Calorie restriction alters the mechanisms of radiation-induced mouse thymic lymphomagenesis

Calorie restriction (CR) suppresses not only spontaneous but also chemical- and radiation-induced carcinogenesis. Our previous study revealed that the cancer-preventive effect of CR is tissue dependent and that CR does not effectively prevent the development of thymic lymphoma (TL). We investigated the association between CR and the genomic alterations of resulting TLs to clarify the underlying resistance mechanism. TLs were obtained from previous and new experiments, in which B6C3F1 mice were exposed to radiation at 1 week of age and fed with a CR or standard (non-CR) diet from 7 weeks throughout their lifetimes. All available TLs were used for analysis of genomic DNA. In contrast to the TLs of the non-CR group, those of the CR group displayed suppression of copy-neutral loss of heterozygosity (LOH) involving relevant tumor suppressor genes (Cdkn2a, Ikzf1, Trp53, Pten), an event regarded as cell division–associated. However, CR did not affect interstitial deletions of those genes, which were observed in both groups. In addition, CR affected the mechanism of Ikzf1 inactivation in TLs: the non-CR group exhibited copy-neutral LOH with duplicated inactive alleles, whereas the CR group showed expression of dominant-negative isoforms accompanying a point mutation or an intragenic deletion. These results suggest that, even though CR reduces cell division–related genomic rearrangements by suppressing cell proliferation, tumors arise via diverse carcinogenic pathways including inactivation of tumor suppressors via interstitial deletions and other mutations. These findings provide a molecular basis for improved prevention strategies that overcome the CR resistance of lymphomagenesis

Preparation of Monoclonal Antibody Against Chicken Apolipoprotein B and Development of Enzyme Liked Immunosolvent Assay (ELISA) Method with the Antibody Aiming at the Optimization of Lipid Metabolism in Chickens

Apolipoprotein B (apoB) containing lipoproteins are major lipoproteins in blood and the elevated concentration in plasma may lead to increased transport of lipoproteins into adipose tissues resulting in the excessive fat deposition. Aims of the present study were to prepare monoclonal antibodies against chicken apoB and to development enzyme linked mmunosolvent assay (ELISA) methods with the antibody which would be provided for studies on lipoprotein metabolism in chickens. A monoclonal antibody, CAB4, was prepared using a purified chicken apoB and specific reactivity of the antibody was verified by Western blot analysis and immuno-precipitation of apoB in plasma and primary hepatocyte cultures in chickens. ELISA method with monoclonal anti-chicken apoB antibody developed in this study was applicable to measure apoB concentration in plasma and cell cultures within the range of 0 to 500ng/ml with the intra- and inter-assay variation of less than 10.6%. Using the present ELISA method, plasma apoB concentrations in broiler chickens starved for 8h were shown to be 0.44mg/ml against 1.02mg/ml in fed chicken counterparts. The monoclonal antibodies against chicken apoB and ELISA method developed in the present study could be potentially used for studies on the lipoprotein metabolism focusing on the optimization of lipid metabolism in chickens

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

The effects of short-term calorie restriction on mutations in the spleen cells of infant-irradiated mice.

The risk of cancer due to exposure to ionizing radiation is higher in infants than in adults. In a previous study, the effect of adult-onset calorie restriction (CR) on carcinogenesis in mice after early-life exposure to X-rays was examined (Shang, Y, Kakinuma, S, Yamauchi, K, et al. Cancer prevention by adult-onset calorie restriction after infant exposure to ionizing radiation in B6C3F1 male mice. Int J Cancer. 2014; 135: 1038-47). The results showed that the tumor frequency was reduced in the CR group. However, the mechanism of tumor suppression by CR is not yet clear. In this study, we examined the effects of CR on radiation-induced mutations using gpt delta mice, which are useful to analyze mutations in various tissues throughout the whole body. Infant male mice (1-week old) were exposed to 3.8 Gy X-rays and fed a control (95 kcal/week/mouse) or CR (65 kcal/week/mouse) diet from adult stage (7-weeks old). Mice were sacrificed at the age of 7 weeks, 8 weeks and 100 days, and organs (spleen, liver, lung, thymus) were harvested. Mutations at the gpt gene in the DNA from the spleen were analyzed by using a gpt assay protocol that detects primarily point mutations in the gpt gene. The results showed that mutation frequencies were decreased in CR groups compared with non-CR groups. Sequence analysis of the gpt gene in mutants revealed a reduction in the G:C to T:A transversion in CR groups. Since it is known that 8-oxoguanine could result in this base substitution and that CR has an effect of reducing oxidative stress, these results indicate that the suppression of oxidative stress by CR is the cause of the reduction of this transversion

Age dependency of Ikaros and Pten alterations in radiation-induced T-cell lymphoma

Background: Radiation carcinogenesis is greatly dependent on the age-at-exposure. Previous studies on the survivors of A-bombing and Chernobyl accident have indicated that there is an age difference in incidence and causal gene involved in both leukemia and thyroid cancer. The data on underlying mechanism of age dependency, however, is still limited. In this study, mouse T-cell lymphoma (TL) model was used for investigating the different effect of ionizing radiation to infant age and young adult age-at-exposure. We aimed to clarify the age dependent change of the affected molecular pathways of radiation-induced mouse T-cell lymphomagenesis.Materials and Methods: We analyzed the three groups of TLs developed after irradiation to female B6C3F1 mice weekly to 1.2 Gy X-ray for 4 consecutive weeks starting at infant (1 week of age), prepubertal (4 weeks of age) and adult (8 weeks of age). Loss of heterozygosity (LOH) analyses on chromosome 11, 12 and 19 were performed by PCR using genomic DNA of TLs. Mutations of the Ikaros and Pten were analyzed by sequencing of cDNA and genomic DNA, Western blotting, and array CGH.Results and Discussion: The frequency of LOH on chromosome 11 in 1W-TLs (27%; 4/15) was lower than those in 4W (46%; 6/13) and 8W-TLs (63%; 5/8). In contrast, the frequency of LOH on chromosome 19 in 1W-TLs (60%; 9/15) was higher than those in 4W-TLs (31%; 4/13) and 8W-TLs (13%; 1/8). Thus, LOH frequency was altered dependent on the age-at-exposure. Array CGH analysis revealed that intragenic deletion within Ikaros locus, which is mapped on chromosome 11, was characteristic in 1W-TLs, which produced aberrant Ikaros isoform, a lack of exon 5. On the other hand, different size deletions were found in each allele in 8W-TLs; one is intragenic deletion and the other is wide deletion including whole Ikaros locus. This type deletion caused null expression of Ikaros. In 4W-TLs, either type of deletion was found. Thus, deletion type in Ikaros region was different depend on the age-at-exposure. Array-CGH analysis revealed that the genomic copy number of the defined region of chromosome 19 in 1W-TLs was unchanged despite LOH, suggesting the LOH was caused by mitotic recombination. Therefore, it is considered that one allele deletion or intragenic mutation was formed at first and then the mitotic recombination occurred, resulting in homozygous deletion or homozygous intragenic mutations in 1W-TLs. Mutation frequency of Pten was higher than that in 4W- and 8W-TLs.Conclusion: Ikaros mutation was more involved in older age-at-exposure for T-cell lymphomagenesis, while Pten mutation was more involved in younger age-at-exposure. These results suggest that a target gene of radiation-induced T-cell lymphomagenesis and molecular mechanism of mutation change as a function of age-at-exposure.Childhood Cancer 2012 -International scientific conference on early exposure and childhood cancer

Age dependent role of Ikaros and Pten mutations in radiation T-cell lymphomagenesis in mice

Radiation carcinogenic risk is greatly dependent on the age at exposure. To find characteristic molecular changes in tumors induced by radiation at young age, we used the mouse T-cell lymphoma (TL) model. TL was induced by fractionated whole-body X-irradiation (four weekly fractions) during infancy (starting at 1 week old), prepuberty (4 weeks old), and young adult (8 weeks old). We assessed the mutation of Ikaros and Pten in the TLs by the analyses of loss of heterozygosity (LOH), array-based comparative genome hybridization, sequencing and Western blotting. LOH at Ikaros loci and mutation of Ikaros were most frequent in the young adult irradiation group. In contrast, the frequency of LOH at Pten loci and mutation of Pten were the highest in the infant irradiation group. Thus, Ikaros mutations were implicated in T-cell lymphomagenesis in adult, while Pten mutations were predominant in infant. Moreover, the mechanisms of allele loss appeared different among these age groups. These results suggest that major tumor suppressor genes involved in radiation T-cell lymphomagenesis and the molecular mechanism of mutation change as a function of age at exposure.Kyoto T Cell Conference (KTCC

Genomic profile of radiation-induced early-onset mouse B-cell lymphoma recapitulates features of Philadelphia chromosome–like acute lymphoblastic leukemia in humans

Epidemiological studies have revealed a radiation-related increase in the risk of developing acute lymphoblastic leukemia (ALL). Our recent study revealed early induction and increased risk of precursor B-cell (pB) lymphomas in mice after radiation exposure. However, the genomic landscape of radiation-induced B-cell lymphomas remains unclear. To identify the relevant genetic alterations in mice, whole-exome sequencing was performed on both early-onset and late-onset B-cell lymphomas that developed spontaneously or after gamma-irradiation. In addition to multiple driver mutations, the data revealed that interstitial deletion of chromosome 4, including Pax5, and missense mutations in Jak3 are unique genomic alterations in radiation-induced, early-onset B-cell lymphomas. RNA sequencing revealed a pB-cell-type gene-expression profile with no involvement of known fusion genes for human ALLs in the early-onset B-cell lymphomas. Activation of Jak3/Stat5 signaling in early-onset B-cell lymphomas was validated using western capillary electrophoresis. Those features were similar to those of Philadelphia chromosome–like ALL. Our data suggest a critical role for Pax5 loss-of-function mutations in initiating B-cell leukemogenesis coupled with activation of Jak3/Stat5 signaling as a basis for the rapid development of radiation-induced pB-ALL. These molecular signatures for radiation-induced cancers will inform both risk assessment and potential targeted therapies for pB-ALL