1993 Fiscal Year Final Research Report Summary
Joint study on human genomic analysis of drug resistance
| Project/Area Number |
03044118
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Kyushu University Faculty of Medicine (1993)
大分医科大学 (1991-1992) |
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Principal Investigator |
KUWANO Michihiko Kyushu University Faculty of Medicine, 医学部, 教授 (80037431)
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| Co-Investigator(Kenkyū-buntansha) |
WADA Morimasa Kyushu University Faculty of Medicine, 医学部, 助手 (20220965)
ONO Mayumi Kyushu University Faculty of Medicine, 医学部, 講師 (80128347)
KOHNO Kimitoshi Oita Medical University, 医学部, 助教授 (00153479)
FOJO Antonio t. National Institute of Health, National Cancer Institute, 主任
LONGO Dan National Institute of Health, National Cancer Institute, フレデリック支部(米国), 部長
SCHLESSINGER David Washington University School of Medicine, 医学部(米国), 教授
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| Project Period (FY) |
1991 – 1993
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| Keywords | MDR1 / Multi drug resistance / DNA topoisomerase / Yeastartifitial chromosome / YAC / Genome analysis / Gene amplification / Gene expression |
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| Research Abstract |
The mechanisms of gene amplification and expression of human multidrug resistance 1(MDR1) gene have been studied during acquisition of drug resistance by using genomic DNA cloned into phase or yeast artificial chromosome(YAC)vector.
1. The promoter region of MDR1 gene was isolated from phage library to clarify the structure of transcriptional regulatory domain. Regulatory elements which respond to anticancer agents and to UV have been identified by deletion analysis. We propose that MDR1 gene is a member of the stress inducible genes and the inducibility is the underlying mechanism by which cells acquire drug resistance after chemotherapy.
2.We tried to construct a map of this region to determine the long range genomic organization of MDR region and to isolate diagnostic probe for drug resistance. Twenty YAC clones around MDR1 gene region were isolated from the total human and the chromosome 7 specific library which was constructed at Washington University. 1.5Mb contigu has been built f
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rom these clones by STS content mapping procedure. Physical map spanning 600kb including MDR1 gene was also constructed using rare cutter enzymes.
3.The YAC clone human containing MDR1 gene was transferred to mouse cell line by polyethylene glycol mediated spheroplast fusion method. Pulsed-field gel electrophoresis and PCR analysis of the fusion line showed intact structure of the YAC clone transferred. The step-wise gene amplification and expression of human MDR1 gene were observed during acquisition of step-wise resistance to vincristine, indicating functional intactness of the clone. In contrast, amplification and expression of endogenous mouse MDRI gene were not observed, suggesting an unknown mechanism which permit, selective expression of human MDR1 gene.
4.The mechanisms of drug resistance to topoisomerase targeting agents were also investigated. Decrease of level of topoisomerase II was observed in etoposide resistant cells. We also found that the topoisomerase II gene is heat inducible. The promoter region of the human topoisomerase II was isolated for further study of topoisomerase II gene expression. Less
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