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A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model
Published on: October 14, 2016
In vitro detection of mdr1 mRNA in murine leukemia cells with 111In-labeled oligonucleotide
Jingming Bai1, Kunihiko Yokoyama, Seigo Kinuya
1Department of Biotracer Medicine (Nuclear Medicine), Kanazawa University Graduate School of Medical Sciences, 13-1 Takara-machi, Kanazawa, 920-8640 Japan. bjingming@nmd.m.kanazawa-u.ac.jp
Purpose:
The feasibility of intracellular mdr1 mRNA expression detection with radiolabeled antisense oligonucleotide (ODN) was investigated in the murine leukemia cell line, P388/S, and its subclonal, adriamycin-resistant cell line, P388/R.
Methods:
The expression level of mdr1 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Existence of the multidrug resistance (MDR) phenomenon was assessed via cellular uptake of 99mTc-sestamibi (MIBI), a known substrate for P-glycoprotein. A 15-mer 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 penta-acetic 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 0.1-17.4 microM were reacted with 111InCl3 at pH 5 for 1 h. The hybridization affinity of labeled ODN was evaluated with size-exclusion high-performance 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 assessed.
Results:
P388/R cells displayed intense mdr1 mRNA expression in comparison with P388/S cells. 99mTc-MIBI uptake in P388/S cells was higher than that in P388/R cells. Specific radioactivity up to 1,634 MBq/nmol was achieved via elevation of added radioactivity relative to ODN molar amount. The hybridization affinity of antisense 111In-ODN was preserved at approximately 85% irrespective of specific activity. Cellular uptake of antisense 111In-ODN did not differ from that of sense 111In-ODN in either P388/S cells or P388/R cells. However, lipid carrier incorporation significantly increased transmembrane delivery of 111In-ODN; moreover, specific uptake of antisense 111In-ODN was demonstrated in P388/R cells.
Conclusion:
Radiolabeling of ODN at high specific radioactivity and specific uptake of antisense 111In-ODN in drug-resistant cells may facilitate future gene imaging of mdr1 mRNA.
