Hong Liu ¹, Kimio Tanaka ², Nanao Kamada ³

Affiliations
1 Department of Hematology, Affiliated Hospital of Nantong Medical College, Nantong, Jiangsu 226001, CN
2 Department of Radiobiology, Institute for Environmental Sciences, Hachazawa 2-121, Takahoko, Rokkasho, Kamikita, Aomori 139-3212, JP
3 Hiroshima Atomic Bomb Relief Foundation, Kurakake, Asakita-ku, Hiroshima 739-1743, JP

Abstract

The function of PML in apoptosis was elucidated in human B lymphocyte- derived cell line. For this purpose, the number of the PML focus and the percentage of apoptotic cells were determined periodically with immunofluorescent staining after ⁶⁰Co γ-ray or γ-interferons (γ-IFN) treatment. On irradiation with ⁶⁰Co γ-ray or treatment with γ-IFN, PML protein was over-expressed. This was measured from PML foci, which peaked at 72 hr and 24 hr, respectively. The B-cell line also contained a greater proportion of apoptotic cells after the treatments. The strongest induction of apoptosis both by ⁶⁰Co- γray irradiation and by γ-IFN treatment was observed 24 hr later than the induction of PML expression. In addition, γ- rays-induced apoptosis and PML expression were mediated through caspase-8 but not through caspase-3. However, caspase-8 was involved in γ-IFN-induced PML expression and apoptosis. While caspase-3 is involved solely in PML expression, and partially in apoptosis. These results suggest that ⁶⁰Co- γ ray irradiation or γ-IFN treatment can induce PML protein expression and apoptosis in the B-cell line as caspase-3 dependently or independently.

Keywords

PML Protein, Apoptosis, ⁶⁰Co-γ Ray, γ-IFN, Caspase Activation

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Hong Liu ¹, Kimio Tanaka ², Nanao Kamada ³

Affiliations
1 Department of Developmental Biology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Minamu-ku, Hiroshima, JP
2 Department of Radiobiology, Institute for Environmental Sciences, Rokkasho , Aomori, JP
3 Hiroshima Atomic Bomb Relief Foundation, Asakita-ku, Hiroshima, JP

Abstract

PML is essential for multiple apoptotic pathways and has an altered expression in human oncogenesis. However, the role of PML in regulating cell growth and apoptosis in aplastic anemia (AA) is still unclear. To investigate the involvement of PML in the pathogenesis of AA, the expression of PML protein and apoptosis in lymphocytes was measured with immunofluorescent staining after 24 hours incubation with serum of healthy individuals (controls, n=10) and patients with severe AA (SAA, n=10). After incubation with serum from SAA patients, PML protein was overexpressed both in peripheral lymphocytes from a normal donor and in a B cell line established from cells of the same individual. The normal lymphocytes contained a greater proportion of apoptotic cells after incubation with serum from SAA patients than serum from controls, which correlated to the serum-induced expression of PML. In addition, the induction of PML expression and apoptosis in lymphocytes by the serum was partly blocked by caspase 8 inhibitor, whereas no significant difference was found before and after caspase 3 inhibitor was added. These results indicate that some components contain of the serum of SAA patients, which was confirmed to γ-IFN by us, acts on lymphocytes not only to upregulate PML protein, but also to selectively activate caspase 8, causing apoptosis. This disregulation may play an important role in the pathogenesis of lymphocytopenia in aplastic anemia.

Keywords

Aplastic Anemia, PML Protein, Apoptosis, Caspase 3, Caspase 8

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Kimio Tanaka ¹, Natarajan Gajendran ², Nanao Kamada ³

Affiliations
1 Department of Radiobiology, Institute for Environmental Sciences, Hachazawa 2-121, Takahoko, Rokkasho, Kamikita, Aomori 039-3213, JP
2 Department of Cancer Cytogenetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, JP
3 Hiroshima Atomic Bomb Relief Foundation, 50-1, 3 Chome Asakita-ku, Hiroshima 739-1743, JP

