Authors: Jingming Bai Kunihiko Yokoyama Seigo Kinuya Kazuhiro Shiba Ryo Matsushita Masaaki Nomura Takatoshi Michigishi Norihisa Tonami
Publish Date: 2004/09/21
Volume: 31, Issue: 11, Pages: 1523-1529
Abstract
The expression level of mdr1 mRNA was analyzed by reverse transcriptionpolymerase chain reaction RTPCR Existence of the multidrug resistance MDR phenomenon was assessed via cellular uptake of 99mTcsestamibi MIBI a known substrate for Pglycoprotein A 15mer phosphorothioate antisense ODN complementary to the sequences located at −1 to 14 of mdr1 mRNA and its corresponding sense ODN were conjugated with the cyclic anhydride of diethylene triamine pentaacetic acid cDTPA via an amino group linked to the terminal phosphate at the 5′ end at pH 8–9 The DTPA–ODN complexes at concentrations of 01–174 μM were reacted with 111InCl3 at pH 5 for 1 h The hybridization affinity of labeled ODN was evaluated with sizeexclusion highperformance liquid chromatography following incubation with the complementary sequence Cellular uptake of labeled ODN was examined in vitro Furthermore enhancing effects of synthetic lipid carriers Transfast on transmembrane delivery of ODN were assessedP388/R cells displayed intense mdr1 mRNA expression in comparison with P388/S cells 99mTcMIBI uptake in P388/S cells was higher than that in P388/R cells Specific radioactivity up to 1634 MBq/nmol was achieved via elevation of added radioactivity relative to ODN molar amount The hybridization affinity of antisense 111InODN was preserved at approximately 85 irrespective of specific activity Cellular uptake of antisense 111InODN did not differ from that of sense 111InODN in either P388/S cells or P388/R cells However lipid carrier incorporation significantly increased transmembrane delivery of 111InODN moreover specific uptake of antisense 111InODN was demonstrated in P388/R cellsThis study was supported by a GrantinAid for Scientific Research B2 13470180 13877137 from the Ministry of Education Science and Culture Japan We are grateful to Dr Hui Li Associate Professor Hideto Yonekura and Professor Hiroshi Yamamoto of the Second Department of Biochemistry Kanazawa University for their technical assistance
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