Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis
Abstract
:1. Introduction
2. Results
2.1. Overall Phosphoproteomic Analysis Reveals Thousands of Phosphorylation Sites
2.2. Combination of Ionizing Radiation and VE-821 Treatment Abrogated Phosphorylation of Checkpoint Kinase-1 on Ser345
2.3. Most of the Regulated Phosphoproteins Were Localized in the Nucleus and Related to Mitosis, Cell Cycle Regulation, DNA Damage Response and Gene Expression
2.4. The Pathways Affected by Co-Treatment Were Involved in Cell Cycle Progression, Stimulus-Based Changes in Gene Expression, DDR and Apoptosis
2.5. Significantly Down-Regulated Phosphorylation Sites Comprised SP/TP Motifs, while SQ/TQ Were amongst Up-Regulated Ones
2.6. Kinase Activity Analyses Predicts Various Kinase Substrates to Be Regulated
3. Discussion
4. Experimental Section
4.1. Cell Culture and Culture Conditions
4.2. Gamma-Ray Irradiation
4.3. Cell Lysis and Protein Digest
4.4. Electrophoresis and Western Blotting
4.5. Hydrophilic Interaction Liquid Chromatography Fractionation and Enrichment of Phosphopeptides
4.6. Mass Spectrometric Analysis
4.7. Data Processing
4.8. Bioinformatic Analysis
5. Conclusions
Supplementary Files
Supplementary File 1Acknowledgments
Author Contributions
Conflicts of Interest
References
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Šalovská, B.; Fabrik, I.; Ďurišová, K.; Link, M.; Vávrová, J.; Řezáčová, M.; Tichý, A. Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis. Int. J. Mol. Sci. 2014, 15, 12007-12026. https://doi.org/10.3390/ijms150712007
Šalovská B, Fabrik I, Ďurišová K, Link M, Vávrová J, Řezáčová M, Tichý A. Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis. International Journal of Molecular Sciences. 2014; 15(7):12007-12026. https://doi.org/10.3390/ijms150712007
Chicago/Turabian StyleŠalovská, Barbora, Ivo Fabrik, Kamila Ďurišová, Marek Link, Jiřina Vávrová, Martina Řezáčová, and Aleš Tichý. 2014. "Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis" International Journal of Molecular Sciences 15, no. 7: 12007-12026. https://doi.org/10.3390/ijms150712007