Abstract

The relative biological effectiveness (RBE) of ²⁵²Cf neutrons at different dose rates (2, 0.2&0.02 cGy/min) was assessed in human lymphocyte and bone marrow cells using cytogenetic end points (by Giemsa staining). The RBE values for 1 Gy of ²⁵²Cf neutrons for chromosomal aberration yield in lymphocyte at the dose rates of 2 and 0.02 cGy/min were 3.3 and 12.0, respectively, in comparison to that of ⁶⁰Co γ - or ¹³⁷Cs γ -rays. RBE values of Cf(n) in human bone marrow were determined for the first time in the present study, and were 6.5 for chromatid-type and 4.4 for chromosome-type aberrations at the dose rate of 2 cGy/min. These values were higher than those of lymphocytes obtained in the present study. As studied by FISH method, the exposure of ²⁵²Cf neutrons and ⁶⁰Co γ - and ¹³⁷Cs γ -rays at different dose rates also revealed the fact that the ratio of the dicentric chromosome yield to the translocation yield was influenced by LET and dose rates. No dose rate effect was found for the induction of dicentric chromosome aberrations. But an increase in the incidence of translocation was observed in the ²⁵²Cf neutrons exposure at a dose rate of 0.02 cGy/min. These results suggest that the formation of dicentrics and translocations differed with source of radiation and dose rate.

Keywords

^252Cf Neutrons, Dose Rate Effect, Fish, Translocations, Low Dose Rate, Biodosimetry, Bone Marrow, Chromosome Aberrations.

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Kimio Tanaka ¹, Mansyur Arif ², Nanao Kamada ³

Affiliations
1 Department of Radiobiology, Institute for Environmental Sciences, Hachazawa 2-121,Takahoko, Rokkasho, Kamikita, Aomori 039-3213, JP
2 Department of Cancer Cytogenetics, Research Institute for Radiation biology and Medicine, Hiroshima University, Kasumi-1-2-3, Minami-ku Hiroshima, JP
3 Atomic Bomb Survivors Relief Foundation, Kurakake, Asakita-ku, Hiroshima 739-1743, IN

Abstract

Peripheral bloods from healthy donors were irradiated by ⁶⁰Co γ-rays and californium (²⁵²Cf) -neutron for 0.5 to 4 Gy and the frequencies of the translocations were detected by two colour fluorescence in situ hybridization (FISH). Chromosomes 1, 2 and 3 and chromosomes 6, 7 and 9 were labeled by rodamine-dUTP and fluorescein-dUTP as viewing red or green signal on metaphase chromosomes, respectively. In separate observation, translocation was detected on the interphase cells by the two color FISH method using whole chromosome painting probes of chromosomes 4 and 9. Detected translocations on the metaphase and interphase cells showed clear dose response relationships. Translocations induced by ⁶⁰Co γ -rays, ²⁵²Cf neutrons, and those in atomic bomb survivors were able to detect easily by both interphase FISH and metaphase FISH using two colour of chromosome subsets. These results suggest that scoring of translocations by two colour FISH of chromosome subsets can serve as more sensitive and applicable biological dosimeter for short or long-term radiation effects.

Keywords

Interphase FISH, Biodosimetry, Atomic Bomb, Neutron, γ Rays

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Kimio Tanaka ¹, Nanao Kamada ²

Affiliations
1 Dept. of Radiobiol., Inst. for Environ. Sci., Hachazawa 2-122, Takahoko, Rokkasho, Kamikita, Aomori 039-3213, JP
2 Hiroshima Atomic Bomb Relief Foundation, 50-1, 3 Chome Asakita-ku, Hiroshima 739-1743, JP

Abstract

Based on experimental evidence using human blood samples we able to suggest that 'common fragile sites' can be targets for different radiation sources. For that we used peripheral blood samples from three healthy adult donors and exposed them to ⁶⁰Co γ-rays (2, 4 Gy) or neutrons (0.8, 2 Gy) at the G₂ stage. In another set, normal bone marrow cells from 19 adult donors were also exposed to ⁶⁰Co γ-rays, tritiated water β-rays, and ²⁵²Cf neutrons and sampled 24 hr later. Chromatid breakpoint sites were identified using Wright's stain G-banding. Analyses of 912 breakpoint sites detected in lymphocytes and 545 breakpoint sites detected in bone marrow cells were within the same bands as 113 reported: (1) 41% - 52% of chromatid breakage sites had a chromosome band of "relative fragile sites" in both bone marrow cells and lymphocytes; (2) the breakage sites induced by different radiation sources were distributed in a similar pattern; (3) significantly higher numbers of breakpoint sites were found at 4q31, 2q35, 3p21, 5q31, 1q32, 2q31, 15q24 and 13q22 in lymphocytes, and at 3p21, 14q24, 17p13, 1q42, 7q22 and 9p11/q11 in bone marrow cells; (4) distribution of breakage sites was similar between lymphocytes and bone marrow cells except for certain breakpoints. Present study also revealed that a B-cell line established from lymphocytes with a history of ⁶⁰Co γ-ray irradiation had more chromosome breakpoints at telomere regions than no-irradiated cell line, which indicating telomere protein might be associated with radiation-induced chromosome instability. This study provides information that will be useful for increasing our understanding of the mechanisms that underlie radiationinduced chromosome aberrations and will aid in assessing genetic and cancer risks in radiation-exposed human populations.

