Abstract 3608: Measurement of kinase activity in cancer cell lines and tumor tissue using a tyrosine kinase peptide substrate array
Authors: Labots Mariette , Gotink Kristy J. , Dekker Henk , van der Mijn Johannes C. , Broxterman Henk J. , Rovithi Maria N. , Jiménez Connie R. , Verheul Henk M.W. 
Published: August 2012

Abstract
Introduction Tyrosine kinases play an important role in tumor biology. Their activity can be measured using a kinase peptide substrate array consisting of 144 Tyr residue-containing peptides (PamChip®, PamGene, Den Bosch, The Netherlands). We evaluated this platform for the measurement of kinase activity in tumor tissue and cancer cell lines under various experimental conditions. Methods Lysates of colorectal and renal cancer cell lines, HCT116 and 786-0 respectively, were made using both Mammalian and Tissue Protein Extraction Reagent (M-PER and T-PER, Thermo Scientific) and Radio-Immunoprecipitation Assay (RIPA, home made) buffer. Lysates from patient-derived tumor tissues were prepared by adding T-PER to several 10 μm cryoslides containing &gt;50% tumor. After lysate incubation with reaction buffer containing a fluorescent labeled antibody against phospo-tyrosine and ATP, kinase activity profiles were determined on kinetics of recorded peptide substrate phosphorylation intensities. The effect of protein and ATP concentration, different lysis buffers and number of freeze-thaw cycles on basal kinase activity was studied. Sunitinib, sorafenib and dasatinib, clinically available tyrosine kinase inhibitors (TKIs), were used to differentially inhibit kinase activity in the lysates. Results Application of 2.5-15 µg protein in 40 µl sample mix per array revealed linearly increasing phosphorylation signal intensities and initial velocities (Vini) of the kinetic curve (R2 = 0.98). Increasing ATP concentrations induced phosphorylation signal intensities, but above 400 µM the curve deviated from linearity. Basal kinase activity profiles of cell lines and tumor tissues were reproducible with CV's below 15%, with good signal-to-background ratios and low aspecific binding. Different lysis buffers resulted in a maximum variation of phosphorylation signal intensity of 47±5.7% in both cell lines without affecting the actual profile. Quadruple freeze-thawing of lysates did not affect signal intensities by more than 10%. Inhibition profiles of treated vs. control lysates were reproducible within and between experiments, showing a higher and differential number of inhibited peptides at increasing TKI concentrations. In contrast to the ATP-independent inhibition of dasatinib, ATP-dependent inhibition for sunitinib and sorafenib was demonstrated by combining a fixed drug concentration with increasing concentrations of ATP up to 800 µM. Conclusion Kinase activity in lysates from cancer cell lines and patient-derived tumor tissue can be reproducibly profiled with a tyrosine kinase peptide substrate array. In addition, TKIs show differential ATP-dependent inhibition profiles on this array. Taken together, we expect that array-based tumor kinase activity profiling may lead to specific TKI-phosphorylation fingerprints for personalized treatment.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3608. doi:1538-7445.AM2012-3608