Cancer prevention by adult-onset calorie restriction after infant exposure to ionizing radiation in B6C3F1 male mice.

Children are especially sensitive to ionizing radiation and chemical carcinogens, and limiting their cancer risk is of great public concern. Calorie restriction (CR) is a potent intervention for suppressing cancer. However, CR is generally not appropriate for children. This study, therefore, examined to see if adult-onset CR influences the lifetime cancer risk in mice after early-life exposure to ionizing radiation. Infant male mice (1-week-old) were exposed to 3.8 Gy X-rays, fed a control 95 kcal/week or CR 65 kcal/week diet from 7 weeks of age (adult stage), and their lifespan and tumor development were assessed. Irrespective of CR, X-rays shortened lifespan by 38%, and irrespective of irradiation CR extended lifespan by 20%. Thymic lymphoma (TL) and early-occurring non-TL were induced by radiation. The liver and Harderian gland were more susceptible to radiation-induced tumors than the lungs and non-thymic lymphoid tissues (late occurring). CR reduced the risk of hepatocellular carcinoma, late-occurring non-TL, lung tumor, Harderian tumor, and hemangioma but had less impact on TL and early-occurring non-TL. Most notably, the effects of X-rays on induction of lung tumors, late-occurring non-TL and hemangioma were essentially canceled by CR. The ability of CR to prevent late-occurring tumors was the same for non-irradiated and irradiated mice, indicating that the mechanism by which CR influences cancer is independent of irradiation. Our results indicate that adult-onset CR significantly inhibits late-occurring tumors in a tissue-dependent manner regardless of infant radiation exposure.

Age dependence of hematopoietic progenitor survival and chemokine family gene induction after gamma irradiation in bone marrow tissue in C3H/He mice.

Age at exposure is a critical factor that influences the risk of radiation-induced leukemia, which arises from hematopoietic stem and progenitor cells. However, little is known about the effect of age on the radiation response of these cells. In this study, we examined the radiation response of hematopoietic stem and progenitor cells in infant (1-week-old), juvenile (3-week-old), and adult (8- and 14-week-old) C3H/He mice, which are susceptible to radiation-induced myeloid leukemia. We first observed an age-dependent increase in the radioresistance of hematopoietic stem and progenitor cells after in vivo irradiation. However, in vitro irradiation of progenitor cells did not show any age differences, suggesting that radiation sensitivity in vivo is dependent on the bone marrow microenvironment rather than to intrinsic properties of progenitors themselves. Expression profiles of bone marrow tissues identified chemokine and cytokine family genes, whose expression differed between infant and adult tissues at time points before and after irradiation. Among the selected thirteen cytokines reported to be radioprotective, we observed increased expression of Csf1, Csf2, Cxcl12, Fgf1, Fgf7, Il1a, Il1b and Kitl after irradiation, mostly in adult tissues. Specifically, Csf2, Fgf1 and Il1b expression, as revealed by qPCR, were significantly enhanced in adult bone marrow tissue after irradiation, but were unresponsive to irradiation in infant tissue. These results suggest that the higher susceptibility of infant hematopoietic stem and progenitor cells to the cell killing effect of ionizing radiation may be attributed to a failure to induce a subset of radioprotective cytokines in the immature bone marrow microenvironment.

[Radiation emergency medical preparedness in Japan--lessons learned from the Fukushima accident].

Although radiation exposure accidents fortunately occur only rarely, potential sources for exposure accidents can be found anywhere. When persons are accidentally exposed to radiation, physicians may be involved in their assessment and care; of course, their early diagnosis and dose assessment are crucial. After the criticality accident at Tokaimura in 1999, the system of radiation emergency medical preparedness has been further strengthened for nuclear facilities in Japan. In the revised system, hospitals involved were classified into three levels, depending on their locations and capabilities. The Great East Japan Earthquake attacked the Pacific coast area of eastern Japan on 11 March 2011. This earthquake and tsunami caused serious damage to the nuclear power plants of Tokyo Electric Power Co.(TEPCO) in Fukushima Prefecture; a large amount of radionuclides such as iodine and cesium were released into the environment. Since the revised system was focused on treatment of heavily exposed patients and knowledge on radiation was not enough for medical staff, many problems were raised at hospitals and fire departments in this disaster.