Keywords

γ-rays, Tritiated Water, β-rays, Neutrons, Fragile Sites, Chromosome Aberrations, Chromosome Instability

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Kimio Tanaka ¹, Ahmed M. Mansoor ², Nanao Kamada ³

Affiliations
1 Department of Radiobiology, Institute for Environmental Sciences, Hachazawa 2-121, Takahoko, Rokkasho, Kamikita, Aomori 039-3213, JP
2 Department of Cancer Cytogenetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima 734-8552, IN
3 Hiroshima Atomic Bomb Relief Foundation, 50-1, 3 Chome Asakita-ku, Hiroshima 739-1743, JP

Abstract

In order to assess the minimal malignant clone (MMC) for the development of leukemia, we studied the bone marrow and lymphocytes of total 44 healthy atomic - bomb survivors by cytogenetic technique. In the cytogenetic study, we compared the frequencies of chromosome aberration and quality of chromosome aberrations detected in the bone marrow and peripheral lymphocytes from 13 and 39 samples from 44 healthy atomic bomb survivors, respectively, relevant to the nonrandom aberrations prevalent in the human leukemic and malignant lymphoma cells. The frequencies of chromosome aberrations in bone marrow cells were slightly lower than those in lymphocytes and a few percent of clone were found in both bone marrow and lymphocytes. Although the completely same type chromosome aberrations as those found in leukemia and lymphomas were not observed, out of 3,846 cells with chromosome aberrations found in 52 samples for 44 survivors, 35 percent were related to the sites of oncogenes involving in the leukemogenesis and lymphomagenesis such as 8q24, 9q34 and 22q11. In addition, molecular genetic studies using PCR techniques for RAS point mutations in 3 survivors, specific chromosome translocations such as t(14;18), and t(7;14) in the bone marrow and peripheral lymphocytes of 3 healthy survivors revealed that any MMC was detected. On the other hand, by in vivo selection assay could detect transforming genes of N-and K-RAS in bone marrow samples from all 4 survivors. Three of the 4 survivors had development cancer or leukemia 7-10 years after present examination. Even though, we couldn't detect any MMC by chromosome analysis, the finding that 20 fragile sites were correlated with higher number of breakpoint of 52 atomic bomb survivors might also significantly an important role MMC in the initiation of radiation-related leukemogenesis and lymphomagenesis process, as seen presently, there is still an increase in the development

Keywords

Atomic Bomb Survivors, Chromosome Aberrations, Bone Marrow, Lymphocytes, Minimal Clone, PCR, RAS Gene

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Kimio Tanaka ¹, Hiroo Dohy ², Nanao Kamada ³

Affiliations
1 Dept. of Radiobiology, Institute for Environmental Sciences, Hachazawa-2-121, Rokkasho, Takahoko, Kamikita, Aomori-039-3213, JP
2 Hiroshima Red Cross Hospital, Senda Machi-1-9-6, Minami-ku, Hiroshima-730-8619, JP
3 Hiroshima Atomic Bomb Relief Foundation-50-1,3 Chome Asakita-ku, Hiroshima 739-1743, JP

