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A Novel FRET-Based Biosensor for the Measurement of BCR-ABL Activity and Its Response to Drugs in Living Cells

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Title: A Novel FRET-Based Biosensor for the Measurement of BCR-ABL Activity and Its Response to Drugs in Living Cells
Authors: Mizutani, Tatsuaki Browse this author
Kondo, Takeshi Browse this author
Darmanin, Stephanie Browse this author
Tsuda, Masumi Browse this author
Tanaka, Shinya Browse this author
Tobiume, Minoru Browse this author
Asaka, Masahiro Browse this author
Ohba, Yusuke Browse this author
Keywords: chronic myeloid leukemia (CML)
fluorescence resonance energy transfer (FRET)
molecular targeted drugs
Issue Date: 1-Aug-2010
Publisher: American Association for Cancer Research
Journal Title: Clinical Cancer Research
Volume: 16
Issue: 15
Start Page: 3964
End Page: 3975
Publisher DOI: 10.1158/1078-0432.CCR-10-0548
PMID: 20670950
Abstract: Purpose: To develop a novel diagnostic method for the assessment of drug efficacy in CML patients individually, we generated a biosensor that enables the evaluation of BCR-ABL kinase activity in living cells using the principle of fluorescence resonance energy transfer (FRET). Experimental Design: To develop FRET-based biosensors, we utilized CrkL, the most characteristic substrate of BCR-ABL, and designed a protein in which CrkL is sandwiched between Venus, a variant of YFP, and ECFP, so that CrkL intra-molecular binding of the SH2 domain to phosphorylated tyrosine (Y207) increases FRET efficiency. After evaluation of the properties of this biosensor by comparison to established methods including western blotting and flow cytometry, BCR-ABL activity and its response to drugs were examined in CML patient cells. Results: After optimization, we obtained a biosensor that possesses higher sensitivity than that of established techniques with respect to measuring BCR-ABL activity and its suppression by imatinib. Thanks to its high sensitivity, this biosensor accurately gauges BCR-ABL activity in relatively small cell numbers and can also detect less than 1% minor drug-resistant populations within heterogeneous ones. We also noticed that this method enabled us to predict future onset of drug resistance as well as to monitor the disease status during imatinib therapy, using patient cells. Conclusion: In consideration of its quick and practical nature, this method is potentially a promising tool for the prediction of both current and future therapeutic responses in individual CML patients, which will be surely beneficial for both patients and clinicians.
Type: article (author version)
Appears in Collections:医学院・医学研究院 (Graduate School of Medicine / Faculty of Medicine) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 大場 雄介

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