| Co-Investigator(Kenkyū-buntansha) |
SERA Koichiro Iwate Medical University School of Medicine, Professor, 医学部, 教授 (00230855)
ISHII Keizo Tohoku University, Institue of Technology, Professor, 工学部, 教授 (00134065)
FUKUDA Mitsuhiro Takasaki Radiation Chemistry Research Establishment Japan Atomic Energy Agency, General manager, イオンビーム開発室, 室長 (60370467)
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| Research Abstract |
To study the interactions between the induction of radiation-induced apoptosis and trace elements kinetics, human leukemia cells were irradiated in vitro by ^<60>Co γ rays, after which the cells were evaluated for the detection of apoptosis and trace element (Fe, Ca, Zn) imaging was carried out. The frequency of apoptosis, i.e. the number of apoptotic bodies per 100 nuclei, was obtained by microscopic assay using TUNEL staining at 400X magnification. The trace element distribution in the cell was determined by micro-PIXE using 2 MeV proton beams. In the early phase of apoptosis, the maximum level of Fe accumulation was observed in the cell stroma. In the mid to end phase, Fe accumulation was diminished, and, instead, Ca accumulation increased and Zn decreased in the nucleus. There appear to be two steps for the development of apoptosis : 1)the signaling from cell stroma to nucleus by Fe,
or an Fe-containing enzyme ; and 2)the degeneration of the nucleus by Ca-dependent enzyme, and relea
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se of Zn from digested nucleus. As the Fe containing enzyme, cytochrome-C was considered. To confirm these, the comparison between high point of Fe by micro PIXE camera and releasing of cytochrome-C from mitochondria was performed. The high point of Fe during apoptosis agreed with releasing of chytochrom-C from mitochondria to nucleus. The inhibition of releasing of cytochrome-C from mitochondria to cytosol inhibited the high point of Fe in cytosol. As for the Ca-dependent enzyme, caspase-3-9 were considered. The accumulation of Ca in nucleus agreed with activity of caspase-3 and -9. The inhibition of caspase-3 and -9 inhibited the Ca accumulation in nucleus. Those phenomena indicated that the kinetics of Fe and Ca may be new markers fro apoptosis.
These phenomena, which was observed in VITRO, was tested IN VIVO in inoculated L1210 leukemia and L1210 radioresistant variant in BALB/c mice. Also IN VIVO, there was the high point of Fe in cytosol in the early phase of apoptosis, which agreed with releasing of cytochrome-C from mitochondoria to cytosol. The Ca accumulation in nucleus in the late phase of apoptosis agreed with the activity of caspase-3 and -9. However intravenous injection of cytochrome-C or Ca did not increase the induction of apoptosis. The one considerable reason for this, cytochrome-C and Ca was not delivered to the irradiated tumor. To solve this problem, the liquid core microcapsules, which can be deoomposed and emit its core by radiation, must be innovated and cytochrome-C and Ca must delivered by the liquid-core microcapsules.
The materials for the liquid core microcpasues was tested by the two chemicals : 1)alginate and 2)hyaluronic acid. Alginate forms the microcapsule by Ca polymerization and hyaluronic acid is denatured by radiation. If liquid core microcapsule is made by the two chemicals, it may emit its liquid core by irradiation. As a result, alginate and hyaluronic acid formed liquid core micorocapsules and emit its core by radiation. The emission of its core was mainly influenced by the rate of alginate and hyaluronic acid. The best emission was observed, when the composition of alginate and hyaluronic acid was 2 ; 1. Less
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