Abstract

Extensive study on Chronic Myelocytic Leukemia (CML) was performed sequentially from the time of early chronic phase to the blastic crisis (BC) phase. A large number of 80-385 metaphases were karyotyped at the time of diagnosis in 16 patients and 13 in BC phase. Three patients were BC phase at diagnosis. All the patients had small percent of clones with additional chromosome aberration in 1.3-12.4% of the observed cells in the early stages of chronic phase. The additional abnormal clones which had existed at the time of diagnosis of early chronic phase remarkably increased in the BC phase of only 5 of 10 patients examined, because 3 of the 13 patients had no additional chromosome aberrations in BC phase. New abnormal clones which were not detected at the time of diagnosis emerged into the BC phase of 5 of the 10 patients. These additional clones were not always associated with the development of the BC. In all 3 patients diagnosed at 10.6 months before developing BC, small sized clone found in early chronic phase related to main clone in BC. Then, detection of additional clones in chronic phase for early detection of BC clone can be useful at only 10.6 months before BC. However, weak correlation between frequency of additional aberrations with Ph chromosome and duration of chronic phase was found. These findings also suggested that leukemic cells harboring Ph chromosome are high chromosome instability, in which are intrinsically labile for the acquisition of various additional chromosome aberrations at any stage, which might be related with resistance to chemotherapy, and therapies of interferon α and imatinib.

Keywords

Chronic Myelocytic Leukemia, Ph Chromosome, Clonal Evolution, Large Amount Karyotype Analysis

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Kimio Tanaka ¹, Masako Minamihisamatsu ², Taichi Kyo ³, Nanao Kamada ⁴

Affiliations
1 Department of Radiobiology, Institute for Environmental Sciences, Takahoko 2-121, Rokkasho, Kamikita, Aomori 039- 3213, JP
2 Radiation Hazards Research Group, National Institute for Radiological Sciences, Anagawa 4-9-1, Chiba 263-8555, JP
3 Fourth Department of Internal Medicine, Hiroshima Red Cross Hospital, Senda-machi, 1-9-6, Minami-ku, Hiroshima 739-1743, JP
4 Hiroshima Atomic Bomb Relief Foundation, 50-1, 3 Chome Asakita-ku, Hiroshima 739-1743, JP

Abstract

As a result of reciprocal translocation between chromosomes 8 and 21, acute myelocyticleukemia (AML) cells contains chimeric gene of AML1 and MTG8/ETO and express fusion proteins. The AML1-MTG8/ETO chimeric gene is considered to have an important role in the pathogenesis of AML FABM2. Among AML M2 patients, about 3 -5% of the patients show complex translocation including chromosome 8;21 and third chromosome. We analyzed metaphases from seven AML M2 patients with complex 8;21 translocation by two color FISH using WCP probes, AML1 probe and several cosmid probes locating near AML1 and MTG 8/ETO locus. All of the 7 patients could show two step translocation (chromosome8-chromosome 21-third chromosome). Seven patients including two insertion 8;21 cases represented two step translocation for formation either between chromosome [der(8); 8q-] and third chromosome or between [der(8); 8q-]and [der(21); 21q+ ] chromosomes . These results suggest that there is at least two step mechanism for the formation of complex 8;21 translocation, following formation of standard 8;21 translocation and AML1-MTG8/ETOchimeric gene. Interestingly, 3 patients diagnosed as AML FABM4, AML M2 transformed from myelodysplastic syndrome (MDS) (MDS-AMLM2) and acute lymphocytic leukemia (ALL) who had t(8;21) translocation had breakpoints proximal of AML1 gene. Other 13hematological disease such as AML or acute lymphocytic leukemia (ALL) patients who had chromosome abnormalities at band 21q22 of chromosome 21, including t(16;21)in 3 patients, had breakpoint at telomeric region of AML1. These results indicate that 21q22 chromosomal region has higher chromosome instability and is genetically extremely unstable.

Keywords

Complex Chromosome Translocation, Acute Myelocytic Leukemia, Chromosome Instability, AML1, FISH

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Kimio Tanaka ¹, Takahiro Shintani ², Masako Minamihisamatsu ³, Taichi Kyo ⁴, Nanao Kamada ⁵

Affiliations
1 Department of Radiobiology, Institute for Environmental Sciences, Hachazawa 2-121, Takahoko, Rokkasho, Kamikita, Aomori 019-3213, JP
2 Department of Cancer Cytogenetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi 1-2-3, Minamiki-ku, Hiroshima 734-8552, JP
3 Radiation Hazards Research Group, National Institute for Radiological Sciences, Anagawa 4-9-1, Chiba 263-8555, JP
4 Fourth Department of Internal Medicine, Hiroshima Red Cross Hospital, Senda-machi, 1-9-6, Minami-ku, Hiroshima 739-1743, JP
5 Hiroshima Atomic Bomb Relief Foundation, 50-1, 3 Chome Asakita-ku, Hiroshima 739-1743, JP

Abstract

Acute myelocytic leukemia (AML), French-American-British (FAB) classification M3 contains variable clinical disease. About 90% of the AMLM3 has specific reciprocal chromosome translocation between chromosomes15 and 17, which resulted in PML-RARα and RARα-PML chimeric genes. We analyzed 39 AMLM3 patients who showed typical clinical M3 symptoms, with simple or complex 15;17 translocation or not, by metaphase and interphase fluorescence in situ hybridization (FISH) methods using both of PML-RARα and RARα-PML chimeric probes or reverse transcriptase PCR of PML-RARα chimeric gene. Thirty-one patients reported positive for PML-RARα gene while eight patients did not. Of interest, 6 of the 31 PML-RARα positive AMLM3 patients (19.4%) did not show RARα-PML chimeric signal, which was considered to be the cases having the deletion of the region locating RARα-PML chimeric gene on der (17) chromosome either after or at the same time of formation of simple 15;17 translocation. Out of 6, 2 patients had complex 15;17 translocation of t(3;15;17) and ins(15;17). Two-way translocation model might be more acceptable to show the mechanisms for formation of complex 15;17 translocation and insertion 15;17 where insertion of a region of chromosome 17 involving RARα into PML region of chromosome 15. These precise FISH analysis also revealed that AMLM3 acquired variable chromosomal instabilities such as deletion of RARα gene and segmental jumping translocation following 15;17 translocation and the FISH analysis will be applicable for classification of AMLM3.

Keywords

15, 17 Chromosomal Translocation, Acute Myelocytic Leukemia (AML), Acute Promyelocytic Leukemia (APL), Chromosome Instability, PML, RARα, FISH

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Kimio Tanaka ¹, Natarajan Gajendran ², Hideki Asaoku ³, Taichi Kyo ³, Nanao Kamada ⁴

Affiliations
1 Department of Radiobiology, Institute for Environmental Sciences, Takahoko 2-121, Rokkasho, Kamikita, Aomori 039-3213, JP
2 Department of Plant Biology & Plant Biotechnology, Presidency College, Chennai-600 005, IN
3 Department of Internal Medicine, Hiroshima Red Cross Hospital
4 Hiroshima Atomic Bomb Relief Foundation, 50-1, 3 Chome Asakita-ku, Hiroshima 739-1743, JP

Abstract

Importance of subtelomeric chromosome rearrangements associated with idiopathic mental retardation and with methylation of gene expression in neoplastic cells has been shown. In order to observe incidence of deletions or translocations involving subtelomere region in leukemias and lymphomas, 41 patients were observed precisely by chromosome metaphase fluorescent in situ hybridization (FISH) using subtelomere probes and so on, specially focus on the end of long arm of chromosome 11 or short arm of chromosome 17. The abnormalities of subtelomere region on chromosome 11 were frequently observed in 7 of 17 patients (41.6%) with 11q22-q25 abnormalities, which were 3 of 9 patients with add(11)(q23-q25) and 3 of 7 patients with del(11)(q22-25), and 16 of 24 patients (66.0%) with 17p13 abnormalities, which were 2 of 6 with translocation between 17 and other chromosome, 6 of 10 with add(17)(p13) and all of 8 with del(17)(p13). Lymphoid disease had slightly higher abnormalities of subtelomere region than myeloid disease in patients with 11q22-25 or 17p13 abnormalities. Chemo- or radiotherapy treated patients had also subtelomeric chromosomal rearrangements. These results indicate that leukemia and lymphomas have many abnormalities at subtelomere region and possible association of chromosome instability, relating to pathogenesis of these diseases especially after therapy. Furthermore, FISH and chromosome analyses were performed on long-term cultured HL-60 leukemic cell line after irradiation using three different sources, and the results confirmed the higher induction of chromosome instability involving subtelomere region after α-rays irradiation.

Keywords

Chromosome Instability, Subtelomere, Telomere, Radiation, α-rays, γ-rays, β-rays, Leukemia Cell Line, FishAnalysis

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