Molecular interaction of X-rays and N-ethyl-N-nitrosourea in thymic lymphomagenesis depends on the interval of two treatments

IntroductionRadiation carcinogenesis in human is considered as a result of the combined effect with other environmental carcinogens such as tobacco and foodstuffs. We previously showed that single exposure to X-rays or N-ethyl-N-nitrosourea (ENU) at high dose efficiently induced thymic lymphoma (TL) and that loss of heterozygosity (LOH) on chromosome 11, which accompanied altered expression and mutation of Ikaros, was characteristic to X-ray-induced TL (>40%), while it was rarely observed in ENU-induced ones. In addition, the combined effect of two carcinogens on the induction of TL depended upon not only the dose of carcinogen(s) but also the order of treatment. The aim of this study was to clarify the effect of the interval between X-rays and ENU on the TL induction and its underlying mechanism.Materials and methods Fifty of 4-week-old female B6C3F1 mice were used in each group. They were exposed to X-rays (1.0 Gy per fraction) for four consecutive weeks, and then were treated with ENU (200 ppm) in drinking water for 4 weeks with the interval of 0, 2, 4, and 8 weeks. The mice were observed until moribundity. Lifespan, incidence of TL and LOH frequency of chromosome 11, together with that of chromosomes12 and 19, were determined. Results and discussion 1. Lifespan of the groups with the treatment interval of 0 or 2 weeks were significantly shorter than that with the interval of 4 or 8 weeks, indicating a significant interaction of the two carcinogens with shorter interval. 2. Incidence of TL after X-irradiation alone was 13%, and that after ENU treatment was around 20% regardless of the age at exposure. The incidences of TL of the groups with the interval of 0, 2, 4, and 8 weeks were 94%, 98%, 58%, and 70%, respectively. Thus, the effect of prior X-irradiation persisted up until 8 weeks, but longer interval was much less effective than shorter one. 3. LOH frequency on chromosome 11 was low for the group with no interval (15%), suggesting less involvement of X-ray-associated LOH in TL induction. However, spit of the two treatments longer than 2 weeks increased the LOH frequency to the level of X-irradiation alone (35-45%). LOH frequencies on chromosomes 12 and 19, however, were little affected by the presence of interval.ConclusionThe interval period between X-rays and ENU was critical for both the frequency of TL and the mechanism of lymphomagenesis involving LOH on chromosome 11, possibly Ikaros mutation.The 22nd Biennial Congress of the European Association for Cancer Researc

Tissue-specific and time-dependent clonal expansion of ENU-induced mutant cells in gpt delta mice.

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N-ethyl-N-nitrosourea (ENU) with the persistence of ENU-induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU-induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis

Profiling Genetic Changes at Common Tumor Suppressor Loci in Carbon Ion Radiation-Induced Thymic Lymphomas

Secondary cancer risk following carbon ion radiotherapy is a key consideration for its use in children over conventional photon radiotherapy. Understanding not only the relative carcinogenic risk of carbon ion therapy, but also the potentially different carcinogenic mechanisms is a primary goal of the Radiobiology for Childrens Health Research Program at the National Institute of Radiological Sciences, Japan.The research program has undertaken a large-scale study over the past 8 years, for which cohorts of irradiated B6C3F1 mice are followed throughout their natural life for tumour development. The study includes groups which vary in their age-at-exposure, as well as radiation source, quality, fractionation and total dose. From this study, a sub-cohort of mice was selected for detailed genetic analysis of radiation-induced thymic lymphomas. This included all mice irradiated with 4 - 4.8 Gy carbon ions starting at one week of age (either in a single exposure, or four weekly fractions), which had been sacrificed in a moribund state with a cause of death at autopsy of thymic lymphoma (n 102 mice). DNA and RNA were isolated from the corresponding thymic lymphoma tissue bank samples. Gross genomic changes at tumour suppressor loci including Pten, Bcl11b and Ikzf1(IKAROS family zinc finger 1) are being assessed by loss of heterozygosity (LOH) analysis using PCR, and amplicon sizing of sites polymorphic for sequence length between the B6 and C3H parental strains. Smaller genetic alterations are being further assessed by exon sequencing of Pten, Bcl11b and Ikzf1 to identify small insertions/deletions and point-mutations. The features observed in the carbon ion-irradiated sub-cohort will be compared to those in a reference population of tumours in mice irradiated with a corresponding regime of gamma-radiation. Preliminary results suggest universal LOH in all carbon ion radiation-induced thymic lymphomas at sites flanking Bcl11b (compared to only 69pc in gamma radiation-induced thymic lymphomas), and approximately twice the frequency of LOH at a site -4 kb downstream of Pten (90pc vs. 43pc). Mechanistic insight into carbon ion-induced carcinogenesis will be vital in assessing the long-term safety of carbon ion radiotherapy for children.平成24年度「個体レベルでのがん研究支援活動」ワークショップ　個体レベルのがん研究による相乗効果　学術的インターラクションから創造

