Hiroko Ishii-Ohba

Involvement of Illegitimate V(D)J Recombination or Microhomology-Mediated Nonhomologous End-Joining in the Formation of Intragenic Deletions of the Notch1 Gene in Mouse Thymic Lymphomas

Deregulated V(D)J recombination-mediated chromosomal rearrangements are implicated in the etiology of B- and T-cell lymphomagenesis. We describe three pathways for the formation of 5′-deletions of the Notch1 gene in thymic lymphomas of wild-type or V(D)J recombination-defective severe combined immune deficiency (scid) mice. A pair of recombination signal sequence-like sequences composed of heptamer- and nonamer-like motifs separated by 12- or 23-bp spacers (12- and 23-recombination signal sequence) were present in the vicinity of the deletion breakpoints in wild-type thymic lymphomas, accompanied by palindromic or nontemplated nucleotides at the junctions. In scid thymic lymphomas, the deletions at the recombination signal sequence-like sequences occurred at a significantly lower frequency than in wild-type mice, whereas the deletions did not occur in Rag2−/− thymocytes. These results show that the 5′-deletions are formed by Rag-mediated V(D)J recombination machinery at cryptic recombination signal sequences in the Notch1 locus. In contrast, one third of the deletions in radiation-induced scid thymic lymphomas had microhomology at both ends, indicating that in the absence of DNA-dependent protein kinase-dependent nonhomologous end-joining, the microhomology-mediated nonhomologous end-joining pathway functions as the main mechanism to produce deletions. Furthermore, the deletions were induced via a coupled pathway between Rag-mediated cleavage at a cryptic recombination signal sequence and microhomology-mediated end-joining in radiation-induced scid thymic lymphomas. As the deletions at cryptic recombination signal sequences occur spontaneously, microhomology-mediated pathways might participate mainly in radiation-induced lymphomagenesis. Recombination signal sequence-mediated deletions were present clonally in the thymocyte population, suggesting that thymocytes with a 5′-deletion of the Notch1 gene have a growth advantage and are involved in lymphomagenesis.

Chemoprevention by dietary dehydroepiandrosterone against promotion/progression phase of radiation-induced mammary tumorigenesis in rats

When pregnant rats received whole body irradiations with 260 cGy gamma-ray at day 20 of pregnancy, and were then implanted with a diethylstilbestrol (DES) pellet for an experimental period of 1 year under feeding of a control diet, a high incidence (96.2%) of mammary tumors was observed. Administration of dietary 0.6% dehydroepiandrosterone (DHEA) together with DES implantation significantly decreased the incidence (35.0%) of mammary tumors. The first appearance of palpable tumors in the DHEA-fed group was 4.5 months later than that in the control group. For clarification of the mechanism of the chemopreventive action, we measured hormone levels in the serum of DHEA-fed rats. In the DHEA diet rats, the concentration of estradiol-17 beta exceeded, by approximately 6-fold, that in the control rats, while the levels of progesterone and prolactin were decreased by 30 and 45%, respectively. Interestingly, DHEA feeding prevented DES-induced hypertrophy of pituitary glands and DES-induced high level of prolactin in pituitary glands detected by immunohistochemical studies, but stimulated the development of mammary glands more than that in control rats treated with DES alone. These findings suggest that DHEA has a potent preventive activity against the promotion/progression phase of radiation-induced mammary tumorigenesis. The mechanism of chemoprevention by change of endocrinological environment is discussed.

Rag-dependent and Rag-independent mechanisms of Notch1 rearrangement in thymic lymphomas of Atm(-/-) and scid mice

The pathways of thymic lymphomagenesis are classified as Rag-dependent or -independent according to their dependence on recombination-activating gene (Rag1/2) proteins. The role of the two-lymphoma pathways in oncogene rearrangements and the connection between lymphoma pathways and rearrangement mechanisms, however, remain obscure. We compared the incidence and latency of thymic lymphomas, and associated rearrangements of the representative oncogene Notch1 among Rag2(-/-), ataxia telangiectasia mutated (Atm)(-/-), and severe combined immune deficiency (scid) mice combined with Rag2 deficiency. Contrary to expectations, Rag2(-/-) mice were prone to thymic lymphoma development, suggesting the existence of a Rag2-independent lymphoma pathway in Rag2(-/-) mice. The lymphoma incidence in Rag2(-/-)Atm(-/-) mice was lower than that in Atm(-/-) mice, but higher than that in Rag2(-/-) mice, indicating that Atm(-/-) mice develop lymphomas through both pathways. Scid mice developed lymphomas with an incidence and latency similar to Rag2(-/-)scid mice, suggesting that Rag2-mediated V(D)J recombination-driven events are not necessarily required for lymphomagenesis in scid mice. Notch1 rearrangement mechanisms were classified as Rag2-dependent or Rag2-independent based on the presence of recombination signal-like sequences at rearranged sites. In Rag2(-/-) lymphomas, Notch1 must be rearranged independently of Rag2 function, implying that Rag2(-/-) mice are susceptible to lymphomagenesis due to the presence of other rearrangement mechanisms. The results in Atm(-/-) mice suggest that Notch1 was rearranged through both lymphoma pathways. In scid mice, the frequency of Rag2-mediated rearrangements was relatively low compared with that in wild-type mice, suggesting that the Rag2-independent lymphoma pathway prevails in the development of thymic lymphomas in scid mice. Thus, two rearrangement mechanisms underlie the lymphoma pathways and constitute the mechanistic bases for lymphomagenesis, thereby providing the molecular criteria for distinguishing between Rag2-dependent and Rag2-independent lymphoma pathways.

Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

SummaryThe localization of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3β-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3β-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3β-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.

Diet-Induced Obesity Modulates Epigenetic Responses to Ionizing Radiation in Mice

Both exposure to ionizing radiation and obesity have been associated with various pathologies including cancer. There is a crucial need in better understanding the interactions between ionizing radiation effects (especially at low doses) and other risk factors, such as obesity. In order to evaluate radiation responses in obese animals, C3H and C57BL/6J mice fed a control normal fat or a high fat (HF) diet were exposed to fractionated doses of X-rays (0.75 Gy ×4). Bone marrow micronucleus assays did not suggest a modulation of radiation-induced genotoxicity by HF diet. Using MSP, we observed that the promoters of p16 and Dapk genes were methylated in the livers of C57BL/6J mice fed a HF diet (irradiated and non-irradiated); Mgmt promoter was methylated in irradiated and/or HF diet-fed mice. In addition, methylation PCR arrays identified Ep300 and Socs1 (whose promoters exhibited higher methylation levels in non-irradiated HF diet-fed mice) as potential targets for further studies. We then compared microRNA regulations after radiation exposure in the livers of C57BL/6J mice fed a normal or an HF diet, using microRNA arrays. Interestingly, radiation-triggered microRNA regulations observed in normal mice were not observed in obese mice. miR-466e was upregulated in non-irradiated obese mice. In vitro free fatty acid (palmitic acid, oleic acid) administration sensitized AML12 mouse liver cells to ionizing radiation, but the inhibition of miR-466e counteracted this radio-sensitization, suggesting that the modulation of radiation responses by diet-induced obesity might involve miR-466e expression. All together, our results suggested the existence of dietary effects on radiation responses (especially epigenetic regulations) in mice, possibly in relationship with obesity-induced chronic oxidative stress.

Subcellular distribution of 3β-hydroxysteroid dehydrogenase-isomerase in bovine and murine adrenocortical tissue: species differences in the localization of activity and immunoreactivity

Key to the production of biologically active steroids is the enzyme 3 beta-hydroxysteroid dehydrogenase-isomerase. Some controversy has arisen concerning the subcellular distribution of this enzyme within steroidogenic cells. The distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase was assessed in subcellular fractions obtained from homogenates of rat, bovine, and mouse adrenal glands in two ways. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was quantitated by measuring the conversion of radiolabeled pregnenolone to radiolabeled progesterone in an aliquot of each of the fractions obtained. The presence of the enzyme was assessed by performing Western analyses on aliquots of each of the fractions obtained with the use of a specific polyclonal antiserum against 3 beta-hydroxysteroid dehydrogenase-isomerase, the characterization of which is described. In control experiments, the degree of contamination of the fractions was determined by assessing the presence of known subcellular fraction markers with Western analysis. In the bovine and mouse adrenal glands, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to be localized solely in the microsomal fraction, while in the rat, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to have dual subcellular distribution: the microsomes and the inner mitochondrial membrane. We conclude that there is a species difference in the subcellular distribution of this important steroidogenic enzyme and that this species difference may be related to the steroidogenic pathway preferred in that species.

Steroid hormone production in testis, ovary, and adrenal gland of immature rats irradiated in utero with 60Co.

