1996 Fiscal Year Final Research Report Summary
The Analysis of Human MDR Gene Family Using YAC Clones.
| Project/Area Number |
06044185
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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 | University of Occupational and Environmental Health (1996)
Kyushu University (1994-1995) |
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Principal Investigator |
KOHNO Kimitoshi School of Medicine University of Occupational and Environmental Health, Japan, Professor, 医学部, 教授 (00153479)
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| Co-Investigator(Kenkyū-buntansha) |
KUWANO Michihiko Faculty of Medicine Kyushu University, Professor, 医学部, 教授 (80037431)
ONO Mayumi Faculty of Medicine Kyushu University, Assistant Professor, 医学部, 講師 (80128347)
WADA Morimasa Faculty of Medicine Kyushu University, Associate Professor, 医学部, 助教授 (20220965)
COLE Susan P.C Queen's University, Professor of Oncology, 教授
DEELEY Roger Cancer Research Labs.Stauffer Research Professor, Queen's University, Director, 癌研究所, 所長
FOJI Antonio Medicine Branch, National Cancer Institute, Senior Investigator, 主任
GREEN Eric National Center for Human Genome Research National Institute of Health, Cheif, 主任
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| Project Period (FY) |
1994 – 1996
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| Keywords | multidrug resistance / P-glycoproteins / chromosome 7 / YAC / amplification / YB-1 / ABCsuperfamily / MOAT / MRP |
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| Research Abstract |
The acquistion of multidrug resistance (MDR) in vitro is commonly associated with the increased expression of the P-glycoproteins which are encoded by small families of the linked genes, located on human chromosome 7. One route to analyzes the function and regulation of the entire large DNA segment could be transferred into other mammalian cells. Transfer of loci this large has been accomplished using yeast antificial chromosome (YAC)-mediated transfection. We have isolated YAC clones containing the MDR genes after screening a YAC library prepared from total human DNA using primer pairs of the MDR1 promoter region as a probe. We have also completed the 1.5Mb YAC contig map around MDR loci. We introduced a modified YAC clone containing the entire human MDR locus into mouse cells and established a series of bincristine-resistant cell lines. We showed the functional expression of human MDR genes. We also showed the amplification unit in MDR cell lines using STS.
We cloned the transacting factor, which bind to the inverted CCAATbox located at the MDR1 promoter region. This clone was idenfical to the Y-box binding protein (YB-1). We generated the specific antibodies and examined the expression in various drug resistant cell lines. YB-1 was overexpressed in all cisplatin-resistant cell lines, which we had established. Thus, YB-1 may protect cells from the cytotoxic effects of agents that induce cross-linking of DNA.We also isolated the genomic clones for YB-1, and identified the chromosomal locus on chromosome 1p34. We isolated the cDNA of a new ATP binding cassette superfamily. A human clone is homologous to rat canalicular multispecific organic anion transporter (cMOAT). MOAT gene is located on chromosome 10q24. We analyzed the multidrug resistance associated protein (MRP) in epipodophyllotoxins resistant cell lines and found that selection for resistance to epipodophyllotoxins preferentially induces overexpression of MRP gene.
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