of retrotransposon. These results suggested that a stimulation of low dose rate gamma rays might activate frequency of the transposon integrations. However, it is possible that low-dose-rate gamma rays activate cellular apoptosis such that the damaged cells are more efficiently killed compared to non-irradiated cells.

View full abstract

The frequency of chromosome aberration (CA) is of the most sensitive biomarkers of ionizing radiation (IR). Dicentric (Dic) and ring (unstable type) are good markers of the effect of IR alone, while translocation (stable type) is considered to represent the total effects of environmental mutagens and also suggested to correlate with cancer incidences epidemiologically. In the research of high background radiation area (HBRA) in China, the Dic + ring could detect the increase of CA frequency by very low dose-rate chronic irradiation less than 3 times of background level. On the other hand, there was no difference between HBRA and control area in the translocation. It was explained that the amount of CAs caused by whole environmental mutagens were much larger than by ionizing radiation and the variation of environmental effects could mask the effects of IR, which seemed to correspond with the epidemiological results that there were no significant cancer effects of high background radiation. To evaluate this possibility we tried to get precise conversion factor between CAs and radiation effects. We measured Dic + ring using human peripheral lymphocytes irradiated with 10, 20, and 40 mGy, and achieved linear dose response curve in this very low dose area. We will discuss the significance of the effects of IR among the environmental mutagens using this conversion factor.

View full abstract

Low dose radiation effects on human being are not clearly results even now. Because it is different situation from animal experiment, so we can't observe low dose radiation effects on human beings except radiation accident. The Radiation Effects Research Foundation (RERF) has been surveyed to atomic bomb survivors in Hiroshima and Nagasaki over long term. They have the cohort of A-bomb survivors so called Life Span Study (LSS). They have been reported many results about dose dependency of cancer mortality from 1950 to 1997. On the other hand, according to the ICRP recommendation, low dose radiation effects extrapolate to linear model for dose response curve. It means existence of risk at low dose radiation. Stewart et al. have been clamed it and they reported U-shape curve for non-cancerous mortality by analysis of RERF data. Also, we analyzed atomic bomb survivors in Nagasaki, and found mortality of low dose group in male less than general one. I would like to discuss about limitation of epidemiology on the low dose radiation effects among human being.

View full abstract

Simultaneous inclusion of dose and dose-rate is required to evaluate the risk of long term irradiation at low dose-rates, since biological responses to radiation are complex processes that depend both on irradiation time and total dose. Consequently, to estimate quantitative dose-response relationship on the biological response to radiation, it is necessary to consider a model including cumulative dose, dose-rate and irradiation time. In this study, we measured micronucleus formation and [3H] thimidine uptake in human cells as indices of biological response to gamma radiation, and analyzed mathematically and statistically the data for quantitative evaluation of radiation risk at low-dose and low dose-rate. Our results suggest that the biological response declined sharply when dose-rate was less than 0.01 Gy/h. Furthermore, for defining statistically no risk level, we adapted some statistical models to data at low dose, and compared them with other statistical indexes corresponding to "threshold limit". The values of statistically no risk levels and their confidence intervals depend on definitions of the value and on statistical models. These results suggest that we should statistically define the term "threshold" and include dose rate effects in the risk assessment model. Moreover, we acquired iterative experimental data of biological reaction to low dose gamma radiation and analyzed them using meta-analysis to evaluate quantitatively the radiation effects at very low dose. Synthesized effect sizes of the iterative experiments showed no significant difference between irradiated mice and controls. Our statistical models support biological observations with sufficient statistical power, and numerical expression of our data showed clear dose-rate effect on biological response.

View full abstract

Sixty-three years have passed since the atomic bombing, and increased cancer risk is continuously observed in A-bomb survivors. Moreover, recent studies have enlightened the existence of new scientific evidences that are increased risks of preleukemic myelodysplastic syndromes and increased incidence of multiple primary cancers among proximally exposed survivors. Nagasaki Global COE symposium will focus on the scientific evidences obtained from previous researches conducted in Hiroshima and Nagasaki, overview the current new findings, and discuss future directions of the A-bomb disease medical research. Current status of the research projects running in the Nagasaki GCOE program will also be introduced.

