A novel FRET-based biosensor for the measurement of BCR-ABL activity and its response to drugs in living cells.
OBJECTIVE
To develop a novel diagnostic method for the assessment of drug efficacy in chronic myeloid leukemia (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).


METHODS
To develop FRET-based biosensors, we used CrkL, the most characteristic substrate of BCR-ABL, and designed a protein in which CrkL is sandwiched between Venus, a variant of YFP, and enhanced cyan fluorescent protein, so that CrkL intramolecular binding of the SH2 domain to phosphorylated tyrosine (Y207) increases FRET efficiency. After evaluation of the properties of this biosensor by comparison with 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 <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.


CONCLUSIONS
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.
Mizutani
Tatsuaki
T
Laboratory of Pathophysiology and Signal Transduction, Hokkaido University Graduate School of Medicine, Kira-ku, Sapporo, Japan.
Kondo
Takeshi
T
Darmanin
Stephanie
S
Tsuda
Masumi
M
Tanaka
Shinya
S
Tobiume
Minoru
M
Asaka
Masahiro
M
Ohba
Yusuke
Y
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001549:Benzamides; D015374:Biosensing Techniques / Q000379:methods*; D015153:Blotting, Western; D002469:Cell Separation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D016044:Fusion Proteins, bcr-abl / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D000068877:Imatinib Mesylate; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis* / Q000188:drug therapy / Q000378:metabolism; D049408:Luminescent Agents; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000627:therapeutic use; D011743:Pyrimidines / Q000627:therapeutic use; D012680:Sensitivity and Specificity
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D001549:Benzamides; C082869:CRKL protein; D049408:Luminescent Agents; D009687:Nuclear Proteins; D010879:Piperazines; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl