Combined effects of X-rays and N-ethyl-N-nitrosourea in murine thymic lymphoma s -(2) Analysis in Ikaros point mutation-

Ikaros is a potent tumor suppressor gene in both human and murine leukemogenesis. We previously showed Ikaros alteration type is differed between X-ray-induced murine thymic lymphoma (TL) and N-ethyl-N-nitrosourea (ENU) -induced TL. Thus, X-ray-induced TL was characterized by altered splicing, null expression and point mutations, which were accompanied by by loss of heterozygosity (LOH). ENU-induced TL harbored only point mutation without LOH. We showed that combined treatment of X-rays (0.8-1.0 Gy x 4 times) and ENU (100, 200ppm) synergistically increased TL incidence. The aim of the curresnt study is to elucidate the underlying mechanism in the lymphomagenesis induced by combined treatment of X-rays and ENU. In this study, we analyzed the mutation status of Ikaros in the TLs induced by combined (X-rays following ENU) treatment. B6C3F1 mice (4-week-old female) were irradiated by X-rays (0.2 - 1.0 Gy a week x 4 times) and then treated by EMU (50 - 200 ppm in drinking water) from 8-week-old. The analyses of LOH on chromosome 11, expression by RT-PCR and western blotting, and mutation by sequence were performed. Ikaros splicing and null expression was observed in X-ray-induced TL was 10 and12%, ENU-induced TL was both 0%, combined-induced TL was 6 and 0%. Point mutation and LOH status were 26%(LOH:73%) in X-ray-induced TL 26%(LOH:0%) in ENU-induced TL. In combined-induced TL, that was increased to 40% (LOH: 20%), it is ENU type. Thus, X-rays plays a promoter for ENU-induced leukemogenesis in the TL induced by X-rays following ENU.第48回日本放射線影響学会／第1回アジア放射線研究会

Role of Exposure Sequence in the Combined Effect of Ionizing Radiation and N-ethyl-N-nitrosourea in Murine T-cell Lymhomagenesis

Murine T-cell lymphomas (TL) can be reproducibly induced by ionizing radiation or chemical carcinogens. This animal model is considered useful in the search for characterization of genes involved in the development of human acute lymphoblastic leukemia. We have been studying this lymphoma model, which is induced by X-rays, carbon-ions or alkylating agents such as N-ethyl-N-nitrosourea (ENU). In the study of X-ray induced thymic lymphomas in B6C3F1 mice, we found a unique locus with a high frequency of loss of heterozygosity (LOH) (50%) in the centromeric region of chromosome 11 [1]. LOH at this locus was very rarely observed in ENU-induced or spontaneously developing T-cell lymphomas, suggesting that it was a radiation-associated molecular change. We also showed that Ikaros was mapped in this frequent LOH locus, suggesting Ikaros as a tumor suppressor gene. Ikaros is a transcription factor that plays a critical role in both lineage commitment and differentiation of lymphocytes. The mutation analysis of Ikaros in the X-ray- or ENU-induced lymphomas revealed that Ikaros mutation frequency and spectrum is carcinogen dependent. Mutation frequency in X-ray-induced TL was around 50%, while that in ENU-induced TL was 20%. Inactivation of Ikaros in X-ray-induced TL was caused by transcriptional silencing, unusual splicing, point mutation and small insertion, most of which were associated with LOH [2]. LOH at Ikaros locus was generated by intra-chromosomal deletion [3]. On the other hand, Ikaros inactivation in ENU-induced TLs was caused by only point mutations not accompanied with LOH [4]. Thus, the mutation of Ikaros was distinguished between X-rays-induced TL and ENU-induced one. Recently, we showed that TL induced in mismatch repair gene Mlh1-deficient mice harbored frequent frameshift mutations in mononucleotide sequence in Ikaros, suggesting Ikaros is also a mutational target in Mlh1-dificient mice [5]. Taken together, Ikaros could be inactivated by several mechanisms. Recently our interest is focused on the effect of combined exposure of radiation and chemical carcinogens. Since we are living in the environment with numerous natural and man-made radiation and chemicals, cancer development in human is considered as a result of interaction with these factors. But, the quantitative assessment and mechanistic understanding of combined effects of radiation and chemical carcinogens are still insufficient. The aim of this study is to elucidate the mode and mechanism of the combined effect of X-rays with ENU on mouse T-cell leukemogenesis. Beginning at 4- or 8-weeks of age, female B6C3F1 mice were exposed weekly to whole body X-irradiation (0.2 ? 2.0 Gy) for consecutive 4 weeks or were given ENU (50 ? 400 ppm) in drinking water for consecutive 4 weeks. The dose-response curve for X-ray- or ENU?induced lymphoma had threshold dose. Combined exposure was carried out by X-irradiation followed by ENU or by reverse sequence ENU followed by X-irradiation. The exposure of high dose X-rays followed by ENU resulted in a synergistic effect, while the effect was antagonistic at low or threshold dose of carcinogens. Thus, combined effect is dose dependent. When the order of exposure was reversed, i.e., ENU followed by X-rays, the mode of combined exposure was additive at any doses. Molecular changes such as LOH and Ikaros mutations in TL after X-irradiation followed by ENU were similar to those in ENU-induced TL. In contrast, TL induced by reverse treatment exhibited mutations, which were similar to those found in X-ray-induced TL. It is concluded that the mode of combined effect is dependent upon the dose and the treatment order of carcinogens. \n1. Shimada Y., M. Nishimura, S. Kakinuma, M. Okumoto, T. Shiroishi, K. H. Clifton, S. Wakana: Radiation-associated loss of heterozygosity at the Znfn1a1 (Ikaros) locus on chromosome 11 in murine thymic lymphomas. Radiat Res 2000;154:293-300.?2. Kakinuma S., M. Nishimura, S. Sasanuma, K. Mita, G. Suzuki, Y. Katsura, T. Sado, Y. Shimada: Spectrum of Znfn1a1 (Ikaros) inactivation and its association with loss of heterozygosity in radiogenic T-cell lymphomas in susceptible B6C3F1 mice. Radiat Res 2002;157:331-340.?3. Yoshida M. A., A. Nakata, M. Akiyama, S. Kakinuma, T. Sado, M. Nishimura, Y. Shimada: Distinct structural abnormalities of chromosomes 11 and 12 associated with loss of heterozygosity in X-ray-induced mouse thymic lymphomas. Cancer Genet Cytogenet 2007: in press.?4. Kakinuma S., M. Nishimura, A. Kubo, J. Y. Nagai, Y. Amasaki, H. J. Majima, T. Sado, Y. Shimada: Frequent retention of heterozygosity for point mutations in p53 and Ikaros in N-ethyl-N-nitrosourea-induced mouse thymic lymphomas. Mutat Res 2005;572:132-141.?5. Kakinuma S., Y. Kodama, Y. Amasaki, S. Yi, Y. Tokairin, M. Arai, M. Nishimura, M. Monobe, S. Kojima, Y. Shimada: Ikaros is a mutational target for lymphomagenesis in Mlh1-deficient mice. Oncogene 2007;26:2945-2949.NIRS-Bfs Internatinal Workshop on Research Applications of the Radiationbiology Archive

Combined effects of X-rays and N-ethyl-N-nitrosourea on carcinogenesis and life-span-shortening in B6C3F1 mice

Radiation carcinogenesis in human is considered as a result of the combined effect of radiation and environment factors. We previously showed that smaller doses of X-rays reduced the incidence of TL induced by N-ethyl-N-nitrosourea (ENU), while large doses of X-rays increased and accelerated the TL development in a synergistic manner. This indicates the mode of combined effect is dependent upon the dose of X-rays. In this study, we aimed to determine the mode of combined effect on tumors other than TL and life-span (LS) shortening.B6C3F1 mice (4-week-old female) were irradiated with X-rays (0.2 - 2.0 Gy a week x 4 times) or treated with ENU for 4weeks (50 - 400 ppm in drinking water) from 8-week-old. For combined treatment, the mice were irradiated with X-rays (0.2 - 1.0 Gy a week x 4 times) and then treated with ENU (50 - 200 ppm).The LS after combined exposure (0.2/0.4 Gy X-rays with 200 ppm ENU) was much longer than that of ENU treatment alone, suggesting small dose of X-rays has antagonistic function. Contrary to the results of TL, incidence of tumors of lung, liver and ovaries increased more than additively after the combination of ENU with X-rays even at small doses. Thus, the role of X-rays in combined exposure was dependent not only the dose used but also the tissues examined.第48回日本放射線影響学会／第1回アジア放射線研究会

Combined effects of ionizing radiation and N-ethyl-N-nitrosourea in murine T-cell lymphomagenesis

We are living in the environment with numerous natural and man-made radiation and chemicals. Cancer development in human is considered as a result of interaction with these factors. But, the quantitative assessment and mechanistic understanding of combined effects of radiation and chemical carcinogens are still insufficient. The aim of this study is to elucidate the mode and mechanism of the combined effect of X-rays with N-ethyl-N-nitrosourea (ENU) on mouse T-cell leukemogenesis, especially at low or threshold dose range. We previously showed that the dose-response curve for X-ray- or ENU-induced thymic lymphoma (TL) had threshold dose. X-ray-induced TL was characteristic for frequent inactivation of Ikaros caused by transcriptional silencing, unusual splicing, point mutation and small insertion, most of which were associated with loss of heterozygosity (LOH). In contrast, ENU-induced tumors had point mutations of Ikaros without LOH. In combination, X-irradiation followed by ENU exposure resulted in the synergistic effect at high dose, while the effect was antagonistic at low or threshold dose. When the order of exposure was reversed, i.e., ENU followed by X-rays, the mode of combined exposure was additive at any doses. Molecular analysis demonstrated that Ikaros mutation in TL after X-irradiation followed by ENU at high doses was predominantly point mutation without LOH (ENU-type). But, after reverse treatment, mutation spectrum of Ikaros was similar to that observed in TL after X-ray exposure alone. It is concluded that the mode of combined effect is dependent upon the treatment order and the dose of carcinogens.The 6th Japan-France Worksho

Combined effects of ionizing radiation and N-ethyl-N-nitrosourea in murine thymic lymphoma

We are living in the environment with numerous natural and man-made radiation and chemicals. Cancer development in human is considered as a result of interaction with these factors. But, the quantitative assessment and mechanistic understanding of combined effects of radiation and chemical carcinogens are still insufficient. The aim of this study is to elucidate the mode and mechanism of the combined effect of X-rays with N-ethyl-N-nitrosourea (ENU) on mouse thymic lymphoma, especially at low or threshold dose range. We previously showed that the dose-response curve for X-ray- or ENU?induced thymic lymphoma had threshold dose. X-ray-induced thymic lymphoma was characteristic for frequent inactivation of Ikaros caused by transcriptional silencing, unusual splicing, point mutation and small insertion, most of which were associated with loss of heterozygosity (LOH). In contrast, ENU-induced tumors had point mutations of Ikaros without LOH. In combination, X-irradiation followed by ENU exposure resulted in the synergistic effect at high dose, while the effect was antagonistic at low or threshold dose. When the order of exposure was reversed, i.e., ENU followed by X-rays, the mode of combined exposure was additive at any doses. Molecular analysis demonstrated that Ikaros mutation in thymic lymphoma after X-irradiation followed by ENU at high doses was predominantly point mutation without LOH (ENU-type). But, after reverse treatment, mutation spectrum of Ikaros was similar to that observed in thymic lymphoma after X-ray exposure alone. It is concluded that the mode of combined effect is dependent upon the treatment order and the dose of carcinogens.13th International Congress of Radiation Researc