Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Anal Bioanal Chem. 2013 Jun;405(16):5489-99. doi: 10.1007/s00216-013-6963-5. Epub 2013 Apr 27.
Kinases play a key role in cellular signaling, and the overactivation or overexpression of these kinases has been linked to a variety of cancers. Tyrosine kinase inhibitors treat the mechanism of these cancers by targeting the specific kinases that are overactive. Some patients, however, do not respond to these inhibitors or develop resistance to these inhibitors during treatment. Additionally, even within cancers of the same tissue type, different kinases may be overactive in different patients. For example, some lung cancers overexpress epidermal growth factor receptor (EGFR) and respond to EGFR inhibitors, whereas other lung cancers do not overexpress EGFR and receive no benefit from this treatment. Even among patients exhibiting EGFR overexpression, some do not respond to EGFR kinase inhibitors because other kinases, such as Met kinase, are also overactivated. Here we describe a quantitative and specific multiplexed microfluidic assay using a hydrogel immobilized substrate for measuring the kinase activity of Met and Abl kinase from cancer cells. We immobilized kinase-specific substrates on macroporous hydrogel micropillars in microchannels. These microchannels were incubated with 6 μl of a kinase reaction solution containing cancer cell lysate, and we measured kinase activity via fluorescence detection of a phosphotyrosine antibody. We showed that the assay can specifically measure the activity of both Met and Abl kinase within one microchannel and has the potential to measure the activity of as many as five kinases within one microchannel. The assay also detected Met kinase inhibition from lysates of cancer cells grown in the Met kinase inhibitor PHA665752.
激酶在细胞信号转导中发挥着关键作用，这些激酶的过度激活或过度表达与多种癌症有关。酪氨酸激酶抑制剂通过针对过度活跃的特定激酶来治疗这些癌症的机制。然而，一些患者对这些抑制剂没有反应，或者在治疗过程中对这些抑制剂产生耐药性。此外，即使在同一组织类型的癌症中，不同的激酶也可能在不同的患者中过度活跃。例如，一些肺癌过度表达表皮生长因子受体 (EGFR)，并对 EGFR 抑制剂有反应，而其他肺癌则不过度表达 EGFR，因此无法从这种治疗中获益。即使在 EGFR 过度表达的患者中，一些患者对 EGFR 激酶抑制剂没有反应，因为其他激酶，如 Met 激酶，也被过度激活。在这里，我们描述了一种使用水凝胶固定化底物的定量和特异性多重微流控分析，用于测量癌细胞中 Met 和 Abl 激酶的激酶活性。我们将激酶特异性底物固定在微通道中的大孔水凝胶微柱上。这些微通道用含有癌细胞裂解物的激酶反应溶液孵育 6 μl，我们通过荧光检测磷酸酪氨酸抗体来测量激酶活性。我们表明，该测定法可以在一个微通道中特异性地测量 Met 和 Abl 激酶的活性，并且有可能在一个微通道中测量多达五种激酶的活性。该测定法还检测到在 Met 激酶抑制剂 PHA665752 中培养的癌细胞裂解物中 Met 激酶的抑制作用。
