Taisei Nomura

Transgenerational transmission of radiation- and chemically induced tumors and congenital anomalies in mice: studies of their possible relationship to induced chromosomal and molecular changes

This article provides a broad overview of our earlier studies on the induction of tumors and congenital anomalies in the progeny of X-irradiated or chemically treated mice and our subsequent (published, hitherto unpublished and on-going) investigations aimed at identifying potential relationships between genetic changes induced in germ cells and the adverse effects manifest as tumors and congenital anomalies using cytogenetic and molecular approaches. The earlier studies document the fact that tumors and congenital anomalies can be induced by irradiation or treatment with certain chemicals such as urethane and that these phenotypes are heritable i.e., transmitted to generations beyond the first generation. These findings support the view that transmissible induced genetic changes are involved. The induced rates of congenital abnormalities and tumors are about two orders of magnitude higher than those recorded in the literature from classical mutation studies with specific locus mutations. The cytogenetic studies addressed the question of whether there were any relationships between induced translocations and induced tumors. The available data permit the inference that gross chromosomal changes may not be involved but do not exclude smaller induced genetic changes that are beyond the resolution of the techniques used in these studies. Other work on possible relationship between visible chromosomal anomalies (in bone marrow preparations) and tumors were likewise negative. However, there were indications that some induced cytogenetic changes might underlie induced congenital anomalies, i.e., trisomies, deletions and inversions were observed in induced and transmissible congenital anomalies (such as dwarfs, tail anomalies). Studies that explored possible relationships between induction of minisatellite mutations at the Pc-3 locus and tumors were negative. However, gene expression analysis of tumor (hepatoma)-susceptible offspring of progeny descended from irradiated male mice showed abnormal expression of many genes. Of these, only very few were oncogenes. This lends some support to our hypothesis that cumulative changes in gene expression of many genes, which perform normal cellular functions, may contribute to the occurrence of tumors in the offspring of irradiated or chemically treated mice.

Microsatellite instability and k-ras, p53 mutations in thyroid lymphoma.

Patho‐epidemiological studies showed that thyroid lymphoma (TL) arises in inflammatory lesions of chronic lymphocytic thyroiditis (CLTH). Replication error (RER) is found in inflammatory lesions and associated cancer, suggesting that chronic inflammation could be a risk factor for neoplastic development through causing RER. To clarify whether RER is involved in the pathogenesis of TL, we examined the microsatellite instability (MSI) in 9 cases with CLTH and 19 with TL, including 10 diffuse large B‐cell lymphoma (DLBL), 4 follicle center cell lymphoma, 3 marginal zone B‐cell lymphoma of extranodal (MALT) type, and 2 lymphoplasmacytic type. Sixteen distinct microsatellite repeats were analyzed. Mutations of p53 and k‐ras genes were also examined. When alterations at 2 or more microsatellite loci were judged as positive, only 5 DLBL cases exhibited MSI. The frequency of MSI in DLBL was significantly higher than that in other types of TL and CLTH (P < 0.05). Four of 19 cases (21.1%) showed point mutation of the k‐ras gene. The k‐ras mutations occurred in the cases with DLBL with RER, and four of five cases with RER had a k‐ras mutation, indicating a close association between RER and k‐ras mutation. p53 mutations were not found in the CLTH. Two of 19 TL cases showed mutations of p53 gene. There was no significant association between RER and p53 mutation. These findings indicate that genomic instability contributes to the progression of TL from low grade to high grade, but not to the development of low grade lymphoma in CLTH lesions.

Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following γ-radiation-induced damage to pMTa4 DNA in E. coli

Purpose: This study was undertaken to understand the roles of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of the process following exposure of E. coli to γ-radiation in vivo. Materials and methods: A plasmid DNA construct, pMTa4, was transformed into isogenic repair proficient (wild) and deficient (recF and recA) E. coli strains and γ-irradiated up to 30 Gy in vivo. The plasmid DNA was isolated under repair non-permissive (R−)and permissive (R+) conditions and analyzed by gel electrophoresis for the yields of single strand breaks (SSB) and double strand breaks (DSB) and their repair. The clonogenic survival of the E. coli was also recorded. The effects of γ-irradiation on recA reconstituted with cell free extract of wild strain or ultra-violet (UV)-irradiation were also monitored. Results: None of the strains used in this investigation showed effects of radiation-induced oxidative base damage. The dose dependent increase in SSB and DSB on pMTa4 in wild and recF mutants in R− condition were abolished upon repair incubation. The recA mutant exhibited a disturbed yield of SSB and DSB along with formation of γ-radiation-induced ‘ladder’. The ‘ladder’ was not observed after repair incubation, UV-irradiation or γ-irradiation in presence of cell-free extract of wild strain. The survival of recA mutants was seriously compromised. Conclusions: Wild, recF and recA strains of E. coli could repair γ-irradiation-induced oxidative damage to base or nucleotide (NT) in vivo. In absence of either RecA or RecF proteins, efficiency of rejoining of strand went down; RecA proteins seemed more critical than RecF in this. High fidelity or correct rejoining of strand breaks, on the other hand, seemed to require simultaneous presence of both RecA and RecF proteins.

TP53 Gene Mutation, an Unfavorable Prognostic Factor for Malignant Lymphomas in Autoimmune Diseases

Objectives: To investigate whether mutations of the TP53 tumor suppressor gene are associated with a poor prognosis in lymphoproliferative disorders (LPD) developing in patients with a history of autoimmune disease (AID). Methods: Fifty patients, 15 males and 35 females ranging in age from 23 to 83 (median, 61) years, were examined. Rheumatoid arthritis (21 cases) is the commonest type of AID followed by systemic lupus erythematosus (10), dermatomyositis (9), progressive systemic sclerosis (4), and autoimmune hemolytic anemia (6). The interval between the diagnosis of AID and LPD ranged from 1 to 660 months (mean 42 months). Histological, immunohistological, and in situ hybridization studies revealed that 37 tumors were B cell lymphomas and 13 were T cell lymphomas with the Epstein-Barr virus genome present in the tumor cells in 24% of cases. Stage of disease was I in 15 cases, II in 5, III in 9, and IV in 21. Results: Polymerase chain reaction-single strand conformation polymorphism followed by direct sequencing revealed TP53 mutations in 45.9% of B cell and 53.8% of T cell lymphomas. The follow-up study revealed an unfavorable prognosis in cases with mutations compared with those without: the 1-year survival rate was 43.5 and 73.0% in B cell and 16.7 and 50% in T cell lymphoma, respectively. Conclusions: The occurrence of a TP53 mutation is an unfavorable prognostic factor not only in B cell but also in T cell LPD in AID.

Low-Dose-Dependent Carcinogenic Potential of 2-Amino-3-Methylimidazo[4,5-f]quinoline in the Immunodeficient (SCID) Mouse Colon

The carcinogenic potential of 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), one of the most potent mutagenic heterocyclic aromatic amines in food, for the colon was assessed in mice with severe combined immunodeficiency (SCID). In Experiment I, 60 male animals, 7-8 weeks old, were administered 300, 100, or 0 ppm IQ in the diet for 20 weeks. The incidence of aberrant crypt foci (ACF), preneoplastic lesions, was 100% in the two IQ-administered groups, whereas no ACF were found in the controls. Larger lesions, at least four aberrant crypts per focus, were noted in the colons of both treated groups. Most ACF were located in the proximal colon, and the bromodeoxyuridine-labeling indexes were elevated in a dose-dependent manner, especially in this region. In Experiment II, IQ was administered in the diet at 50, 10, 2, or 0 ppm to 60 female and male 7- to 8-week-old SCID mice for 30 and 23 weeks, respectively. The incidence of ACF was dose dependent in both sexes: 100%, 100%, and 63% in the females administered 50, 10, and 2 ppm, respectively, and 100%, 83%, and 38%, respectively, in the males. Lesions of at least four aberrant crypts per focus were again evident with the 50-ppm dose. The long-term or higher dose administration of IQ in the diet might thus be applied to elucidate colon carcinogenesis in the SCID mouse.

Epstein-Barr virus associated B-cell lymphoma of brain developing in myelodysplastic syndrome with c-kit mutation (Try-557 stop)

The first case of B‐cell lymphoma of brain in a patient with myelodysplastic syndrome (MDS) was reported. A 68‐year‐old man was admitted because of anemia, fever, and thrombocytopenia and was diagnosed as having MDS (refractory anemia with excess of blasts) on the basis of the findings of bone marrow aspiration and chromosomal analysis. The patient was followed up without chemotherapy, but a brain tumor appeared after 3 years. Histologic and immunohistologic examinations revealed diffuse large B‐cell lymphoma. Mutations of the c‐kit proto‐oncogene (stem cell factor receptor) and the p53 tumor‐suppressor gene were examined in the MDS lesion and malignant lymphoma (ML) by the polymerase chain reaction‐single‐strand conformational polymorphism (PCR‐SSCP) method followed by direct sequencing. The p53 mutation was not found in either MDS or ML, but a nonsense mutation (Try‐557 → stop) in exon 11 of the c‐kit, which might lead to dysfunction of tyrosine kinase activity, was detected in MDS. This is the first report of c‐kit mutation in MDS. Epstein‐Barr virus (EBV) genome was demonstrated in the nucleus of brain ML cells by in situ hybridization with EBV‐encoded RNA‐1 probe. Immunohistochemistry showed that the tumor cells expressed latent infection gene products, including EBV nuclear antigen‐2 and latent membrane protein‐1. This pattern of latent gene expression was Lat III, which is usually found in malignant lymphomas developing in immunocompromised hosts. These findings suggest that a profound pancytopenia in MDS resulted in an immunodeficient condition, after which EBV‐positive B‐cell lymphoma of brain developed. Am. J. Hematol. 65:234–238, 2000.

Dose and dose rate effect in mutagenesis, teratogenesis and carcinogenesis

Abstract In utero exposure to X-rays, 60Co γ-rays, 252Cf neutron, and 3H water resulted in the linear increase of in vivo somatic mutation in PTHTF1 mice, while tumors were induced in the offspring after the postnatal treatment with tumor promoter. Apparent dose rate effects were observed in X-ray-induced mutation, malformation and tumor. In the young adult mice exposed to 0.4–6.8 Gy of γ-rays at the dose rates from 0.04 to 1189 mGy/min at the same age, a large and significant reduction of leukemia was observed by the low dose rate exposure, while dose rate effects were not detected in solid tumors at high doses. However, a significant reduction in the incidences of solid tumors was observed at low dose (0.4 Gy) and low dose rate (0.04 mGy/min) exposure. Double-strand break repair deficient SCID (severe combined immunodeficient) mice were extremely sensitive to radiation, and no dose rate effects were observed in γ-ray-induced mortality, malformation and leukemia. The double-strand break repair plays an essential role in homeostasis of the organism. In the normal human tissues maintained in the improved SCID mice, daily UV-light B exposure-induced mutation, keratosis and skin cancer. However, cancer has not been induced in the human thyroid gland below 60 Gy of X-rays, although p53 and c-kit mutations were detected at lower doses.

Chapter 12:Transmissible Genetic Risk Causing Tumours in Mice and Humans

Germinal exposure to radiation and chemicals may cause various adverse effects in the offspring. We conducted the first and largest experiments with ICR mice between 1967 and 1981 and these were subsequently extended to N5 and other strains of mice. The main aim of all these studies was to discover ...

Spontaneous and radiation-induced tumorigenesis in p53-deficient mice

Abstract To investigate the spontaneous and radiation induced tumorigenesis, tumor development and molecular changes were examined in p53 gene-deficient mice. All of the null (p53−/−) animals died of leukemia, sarcoma, etc. before 12 months of age, and predominantly of lymphocytic leukemia at the early age of 3–5 months, while heterozygous (p53+/−) and wild type (p53+/+) animals survived more than 30 and 40 months, respectively. Half of the heterozygotes developed sarcomas (osteosarcomas, rhabdomyosarcomas, hemangiomas), and some developed tumors in the lung and liver; however, a lower incidence of leukemia was found. The onset of the tumor was late in their lives in the heterozygotes, though the tumors grew rapidly. In the wild type mice, leukemia and sarcomas were rarely observed. The 137Cs γ-irradiation shortened the lifespan of the heterozygotes with an earlier tumor development, though this effect was not evident in the null mice. Among the 54 spontaneous tumors studied in the heterozygotes, 12 (22.2%) showed loss of the wild type p53 allele (loss of heterozygosity, LOH) and one functional mutation was found at exon 6 of the p53 gene. All the LOH was found in leukemia and sarcomas, except in one breast carcinoma.