Pregnant rats received whole-body irradiation at 20 days of gestation with 2.6 Gy lambda rays from a 60Co source. Endocrinological effects before maturation were studied using testes and adrenal glands obtained from male offspring and ovaries from female offspring irradiated in utero. Seminiferous tubules of the irradiated male offspring were remarkably atrophied with free germinal epithelium and containing only Sertoli cells. Female offspring also had atrophied ovaries. Testicular tissue obtained from intact and 60Co-irradiated rats was incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, and androstenedione as a substrate. Intermediates for androgen production and catabolic metabolites were isolated after the incubation. The amounts of these metabolites produced by the irradiated testes were low in comparison with the control. The activities of delta 5-3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, C17,20-lyase, and delta 4-5 alpha-reductase in the irradiated testes were 30-40% of those in nonirradiated testes. Also, the activities of 17 beta- and 20 alpha-hydroxysteroid dehydrogenases were 72 and 52% of the control, respectively. In adrenal glands, the 21-hydroxylase activity of the irradiated animals was 38% of the control, but the delta 5-3 beta-hydroxysteroid dehydrogenase activity was comparable to that of the control. On the other hand, the activity of delta 5-3 beta-hydroxysteroid dehydrogenase of the irradiated ovary was only 19% of the control. These results suggest that 60Co irradiation of the fetus in utero markedly affects the production of steroid hormones in testes, ovaries, and adrenal glands after birth.

Absence of Linkage between Radiosensitivity and the Predisposing Atp7b Gene Mutation for Heritable Hepatitis in the LEC Rat

Abstract Ogiu, T., Nishimura, M., Watanabe, F., Ukai, H., Ishii-Ohba, H., Shimada, Y., Tsuji, H., Sakurai, J. and Hino, O. Absence of Linkage between Radiosensitivity and the Predisposing Atp7b Gene Mutation for Heritable Hepatitis in the LEC Rat. The LEC rat is known to be a mutant strain that spontaneously develops heritable hepatitis due to copper accumulation, caused by mutation of the copper-transporting ATPase gene (Atp7b). Immunodeficiency and radiosensitivity have also been observed. Hayashi et al. extensively examined the radiosensitivity of the LEC rat and concluded that its hypersensitivity is controlled by a single autosomal gene. Furthermore, they suggested the possibility that it correlates to copper accumulation due to the Atp7b gene mutation, because ionizing radiation-induced hydroxyl radicals might act in concert with copper-induced hydroxyl radicals. In the present experiment, we analyzed linkage between radiosensitivity and the mutation responsible for hepatitis in F1 animals of a cross with the F344 rat. Our results clearly demonstrated an absence of any significant association. In addition, partial dominance for radiosensitivity was observed, and radiosensitive (F1 × LEC) backcross rats were twice as numerous as their radioresistant counterparts, suggesting the possibility of control by two or more recessive genes.

Nature of nontargeted radiation effects observed during fractionated irradiation-induced thymic lymphomagenesis in mice

Changes in the thymic microenvironment lead to radiation-induced thymic lymphomagenesis, but the phenomena are not fully understood. Here we show that radiation-induced chromosomal instability and bystander effects occur in thymocytes and are involved in lymphomagenesis in C57BL/6 mice that have been irradiated four times with 1.8-Gy γ-rays. Reactive oxygen species (ROS) were generated in descendants of irradiated thymocytes during recovery from radiation-induced thymic atrophy. Concomitantly, descendants of irradiated thymocytes manifested DNA lesions as revealed by γ-H2AX foci, chromosomal instability, aneuploidy with trisomy 15 and bystander effects on chromosomal aberration induction in co-cultured ROS-sensitive mutant cells, suggesting that the delayed generation of ROS is a primary cause of these phenomena. Abolishing the bystander effect of post-irradiation thymocytes by superoxide dismutase and catalase supports ROS involvement. Chromosomal instability in thymocytes resulted in the generation of abnormal cell clones bearing trisomy 15 and aberrant karyotypes in the thymus. The emergence of thymic lymphomas from the thymocyte population containing abnormal cell clones indicated that clones with trisomy 15 and altered karyotypes were prelymphoma cells with the potential to develop into thymic lymphomas. The oncogene Notch1 was rearranged after the prelymphoma cells were established. Thus, delayed nontargeted radiation effects drive thymic lymphomagenesis through the induction of characteristic changes in intrathymic immature T cells and the generation of prelymphoma cells.

Development and molecular analysis of thymic lymphomas induced by ionizing radiation in Scid mice

Abstract The Scid mouse is well known to be immunodeficient and also radiosensitive because of point mutation in the DNA-dependent protein kinase catalytic subunits (DNA-PKcs) gene. To analyze the effects of Scid mutation on radiation carcinogenesis, Scid (Scid homozygote), C.B-17 (wild-type) and their (C.B-17×Scid) F1 hybrid (Scid heterozygote) were used. These strains of mice were first examined for acute effects, and then irradiated with 1–3 Gy gamma rays for carcinogenesis experiments. Scid mice are extremely susceptible to the induction of thymic lymphomas by ionizing radiation. Molecular analyses demonstrated that mutation of the Ras gene contributed less to the induction of thymic lymphomas, although Notch1 mutation played a role. These results suggest a close relationship between radiosensitivity and the development of thymic lymphomas in Scid mice, and correlation of reduced DNA-PK activity with an increase in the yield of deletions or insertions in oncogene(s) rather than point mutations.