View full abstract

Excess risk of chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) reached the peak in 1950ies and seemed disappeared. However, preliminary analysis by RERF suggests a persistence of AML risk and epidemiological study of Nagasaki University 21Century COE program indicated that along with aging of the atomic bomb survivors, myelodysplastic syndromes (MDS) are developing recent two decades, with a relative risk of 4 between proximally (less than 1.5 km) exposed group and distal (3.0 km or more) group. Approximately 25% of MDS cases eventually developed into AML. Dose response study is now being conducted for MDS cases within RERF-LSS cohort. If proved positive, MDS/AML is persistently occurring when the young survivors at the time of bombing became elderly, suggesting a life-long effect of atomic bomb radiation on human body. CML is characterized by a simple chromosomal translocation t(9;22) that includes BCR/ABL fusion gene. On the other hand, MDS and AML from MDS are characterized by complicated chromosome abnormalities. From the recent advance in hematology, it is clearly elucidated that CML and MDS, and most AML also, occur in a multi-potent hematopoietic stem cell. The research on the atomic bomb-induced leukemia suggests that there are two major processes in the malignant transformation of hematopoietic stem cells; CML type with short latency and MDS type with extremely long latency. Among proximally exposed healthy survivors, it is still possible to observe chromosome abnormalities in blood cells; some of them are clonal. Whether these abnormalities are mere consequences of radiation injury to chromosomes in 1945 or representing chromosomal instability reflecting leukemogenic process in stem cells is an important question of scientific importance.

View full abstract

1) Susceptibility of ionizing radiation from atomic bomb (A-bomb): Differences on sensitivity to A-bomb radiation are suggested among the survivors. We demonstrated that a rat strain possessing sympathetic hyperfunction showed a lower survival rate, a higher weight loss, and a severer intestinal injury than control animals after a whole body irradiation. Disturbance of the autonomic nervous system, particularly sympathetic hyperfunction might be associated with increased sensitivity to acute radiation injury in human. 2) Multiple primary cancers (MPC) in A-bomb survivors: An increased risk of cancer has continued for decades. We have recently described a higher incidence rate of MPC in the survivors, particularly for those who were exposed at a younger age and at a closer distance from the hypocenter. The occurrence of MPC is considered to be a reflection of systematic exposure to environmental carcinogens or of a predisposition to cancer. These results provide evidence for the involvement of A-bomb radiation in MPC among the survivors. A higher risk of cancer, as a late effect of A-bomb radiation, still persists in survivors. 3) Biological resources from A-bomb survivors: The epidemiologic and molecular analyses regarding carcinogenesis in the survivors require clinical data of individuals and biological materials with pathologic data of tumors. Biological materials for solid tumors from Nagasaki survivors have been mainly kept as formalin-fixed paraffin-embedded tissues at the hospitals in the Nagasaki city. Recently, because of the technical advance, several important molecular data about radiation-related tumorigenesis with archival materials from the survivors have increasingly reported. We are definitely required to collect and reserve these biological resources from the survivors for future studies.

View full abstract

Research on the health effects of atomic bomb radiation has been conducted at RERF (or its predecessor, ABCC) since 1947. Early studies focused on acute health effects and the potential for heritable genetic effects, but in the 1950s the major prospective cohorts, that have been followed-up to the present, were formed: the Life Span Study (LSS) of mortality and cancer incidence among 120,000 A-bomb survivors and the Adult Health Study (AHS) biennial clinical examination program for a sub-cohort of about 20,000 survivors. Subsequently, cohorts were created of about 3,600 survivors who were in utero at the time of the bombing and 77,000 children of A-bomb survivors (F1 study), including 12,000 F1 individuals receiving clinical examinations. An important feature is that dose reconstructions permit reasonably precise individual A-bomb dose estimates to be assigned to most cohort members. A major finding has been that rates of most major types of cancer are associated with radiation dose, and that the association extends to the low dose range. More recently, we have found associations of radiation dose with cardiovascular and cerebrovascular disease mortality rates as well, but another 20+ years is required to fully characterize these risks. These diseases and other non-cancer effects (e.g., cataract, chronic inflammation), are being more thoroughly documented through clinical examinations and laboratory studies in the AHS. The F1 studies have been negative to date, but that cohort is only about 50 years old on average, so another 30+ years will be required to fully document their morbidity and mortality risk. The longitudinal biological samples that have been collected in the AHS are, and will continue to be, an unparalleled resource for learning about the mechanisms of radiation-induced human disease.

View full abstract

UV radiation shows biological effects not only through direct photochemical reactions with biomolecules but also in indirect ways mediated by photosensitizing reactions, one of which induces oxidative stress through production of reactive oxygen species. It has been demonstrated that UV produces oxidative damage in DNA and causes structural abnormalities of constituent proteins through the oxidative stress in the tissues like lens and skin, which are exposed to the environmental UV. This workshop aims to evaluate the significance of the biological effects by the oxidative stress UV produces, in comparison with the direct effects by its photochemical reactions. We are focusing on the main components of the genome and the biological structure, DNA and protein, and discussing the influences for them of the oxidative stress as well as the UV photochemistry in terms of not only cells but also tissues such as skin, intestine, and lens.

View full abstract

UVA induces the formation of 8-hydroxy-2'-deoxyguanosines (8-OH-dG) and cyclobutane pyrimidine dimers (CPD) in the cellular genome. However, the relative contribution of each type of damage to the in vivo genotoxicity of UVA has not been clarified. We irradiated living mouse skin with black light lamps (FL20S.BLB, Toshiba, Japan), which emit UVA2 mainly, and with a 364-nm UVA1 laser irradiator (National Institute for Basic Biology, Okazaki, Japan: This study was carried out under the NIBB Cooperative Research Program for the Okazaki Large Spectrograph; 4-507, 5-507, 6-511, 7-509 and 8-501.), and analyzed the DNA damage formation and/or mutation induction in the epidermis and dermis. Whereas dose-dependent increases were observed for both 8-OH-dG and CPD after UVA1 laser irradiation, the mutation induction in the skin was found to result specifically from the CPD formation, based on the induced mutation spectra in the skin genome: the dominance of C → T transition at a dipyrimidine site. Thus, it is the CPD formation, not the oxidative stress, that effectively brings about the genotoxicity in normal skin after UVA exposure. No induction of oxidative stress-specific mutations despite of the UVA-dose dependent 8-OH-dG formation suggests that detoxication processes against oxidative DNA damage, such as DNA repair and cellular apoptosis, are highly efficient in the normal mouse skin. Moreover, the efficient induction of CPD formation by UVA, even by the UVA1 laser light, has challenged the authorized model for UV-induced CPD formation.

View full abstract

Reactive oxygen species, produced by cellular metabolisms and by exposure to ionizing radiation or ultraviolet-light, attack DNA and its precursor nucleotides. Consequently, various modified bases are introduced into the DNA of normally growing cells. One such modified base, 8-oxo-7, 8-dihydroguanine (8-oxoG) is highly mutagenic; 8-oxoG can pair with adenine as well as cytosine, causing G:C to T:A transversion during DNA replication. 8-Oxo-dGTP can be incorporated opposite adenine or cytosine in the template strand, thus inducing both A:T to C:G and G:C to T:A transversions. Mammals have three enzymes to prevent the 8-oxoG-related mutagenesis. OGG1 removes 8-oxoG paired with cytosine, and MUTYH excises adenine incorporated opposite 8-oxoG. MTH1 hydrolyses 8-oxo-dGTP, thus avoiding the incorporation of 8-oxo-dGTP into DNA. We generated mutant mice lacking each of these three enzymes by gene targeting, and analyzed mutagenesis and tumorigenesis in these animals. Our data indicate the significance of these enzymes in the prevention of mutagenesis and tumorigenesis. Recently, we developed a new system using the cells derived from the rpsL transgenic mouse, and analyzed the mutagenesis caused by 8-oxo-dGTP. The introduction of 8-oxo-dGTP into the cells drastically induced A:T to C:G transversions but not G:C to T:A transversions. These results suggest that the mutations caused by the incorporated 8-oxoG opposite cytosine were efficiently suppressed by DNA repair systems, while the incorporated 8-oxoG opposite adenine were poorly processed in mammalian cells.

View full abstract

Chronic UV exposure causes photoaging including skin cancer, solar lentigo and wrinkle. UV-induced oxidative stress is closely related to photoaging. Thus far, UVB-induced DNA damage has been paid much attention in view of photocarcinogenesis. Direct absorption of UVC or UVB leads to formation of cyclobutane-type pyrimidine dimers and pyrimidine pyrimidone (6-4) photoproducts at adjacent pyrimidines. Previous studies suggested that pyrimidine photoproducts are the major classes of UV-induced DNA lesions involved in the cytotoxicity and mutagenesis. Pyrimidine photoproducts induce preferentially transition-type base changes especially at dipyrimidine sites, sometimes called as UV-signature mutations. However, recent reports indicate the involvement of other molecules. UVB induces not only pyrimidine photoproducts but also oxidative DNA damage. Several reports pointed out that types of mutations that are not theoretically caused by pyrimidine photoproducts are frequently observed in the human skin cancers of sun-exposed areas and UVB-induced murine skin cancers. Recent studies have shown that not only UVB but also UVA is involved in photocarcinogenesis based on animal experiments. UVA can induce indirect DNA damage via ROS and lipid peroxidation. ROS have been associated not only with initiation, but promotion and progression in the multistage carcinogenesis model. We have demonstrated that Ogg1 knockout mice, which cannot repair 8-oxoG, representative oxidative DNA damage, is susceptible to photocarcinogenesis. Photoaging is induced via response of epidermal cells or dermal fibroblasts to both UVB and UVA and its signal transduction in the epidermal cells and dermal cells develop photoaging. In order to reduce photoaging we should clarify the mechanisms how UVA and UVB cause cellular response.

View full abstract

Although protein consist exclusively of L-amino acids in living tissues, biologically uncommon D-aspartyl (D-Asp) residues have been detected in various proteins of metabolically inert tissues where the oxidative stress and the effect of UV irradiation are present such as eye lenses, the eye macular, skin, ligament, aorta, brain and lung. We found that Asp58 and Asp151 residues highly were inverted to D-isomers in elderly donors αA-crystallin from lenses. D-Asp occurs via a succinimide intermediate by attack of the nitrogen of the amino acid residue following the Asp residue. When the neighboring amino acid of the Asp residue has a small side chain, the formation of succinimide occurs easily because there is no steric hindrance. Therefore, L-Asp easily converts to D-Asp when the next amino acids are glycine, alanine or serine. Actually, the next amino acids of the two different sites of D-Asp in αA-crystallin were alanine and serine residues, respectively. Recently, we also found D-Asp in protein of the elastic fibers of skin from elderly donors and that the formation of D-Asp in protein is accelerated by sunlight exposure. The appearance of D-Asp in protein would induce a big change of the higher order structure of the protein because the configuration of the Asp residue would be opposite against peptide plane. In addition to D-Asp formation, beta-linkage of Asp formation affects a quaternary structure of protein because the main chain of the protein would be elongated. In this presentation, we demonstrate that this uncommon configuration of Asp by oxidative stress is considered to be one of the trigger of the abnormal aggregation of protein and it induces the related disease, such as cataract, age-related maculopathy, arteriosclerosis, solar elastosis, and Alzheimer's disease.

View full abstract

It has been generally believed that biological significance of DNA damage checkpoint is time-sparing for DNA damage repair. However, some lines of evidence, which is obtained from radiation biology, casts doubt on the well-known paradigm. The first evidence is: there is no significant difference in the repair kinetics of DNA double-strand breaks within 2 h after X-irradiation between normal human cells and ataxia-telangiectasia (AT) cells, which cannot induce DNA damage checkpoint, and approximately 90% of initial DNA double-strand breaks are rejoined in AT cells in the end (Kühne, et al., Cancer Res. 64, 500-508, 2004). The second evidence is: seventy to eighty percent of DNA double-strand breaks produced in G0/G1 phase is rejoined within 2 h after X-irradiation, while G1 checkpoint is induced much more slowly, and its induction requires at least 6-8 h after irradiation (Riballo, et al., Mol. Cell 16, 715-724, 2004; Kastan, et al., Cancer Res. 51, 6304-6311, 1991; Di Leonardo, et al., Genes Dev. 8, 2540-2551, 1994). In this workshop, it will be discussed whether the "time-sparing theory" of DNA damage checkpoint is right or not by distinguished speakers.

View full abstract

In general, DNA damage checkpoint is believed to be induced in all irradiated cells with DNA double-strand breaks (DSBs), and be released when DSBs are rejoined. However, we recently obtained data that: fraction of cells which undergo G1 arrest is dose-dependent, and approx. 40% of cells do not undergo G1 arrest in 1 Gy-irradiated G1 cells. This result suggests that DSB itself, which is generated immediately after irradiation, does not trigger G1 checkpoint. Therefore, we hypothesized that the bona fide role of G1 checkpoint is not providing time for DNA repair, but termination of proliferation of cells with some structural aberration of chromatin derived from DSBs. In the present study, we tested the hypothesis. We previously demonstrated that Ser1981-phosphorylated ATM foci, which form immediately after irradiation, decrease in number time-dependently, however, persistent foci grow, and the foci growth is essential for signal amplification and elicitation of G1 checkpoint. In the present study, we examined the difference of frequency and spectrum of chromosome aberration when foci growth and G1 arrest are inhibited or not. We found significant difference in the frequency of dicentric with fragment and chromosome fragment. This result indicates that it is misrejoined-, or unrejoined DSBs that trigger foci growth of phosphorylated ATM and G1 arrest, and termination of proliferation of cells with such structural aberration of chromatin is a bona fide role of G1 checkpoint.

View full abstract

DNA damage checkpoint prevents the proliferation of cells with irreparable DNA damage by inducing senescence or apoptosis. As shown in the evidence that a wide variety of human cancers has p53 mutations, it has been demonstrated that p53 protein suppresses tumor formation by inducing senescence and apoptosis both in vitro and in vivo. Intriguingly, however, the fraction of cancer cells possessing p53 mutation varies among the types of tumor; the percentage of cells with p53 mutation is low in leukemias and soft tissue sarcomas commonly seen in childhood, whereas the percentage is high in epithelial carcinomas commonly seen in adulthood. I propose to view that the former is child-type tumors, which show short latent periods, a clonal expansion of cells with specific chromosome translocations, and no telomere crisis, and that the latter is adult-type tumors, which show long latent periods, random chromosome aberrations, and telomere crisis due to the extensive telomere erosions. In general, the function of the DNA damage checkpoint is not critical for the tumor suppression in child-type tumors because the formation of chromosome translocations, a crucial step in carcinogenesis, cannot be sensed by the checkpoint. In contrast, the function of the DNA damage checkpoint is critical for the tumor suppression in adult-type tumors because the extensively eroded telomeres can be sensed by the checkpoint, resulting in inducing senescence, which acts as an effective anti-tumor barrier. This may be the reason why the percentage of cells with p53 mutation is higher in adult-type tumors than in child-type tumors.

View full abstract

Earlier studies on the biological effects of ionizing radiation at the cellular level revealed that the cell cycle progression was influenced by irradiation, which provided the bases for the concept of "cell cycle checkpoints". On the other hand, the reduction of cell killing was observed when cells were kept under non-growth conditions. The phenomena were named "Liquid Holding Recovery" or "Potentially Lethal Damage Recovery". Studies with variant cell lines also demonstrated that transient cell cycle arrest would rescue the irradiated cells. Taken together with the results on the relationship between the radiation-induced DNA damage and its repair and cell death and its modification, it is suggested that the radiation induced transient cell cycle arrest would afford the cells opportunity to repair certain types of DNA damage, which would cause cell death, and contribute to the cellular recovery.

View full abstract

When cells are exposed to agents that interfere with DNA replication, they either activate a cell cycle checkpoint - related phosphorylation cascade that inhibits further replication to prevent DNA damage, or decrease the rate of DNA synthesis without activating the checkpoint response. Here we report that exposure to low doses of the DNA polymerase inhibitor aphidicolin, which were below the activation threshold for cell cycle checkpoints, induced a transient surge of DNA breaks that required the Bloom (BLM) helicase and the Mus81 nuclease. Cells with functional BLM and Mus81 continued to replicate DNA at a slow pace in the presence of the drug after resolution of the transient DNA breaks, but cells that lacked BLM or Mus81 did not exhibit transient breaks and completely halted replication after exposure to low levels of APH. In such cells, prolonged exposure to APH led to the accumulation of persistent DNA breaks and loss of viability. These observations suggest that the BLM helicase and the Mus81 nuclease cooperate to convert perturbed replication forks to double strand DNA breaks, which can be repaired by NHEJ and allow DNA replication to resume at a slower pace.

View full abstract

Radioresponse at the preimplantation stage was analyzed for mouse embryos fertilized by 6 Gy irradiated sperm. The p53 dependent transactivation of a p53 dependent lacZ reporter microinjected into in female pronucleus was observed in these embryos. DNA synthesis of sperm irradiated zygotes was suppressed p53 dependently in both male and female pronuclei. Even with these p53 activation, the sperm irradiated zygotes did not arrest at G2/M and progressed normally to 2 cell stage. They further cleaved until day 3.5 when they showed a sign of cell cycle arrest. RT-PCR analyses revealed that p21 was not activated in sperm irradiated embryos of day 1.5 to day 3.5. Direct irradiation of embryos also failed to activate p21 until they progressed to day 3.5. Phosphorylation of histone H2AX were inefficient in zygotes and two cell stage embryos. In contrast, prompt phosphorylation of H2AX was found to take place when the embryos were over day 3.5. Apoptosis was only detected in the cells of inner cell mass of the blastcyst stage embryos. Altogether, our unusual observations demonstrate that the damage response of early embryogenesis does not follow the general pattern conserved throughout eukaryotes. The exceptional damage response is likely to have a protective role in embryogenesis. Indeed, it has long been shown in mice that irradiation of preimplantation stages results in higher rate of embryonal death leading while live born pups are devoid of deleterious outcome such as malformation.

View full abstract

Excellent biological effectiveness and dose conformity represent a rationale for heavy-ion therapy. Heavy-ion therapy which is currently available at three facilities (e.g., Natl. Inst. Radiol. Sci) has so far achieved good cancer controllability while sparing critical normal organs. Several other facilities (e.g., Gunma, Univ.) are now under construction or in the planning phase, so that heavy-ion therapy is becoming more widely available. Nevertheless, the biological mechanism of heavy-ion action is incompletely understood. This workshop aims to summarize the current knowledge and discuss future perspectives. The topic includes repair of heavy ion-induced complex DNA lesions, overcoming tumor radioresistance with heavy ions, potential biological approaches to sensitize tumor cells to heavy ions, and differences in biological actions of photons and heavy ions.

View full abstract

From June 1994 to August 2008, a total of 4,126 patients were enrolled into clinical studies using carbon ion beams generated by HIMAC. Carbon ion radiotherapy of these patients was carried out by more than 40 different phase I/II or phase II protocols and highly advanced medical technology. We treated 642 new patients in 2007. Lung, head and neck, prostate, liver and bone and soft tissue tumors are the leading 5 tumor types in the studies. Local tumor control rate and survival in five major tumor sites, head and neck, lung, liver, prostate, and bone and soft tissue tumors are summarized.

View full abstract

Developments in radiation technology have lead to the clinical improvement of Carbon ion (C-ion) irradiation with well-localized energy distributions (high LET). While the development of high LET has a number of advantages over the more traditional radiation technology for the treatment of cancer, the mechanisms underlying the superior biological effectiveness of C-ion irradiation is not fully understood. Several studies have investigated the biological mechanism of C-ion irradiation in vitro. The main disadvantage of these in vitro studies includes significant differences in the gene expression profile between cell lines in vitro and the same cell lines growing in vivo and the effect of other factors related to the cellular microenvironment on the gene expression profile. Previous studies suggest that more appropriate in vivo tumor models allowing translation of findings to the clinical setting are required. We used microarray technology to examine the fate of multiple C-ion-irradiated tumors in vivo to gain a comprehensive overview of the changes in gene expression induced by C-ion irradiation. Four murine tumors were irradiated in vivo with C-ions at a single dose, and γ-rays were used as a reference beam. A hypothetical C-ion-induced pathway, which is construced through bioinfo-tools, would be presented with the expression changes of a well known radiation-induced genes. We also investigated antimetastatic effects of local C-ion irradiation in highly metastatic murine model with comparative expression analysis between local primary tumors and metastatic lung nodules. Finally, the expression changes of candidate C-ion responsive genes were finally evaluated using clinical situation, in which sequential biopsies were taken from cervical cancer patients, who were treated with C-ion therapy.

View full abstract

The increased biological effectiveness associated with high LET radiation when compared to low LET X-rays is thought to be one of the important causes for the successful clinical outcome of heavy-ion radiotherapy. DNA double strand breaks (DSBs) are considered to be the most crucial lesion induced by ionizing radiation, and the repair of DSBs is much slower in cells irradiated with high LET heavy ions than that with X-rays. This was reflected at the level of chromosomes; for example, the remaining number of chromosome breaks after a certain post-irradiation period as measured by premature chromosome condensation (PCC) is significantly higher in cells irradiated with high LET radiation than cells with X-rays. The phosphorylation status of DNA-PKcs, a non-homologous end joining (NHEJ) protein, was markedly different in cells irradiated with high LET radiation when compared with that with X-rays. HiCEP method, a sensitive gene expression profiling technique developed at NIRS, indicated there are a few genes characteristic specifically for heavy ion irradiation. Using the synchronized CHO and its repair defective mutant cells we investigated the cell survival levels throughout the cell cycle with carbon ions, and the result was compared with that with X-rays. Our data indicated that much less variation in the cell survival level was observed when carbon ions were used to irradiate cells. Although this cell cycle effect has been known for many years, our new experiments seem to give novice insight in the field of heavy-ion radiobiology.

View full abstract