Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Research Institution||Kyushu University Faculty of Medicine(1993)|
KUWANO Michihiko Kyushu University Faculty of Medicine, 医学部, 教授 (80037431)
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
DANLONGO ロンゴ 米国, 国立癌研究所・フレデリック支部, 所長
DAVID Schles 米国ワシントン大学, 医学部, 教授
SCHLESSINGER デーヴイド ワシントン大学, 医学部(米国), 教授
|Project Period (FY)
1991 – 1993
Completed(Fiscal Year 1993)
|Budget Amount *help
¥8,000,000 (Direct Cost : ¥8,000,000)
Fiscal Year 1993 : ¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1992 : ¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1991 : ¥3,000,000 (Direct Cost : ¥3,000,000)
|Keywords||MDR1 / Multi drug resistance / DNA topoisomerase / Yeastartifitial chromosome / YAC / Genome analysis / Gene amplification / Gene expression / ヒト第7番染色体 / 多剤耐性 / プロモーター / 多剤耐性遺伝子ー1 / 抗癌剤耐性 / ヒトゲノム解析 / YACベクタ-|
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
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