Laboratory of Pathophysiology and Signal Transduction, Hokkaido University Graduate School of Medicine, Kira-ku, Sapporo, Japan.
Clin Cancer Res. 2010 Aug 1;16(15):3964-75. doi: 10.1158/1078-0432.CCR-10-0548.
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).
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.
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.
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.
为了开发一种新的诊断方法来评估慢性髓性白血病（CML）患者的药物疗效，我们开发了一种生物传感器，该传感器利用荧光共振能量转移（FRET）的原理来评估活细胞中的 BCR-ABL 激酶活性。
为了开发基于 FRET 的生物传感器，我们使用了 CrkL，它是 BCR-ABL 的最典型底物，并设计了一种蛋白质，其中 CrkL 被 Venus（YFP 的一种变体）和增强型青色荧光蛋白夹在中间，因此 CrkL 分子内结合 SH2 结构域到磷酸化酪氨酸（Y207）增加了 FRET 效率。通过与包括 Western blot 和流式细胞术在内的现有方法进行比较，评估了该生物传感器的性能后，我们在 CML 患者细胞中检查了 BCR-ABL 活性及其对药物的反应。
经过优化，我们获得了一种生物传感器，与现有技术相比，该传感器在测量 BCR-ABL 活性及其对伊马替尼的抑制作用方面具有更高的灵敏度。由于其高灵敏度，该生物传感器可以准确地衡量相对较少细胞数量中的 BCR-ABL 活性，并且还可以检测到异质群体中 <1%的小耐药亚群。我们还注意到，该方法使我们能够预测未来的耐药发生，并使用患者细胞监测伊马替尼治疗期间的疾病状态。
考虑到其快速实用的性质，该方法有望成为预测个体 CML 患者当前和未来治疗反应的有前途的工具，这对患者和临床医生肯定是有益的。