DNA copy number aberrations and disruption of the p16Ink4a/Rb pathway in radiation-induced and spontaneous rat mammary carcinomas.

Chromosomal amplifications and deletions are thought to be important events in spontaneous and radiation-induced carcinogenesis. To clarify how ionizing radiation induces mammary carcinogenesis, we characterized genomic copy number aberrations for gamma-ray-induced rat mammary carcinomas using microarray-based comparative genomic hybridization. We examined 14 carcinomas induced by gamma radiation (2 Gy) and found 26 aberrations, including trisomies of chromosomes 4 and 10 for three and one carcinomas, respectively, an amplification of the chromosomal region 1q12 in two carcinomas, and deletions of the chromosomal regions 3q35q36, 5q32 and 7q11 in two, two and four carcinomas, respectively. These aberrations were not observed in seven spontaneous mammary carcinomas. The expression of p16Ink4a and p19Arf, which are located in the chromosomal region 5q32, was always up-regulated except for a carcinoma with a homozygous deletion of region 5q32. The up-regulation was not accounted for by gene mutations or promoter hypomethylation. However, the amounts of Rb and its mRNA were down-regulated in these carcinomas, indicating a disruption of the p16Ink4a/Rb pathway. This is the first report of array CGH analysis for radiation-induced mammary tumors, which reveals that they show distinct DNA copy number aberration patterns that are different from those of spontaneous tumors and those reported previously for chemically induced tumors.

Genomic and gene expression signatures of radiation in medulloblastomas after low-dose irradiation in Ptch1 heterozygous mice.

Accurate cancer risk assessment of low-dose radiation poses many challenges that are partly due to the inability to distinguish radiation-induced tumors from spontaneous ones. To elucidate characteristic features of radiation-induced tumors, we analyzed 163 medulloblastomas that developed either spontaneously or after X-ray irradiation at doses of 0.05-3 Gy in Ptch1 heterozygous mice. All spontaneous tumors showed loss of heterozygosity in broad regions on chromosome 13, with losses at all consecutive markers distal to Ptch1 locus (S-type). In contrast, all tumors that developed after 3 Gy irradiation exhibited interstitial losses around Ptch1 with distal markers retained (R-type). There was a clear dose-dependent increase in the proportion of R-type tumors within the intermediate dose range, indicating that the R-type change is a reliable radiation signature. Importantly, the incidence of R-type tumors increased significantly (P = 0.007) at a dose as low as 50 mGy. Integrated array-comparative genomic hybridization and expression microarray analyses demonstrated that expression levels of many genes around the Ptch1 locus faithfully reflected the signature-associated reduction in genomic copy number. Furthermore, 573 genes on other chromosomes were also expressed differently between S-type and R-type tumors. They include genes whose expression changes during early cerebellar development such as Plagl1 and Tgfb2, suggesting a recapitulation of gene subsets functioning at distinct developmental stages. These findings provide, for the first time, solid experimental evidence for a significant increase in cancer risk by low-dose radiation at diagnostic levels and imply that radiation-induced carcinogenesis accompanies both genomic and gene expression signatures.

Complicated biallelic inactivation of Pten in radiation-induced mouse thymic lymphomas.

Inactivation of the phosphatase and tensin homolog gene (Pten) occurs via multiple tissue-dependent mechanisms including epigenetic silencing, point mutations, insertions, and deletions. Although frequent loss of heterozygosity around the Pten locus and plausible involvement of epigenetic silencing have been reported in radiation-induced thymic lymphomas, the proportion of lymphomas with inactivated Pten and the spectrum of causal aberrations have not been extensively characterized. Here, we assessed the mode of Pten inactivation by comprehensive analysis of the expression and alteration of Pten in 23 radiation-induced thymic lymphomas developed in B6C3F1 mice. We found no evidence for methylation-associated silencing of Pten; rather, complex structural abnormalities comprised of missense and nonsense mutations, 1- and 3-bp insertions, and focal deletions were identified in 8 of 23 lymphomas (35%). Sequencing of deletion breakpoints suggested that aberrant V(D)J recombination and microhomology-mediated rearrangement were responsible for the focal deletions. Seven of the 8 lymphomas had biallelic alterations, and 4 of them did not express Pten protein. These Pten aberrations coincided with downstream Akt phosphorylation. In conclusion, we demonstrate that Pten inactivation is frequently biallelic and is caused by a variety of structural abnormalities (rather than by epigenetic silencing) and is involved in radiation-induced lymphomagenesis.

Radiation-induced mammary carcinogenesis in rodent models: what's different from chemical carcinogenesis?

Ionizing radiation is one of a few well-characterized etiologic factors of human breast cancer. Laboratory rodents serve as useful experimental models for investigating dose responses and mechanisms of cancer development. Using these models, a lot of information has been accumulated about mammary gland cancer, which can be induced by both chemical carcinogens and radiation. In this review, we first list some experimental rodent models of breast cancer induction. We then focus on several topics that are important in understanding the mechanisms and risk modification of breast cancer development, and compare radiation and chemical carcinogenesis models. We will focus on the pathology and natural history of cancer development in these models, genetic changes observed in induced cancers, indirect effects of carcinogens, and finally risk modification by reproductive factors and age at exposure to the carcinogens. In addition, we summarize the knowledge available on mammary stem/progenitor cells as a potential target of carcinogens. Comparison of chemical and radiation carcinogenesis models on these topics indicates certain similarities, but it also indicates clear differences in several important aspects, such as genetic alterations of induced cancers and modification of susceptibility by age and reproductive factors. Identification of the target cell type and relevant translational research for human risk management may be among the important issues that are addressed by radiation carcinogenesis models.JRRS Incentive Award in 2009.

Microarray-based global mapping of integration sites for the retrotransposon, intracisternal A-particle, in the mouse genome.

Mammalian genomes contain numerous evolutionary harbored mobile elements, a part of which are still active and may cause genomic instability. Their movement and positional diversity occasionally result in phenotypic changes and variation by causing altered expression or disruption of neighboring host genes. Here, we describe a novel microarray-based method by which dispersed genomic locations of a type of retrotransposon in a mammalian genome can be identified. Using this method, we mapped the DNA elements for a mouse retrotransposon, intracisternal A-particle (IAP), within genomes of C3H/He and C57BL/6J inbred mouse strains; consequently we detected hundreds of probable IAP cDNA-integrated genomic regions, in which a considerable number of strain-specific putative insertions were included. In addition, by comparing genomic DNAs from radiation-induced myeloid leukemia cells and its reference normal tissue, we detected three genomic regions around which an IAP element was integrated. These results demonstrate the first successful genome-wide mapping of a retrotransposon type in a mammalian genome.

Upregulation of c-myc gene accompanied by PU.1 deficiency in radiation-induced acute myeloid leukemia in mice.

OBJECTIVE: High-dose radiation exposure induces acute myeloid leukemia (AML) in C3H mice, most of which have a frequent hemizygous deletion around the D2Mit15 marker on chromosome 2. This region includes PU.1, a critical candidate gene for initiation of leukemogenesis. To identify novel cooperative genes with PU.1, relevant to radiation-induced leukemogenesis, we analyzed the copy number alterations of tumor-related gene loci by array CGH, and their expressions in primary and transplanted AMLs. MATERIALS AND METHODS: For the induction of AMLs, C3H/He Nrs mice were exposed to 3 Gy of x-rays or gamma-rays. The genomic alterations of 35 primary AMLs and 34 transplanted AMLs obtained from the recipient mice transplanted the primary AMLs were analyzed by array CGH. According to the genomic alterations and mutations of the 235th arginine of PU.1 allele, we classified the radiogenic AMLs into three types such as Chr2(del) PU.1(del/R235-) AML, Chr2(del) PU.1(del/R235+) AML and Chr2(intact) PU.1(R235+/R235+) AML, to compare the expression levels of 8 tumor-related genes quantitatively by real-time polymerase chain reaction and cell-surface antigen expression. Results. In addition to well-known loss of PU.1 with hemizygous deletion of chromosome 2, novel genomic alterations such as partial gain of chromosome 6 were recurrently detected in AMLs. In this study, we found similarity between cell-surface antigen expressions of bone marrows and those of spleens in AML mice and significantly higher expressions of c-myc and PU.1 expression, especially in the PU.1-deficient (Chr2(del) PU.1(del/R235-)) AML and Chr2(del) PU.1(del/R235+) compared to Chr2(intact) PU.1(R235+/R235+) AMLs. CONCLUSION: The new finding on upregulation of c-myc and PU.1 in both and hemizygous PU.1-deficient AMLs and different genomic alterations detected by array CGH suggests that the molecular mechanism for development of radiation-induced AML should be different among three types of AML.

