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European Journal of Nuclear Medicine and Molecular Imaging

Erschienen in:

01.11.2004 | Molecular Imaging

In vitro detection of mdr1 mRNA in murine leukemia cells with ¹¹¹In-labeled oligonucleotide

Erschienen in: European Journal of Nuclear Medicine and Molecular Imaging | Ausgabe 11/2004

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Abstract

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 μM 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.

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Titel: In vitro detection of mdr1 mRNA in murine leukemia cells with 111In-labeled oligonucleotide
Publikationsdatum: 01.11.2004
Erschienen in: European Journal of Nuclear Medicine and Molecular Imaging / Ausgabe 11/2004
Print ISSN: 1619-7070
Elektronische ISSN: 1619-7089
DOI: https://doi.org/10.1007/s00259-004-1666-y

Springer Medizin

Abstract

Purpose

Methods

Results

Conclusion

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