| Co-Investigator(Kenkyū-buntansha) |
KITAMURA Ryuji Hokkaido University, Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学部, 助手 (40221212)
ITOH Susumu Hokkaido University, Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学部, 助手 (70223154)
YOKOI Tsuyoshi Hokkaido University, Faculty of Pharmaceutical Sciences, Associated Professor, 薬学部, 助教授 (70135226)
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| Research Abstract |
1.A pM56 expression vector, which was ligated with P-450IIIA7 cDNA downstream of SRalpha promoter, was constructed. The pM56 was introduced into mammary tumor cell line, MCF-7, two transformants, M21 and M27 were obtained. We examined whether or not these transformants were capable of activating aflatoxin B_1, and lead to the cell death. The 50% lethal dose of aflatoxin B_1 for MCF-7 was 17.0 mug/ml, while that for M21 and M27 was 1.4 mug/ml. This result indicated that M21 and M27 became thirtween times more sensitive to aflatoxin B_1 than MCF-7. It was suggested that P-450IIIA7 protein expressed in M21 and M27 activated the the micotoxin to result in the cell death.
2.We tried to express P-450lllA7 protein in an insect cell to obtain a larger amounts of P-450IIIA7 protein. The construction procedure was as follows ; P-450IIIA7 cDNA was inserted to the polyhedorin gene of nuclear polyhedorosis virus (NPV), and recombinant Virus (NPVHP1) was prepared. The insect cell (Sf9 cell) was infected with the NPVHP1. Then, the whole cell extracts from the infected Sf9 cells were prepared. One miligram of the whole extracts contained 1.10 nmol P-450IIIA7 protein. The umu test using the cell lysates showed that P-450IIA7 expressed in Sf9 cells converted aflatoxin B_1 to a mutagen.
3.P-450IIIA4 (adult form) and P-450IIIA7 (fetal form) genes were isolated from human genomic library, and the sequences analyzed. Both genes had 13 exons over 20kb. Moreover, 5'flanking sequences between both genes had high similarity (91%).
4.We isolated cDNAs of mouse P-450IIIA, which were termed as P-450IIIA_<M1> (P-450IIIA11) and P-450IIIA_<M2>, from liver cDNA library of ddY strain mouse. P-450IIIA_<M1> (P-450IIIA11) and P-450IIIA_<M2>, from liver cDNA library of ddY strain mouse. P-450IIIA_<M1> was constitutively expressed in mouse livers and induced by dexamethasone, while P-450IIIA_<M2> was expressed at a very low level.
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