Analysis of changes in DNA copy number in radiation-induced thymic lymphomas of susceptible C57BL/6, resistant C3H and hybrid F1 Mice.

Radiation-induced thymic lymphoma in mice is a useful model for studying both the mechanism of radiation carcinogenesis and genetic susceptibility to tumor development. Using array-comparative genomic hybridization, we analyzed genome-wide changes in DNA copy numbers in radiation-induced thymic lymphomas that had developed in susceptible C57BL/6 and resistant C3H mice and their hybrids, C3B6F1 and B6C3F1 mice. Besides aberrations at known relevant genetic loci including Ikaros and Bcl11b and trisomy of chromosome 15, we identified strain-associated genomic imbalances on chromosomes 5, 10 and 16 and strain-unassociated trisomy of chromosome 14 as frequent aberrations. In addition, biallelic rearrangements at Tcrb were detected more frequently in tumors from C57BL/6 mice than in those from C3H mice, suggesting aberrant V(D)J recombination and a possible link with tumor susceptibility. The frequency and spectrum of these copy-number changes in lymphomas from C3B6F1 and B6C3F1 mice were similar to those in C57BL/6 mice. Furthermore, the loss of heterozygosity analyses of tumors in F(1) mice indicated that allelic losses at Ikaros and Bcl11b were caused primarily by multilocus deletions, whereas those at the Cdkn2a/Cdkn2b and Pten loci were due mainly to uniparental disomy. These findings provide important clues to both the mechanisms for accumulation of aberrations during radiation-induced lymphomagenesis and the different susceptibilities of C57BL/6 and C3H mice.

Array-CGH analyses of murine malignant lymphomas: genomic clues to understanding the effects of chronic exposure to low-dose-rate gamma rays on lymphomagenesis.

We previously reported that mice chronically irradiated with low-dose-rate gamma rays had significantly shorter mean life spans than nonirradiated controls. This life shortening appeared to be due primarily to earlier death due to malignant lymphomas in the irradiated groups (Tanaka et al., Radiat. Res. 160, 376-379, 2003). To elucidate the molecular pathogenesis of murine lymphomas after low-dose-rate irradiation, chromosomal aberrations in 82 malignant lymphomas from mice irradiated at a dose rate of 21 mGy/day and from nonirradiated mice were compared precisely by microarray-based comparative genomic hybridization (array-CGH) analysis. The array carried 667 BAC clones densely selected for the genomic regions not only of lymphoma-related loci but also of surface antigen receptors, enabling immunogenotyping. Frequent detection of the apparent loss of the Igh region on chromosome 12 suggested that most lymphomas in both groups were of B-cell origin. Array-CGH profiles showed a frequent gain of whole chromosome 15 in lymphomas predominantly from the irradiated group. The profiles also demonstrated copy-number imbalances of partial chromosomal regions. Partial gains on chromosomes 12, 14 and X were found in tumors from nonirradiated mice, whereas losses on chromosomes 4 and 14 were significantly associated with the irradiated group. These findings suggest that lymphomagenesis under the effects of continuous low-dose-rate irradiation is accelerated by a mechanism different from spontaneous lymphomagenesis that is characterized by the unique spectrum of chromosomal aberrations.

Conserved expression control and shared activity between cognate T-box genes Tbx2 and Tbx3 in connection with Sonic hedgehog signaling during Xenopus eye development.

Tbx2 and Tbx3 are considered to be cognate genes within a Tbx2/3/4/5 subfamily of T-box genes and are expressed in closely overlapping areas in a variety of tissues, including the eye. Herein, we show that misexpression of Tbx2 and Tbx3 in Xenopus embryos gave rise to defective eye morphogenesis, which was reminiscent of the defect caused by attenuated Sonic hedgehog (Shh) signaling. Indeed, Tbx2/3 misexpression suppressed Gli1, Gli2, Ptc2 and Pax2, mediators or targets of Hedgehog (Hh) signals. From these data, Tbx2/3 may have a shared function in inhibiting Gli-dependent Shh signaling during eye development. Conversely, the expression of Tbx2/3 was severely affected by both Shh and a putative dominant negative form of Hh, as well as by both transactivator and transrepressor forms of Gli-fusion proteins, suggesting that the expression of Tbx2/3 may be regulated by a Gli-dependent Hh signal transduction pathway. Because the Shh signal has been considered to play crucial roles in the formation of the proximal-distal and dorsal-ventral axes in the eyes, these findings about the mutual regulatory mechanism between Tbx2/3 and Gli-dependent Hh signaling provide valuable insight into the cause of the localized expression of Tbx2/3 and their role during the formation of these axes. In addition, our findings also imply the conserved regulation and shared activity between the cognate genes of Tbx2 and Tbx3.

Improved mRNA electroporation method for Xenopus neurula embryos.

Electroporation has led to new approaches to the analysis of gene regulation of the chick embryonic system. However, application of this method to Xenopus, another model organism of embryology, has left many difficulties to be overcome. The specially devised electrodes, the examination of luciferase activities expressed, and the direct visualization of green fluorescence protein allow us to optimize the conditions of electroporation for Xenopusembryos. The use of mRNA rather than DNA improved the expression efficiency 120 times more than for the case of plasmid DNA, and the effect emerged more immediately after electroporation. The noncontact electroporation adopted here caused less damage to cells and tissues than with the needle type electrode, making it practical for efficient application to early embryos. Furthermore, the mRNA electroporation technique is applicable for other systems in which the DNA electroporation has not had any significant effect because of its low expression efficiency.

Axes establishment during eye morphogenesis in Xenopus by coordinate and antagonistic actions of BMP4, Shh, and RA.

We have examined the roles of BMP4, Shh, and retinoic acid in establishing the proximal-distal and dorsal-ventral axes in the developing Xenopus eye. Misexpression of BMP4 caused the absence of an optic stalk and the expansion of dorsal and distal markers, tbx2/3/5, and pax6, at the expense of ventral and proximal markers vax2 and pax2. When Shh or Noggin, an antagonist of BMPs, was misexpressed, the reverse expression patterns of these marker genes were observed. These results suggest that BMP4 is involved in the specification of not only dorsal in the optic cup but also distal in the optic vesicle. Because Shh did not suppress bmp4 expression, unlike Noggin, Shh and BMP4 may antagonistically regulate common downstream genes in developing eye. We also found the difference between the effects of Shh and retinoic acid, another possible ventralizing factor, suggesting that Shh could promote ventralization independently of retinoic acid. These findings provide important clues to the coordinate and antagonistic actions of BMP4, Shh, and retinoic acid in axes specifications of Xenopus eyes.

Metamorphic change in EP37 expression: members of the ßγ-crystallin superfamily in newt.