The process of shape-configuration play for a 2.3 year old child using origami: Origami as a tool for instruction to develop a child?s ability to configure shapes

This study treated origami like pieces of a puzzle, by creating simple and regular origami forms, which required folds into point-symmetric shapes (parallelograms) and other folds into line-symmetric shapes (isosceles triangles). The purpose of this research was to examine how access to an existing tradition of origami, as tool for instruction, could be used to create and configure of shapes through child’s play and practice. Observation research was conducted on 26 infant subjects aged between 30 and 44 months of age at a nursery school. First, we examined the way in which the 2 to 3 year old children play with origami. The results were broadly classified into 3 patterns: (1) movement constitution type; (2) insertion type; (3) transformation type. Next, we investigated the difference in development of play by different ages. The results were that the lower the age they connected the pieces using their unchanged forms. As the age increased, it was seen that they developed from playing with many pieces to connecting them using their unchanged forms and by inserting a few pieces through playing, and by modifying a few pieces. The results showed that the 2 to 3 year old child had the opportunity to experiment using origami techniques to fold the paper into line-symmetric shapes and point-symmetric shapes through a form of play, wherein the child formed and created new shapes using his/her own methods

Genetic Profiling of Carbon Ion Radiation-Induced Thymic Lymphomas

Secondary cancer risk following carbon ion radiotherapy is a key consideration for its use in children over conventional photon radiotherapy. Understanding not only the relative carcinogenic risk of carbon ion therapy, but also the potentially different carcinogenic mechanisms is a primary goal of the Radiobiology for Children\u27s Health Program at the National Institute of Radiological Sciences, Japan.The research program has undertaken a large-scale study over the past 9 years, for which cohorts of irradiated B6C3F1 mice are followed throughout their natural life for tumour development. The study includes groups which vary in their age-at-exposure, as well as radiation source, quality, fractionation and total dose. From this study, a sub-cohort of mice was selected for detailed genetic analysis of radiation-induced thymic lymphomas. This included all mice irradiated with 4 or 4.8 Gy carbon ions (HIMAC: mono-energetic beam, 290 MeV/n, average LET 13 keV/micron) starting at one week of age (either in a single exposure, or four weekly fractions), which had been sacrificed in a moribund state with a cause of death at autopsy of thymic lymphoma (n = 102 mice). Gross genomic changes at common thymic lymphoma tumour suppressor loci including Pten, Bcl11b, Trp53 and Ikzf1 are being assessed by loss of heterozygosity (LOH) analysis using PCR of sites polymorphic for sequence length between the parental strains. High-resolution copy number variation analysis is being conducted on selected tumour samples using a customized comparative genome hybridization array to identify further sites of genomic loss/gain. Smaller genetic alterations are being further assessed by exon sequencing of Pten, Bcl11b, Trp53 and Ikzf1 to identify small insertions/deletions and point-mutations. The features observed in the carbon ion-irradiated sub-cohort are being compared to those in a reference population of tumours in mice irradiated with corresponding regimes of gamma-radiation.Mechanistic insight into carbon ion-induced carcinogenesis will be vital in assessing the long-term safety of carbon ion radiotherapy for children.The 59th Annual Meeting of the Radiation Research Societ

Gene signatures in radiation-induced T cell lymphomas.

T cell lymphomas in mice can overcome natural tumour barriers via several routes, including deregulation of Bcl11b, Notch, Pten or Ikaros via gene mutation, amplification or loss. Our research has shown that the etiology of T cell lymphomas (e.g. radiation or chemical mutagenesis) is correlated with a pattern in the disruption of the above pathways and how they are activated or inactivated. Carbon ion radiotherapy may be associated with different risks of secondary cancer than conventional photon therapies. A sub-cohort of mice irradiated with 4 or 4.8 Gy carbon ions starting at one week of age (either in a single exposure, or four weekly fractions), was selected for detailed genetic analysis of radiation-induced thymic lymphomas (n = 102 mice). Gross genomic changes at tumour suppressor loci including Pten, Bcl11b and Ikzf1 are being assessed by loss of heterozygosity (LOH) analysis and array-based comparative genome hybridisation. Smaller genetic alterations are being further assessed by exon sequencing of Pten, Bcl11b and Ikzf1 to identify small insertions/deletions and point-mutations. Preliminary results show that although the same pathways are involved in both carbon- and photon radiation-induced tumours, how these pathways are disrupted may be different between radiation types. Mechanistic insight into carbon ion-induced carcinogenesis will be vital in assessing the long-term safety of carbon ion radiotherapy for children.The 6th International Workshop of the Kyoto T Cell Conferenc