EP37 is an epidermis-specific protein found in the developing embryo of the Japanese newt, Cynops pyrrhogaster. Our previous study predicted the presence of genes homologous to EP37, which show temporary shared expression at the turn of metamorphosis. In this study, we isolated and characterized three cDNAs encoding novel EP37 homologues; two from the skin of an adult newt and the other from swimming larva. Conceptual translation of the open reading frames of these cDNAs predicted proteins carrying ßγ-crystallin motifs and putative calcium-binding sites, both of which are features shared by the originally identified EP37 (EP37L1), as well as a spore coat protein of Myxococcus xanthus, protein S. Immunoblot analyses and immunohistochemical studies indicated that two of the EP37 proteins, EP37L1 and EP37L2, are exclusively expressed in the epidermis (skein cells) including the figures of Eberth at premetamorphic stages. During and after metamorphosis, the expression of EP37 proteins was mainly observed in cutaneous glands, and a molecular transition to the adult types of EP37, EP37A1 and EP37A2, occurred. These observations suggest that EP37 proteins play an important role in construction of integumental tissues and adaptation to the aquatic or amphibious environment.

pax-6 gene expression in newt eye development.

pax-6 is thought to be a master control gene of eye development in species ranging from insects to mammals. We have isolated a pax-6 cDNA homolog of the newt, Cynops pyrrhogaster. RT-PCR and sequence analyses predicted four alternatively spliced forms derived from inclusion or exclusion of the region corresponding to exons 5a and 12 in the human pax-6 ortholog. This gene shared extensive sequence identitiy and similar expression patterns with those of mouse and zebrafish. pax-6 signal was first detected at the anterior ridge of the neural plate, and later at the eye and nasal primordium and in the central nervous system - except for the midbrain. The injection of sonic hedgehog (shh) RNA inhibited the expression of pax-6 within the optic vesicle and disturbed eye cup formation. A similar suppressive effect of shh was also observed in the conjugation of the animal caps preloaded with exogenous shh and noggin mRNA, which was used as an inducer of pax-6. In contrast, shh injection had no effect on the expression of pax-6 in the surface ectoderm overlying the optic cup, suggesting that the expression of pax-6 in the surface ectoderm is not regulated by shh in vivo. Moreover, we found transient activation of pax-6 in animal cap explants at the sibling stage of mid-late gastrula. This observation raises the possibility that the ectoderm is competent to the lens-inducing signal at a stage as early as mid gastrula.

Cloning of an Epidermis-specific Cynops cDNA from Neurula Library.

A cDNA encoding one of the epidermis-specific proteins designated as the spot 6 was isolated from the Cynops embryo. Cynops neurula cDNA library was constructed with the plasmid vector containing the promoter sequence for SP6 RNA polymerase. After transcription and translation in vitro the final protein products were screened for the presence of spot 6 by two-dimensional gel electrophoresis. The total library was digested by 11 restriction endonucleases selected not to destroy the sequence in both the vector and the insert encoding spot 6 protein. The ATP-dependent DNase digestion eliminated the cDNA population sensitive to these endonucleases. These steps effectively enriched the sequence for spot 6 protein. The resultant sublibrary was repeatedly divided into smaller pools and was screened. The tryptic peptide analysis showed that the isolated clone produced the protein identical to the spot 6 protein originally defined in vivo. Northern analyses showed that the cloned gene was expressed as expected from the developmental behavior of the spot 6 in vivo.

Protein Synthesis during Neural and Epidermal Differentiation in Cynops Embryo: (Cynops pyrrhogaster/protein synthesis/neural induction/concanavalin A/epidermal differentiation).

Two-dimensional gel electrophoresis was used to analyze protein synthesis in relation to neural and epidermal differentiation in Cynops pyrrhogaster embryo. Various regions of embryos at different developmental stages, from late morula to early neurula stages, were excised, radiolabelled with 35 S-methionine, and the pattern of protein synthesis were compared. The following four types of protein spots were observed: (1) six proteins synthesized characteristically in the epidermal region of the embryo after gastrulation, (2) two proteins synthesized in both epidermal and endodermal regions, but not in other regions, after gastrulation, (3) a protein first detected at early blastula stage, of which expression was nearly constant in presumptive epidermis region but declined in the other regions, (4) the candidate for neural plate specific protein synthesized at a very high level in ectoderm explants treated with concanavalin A, a substance which evokes neural induction.