Analyzing the influence of kinase inhibitors on DNA repair by differential proteomics of chromatin-interacting proteins and nuclear phospho-proteins

Abstract

The combination of radiotherapy and pharmacological inhibition of cellular signal transduction pathways offers promising strategies for enhanced cancer cell inactivation. However, the molecular effects of kinase inhibitors especially on DNA damage detection and repair after X-irradiation have to be understood to facilitate the development of efficient and personalized treatment regimens. Therefore, we applied differential proteomics for analyzing inhibitor-induced changes in either chromatin-bound or phosphorylated nuclear proteins. The effect of the multi kinase inhibitor sorafenib on DNA repair, chromatin binding and phosphorylation of nuclear proteins was analyzed in UT-SCC 42B head and neck cancer cells using metabolic labeling based differential proteomics (SILAC). Sorafenib significantly inhibited DNA repair but failed to significantly affect chromatin interactions of 90 quantified proteins. In contrast, analyzing nuclear phospho-proteins following sorafenib treatment, we detected quantitative changes in 9 out of 59 proteins, including DNA-repair proteins. In conclusion, the analysis of nuclear phospho-proteins by differential proteomics is an effective tool for determining the molecular effects of kinase inhibitors on X-irradiated cells. Analyzing chromatin binding might be less promising.

Mass spectrometric analysis of PTM dynamics using stable isotope labeled metabolic precursors in cell culture

“…A complete labeling of the protein pool is generally achieved after six to seven cell divisions. [45][46][47] The lysates of the light and heavy cells are combined and the proteolytic peptides are measured by MS. Due to the mass shift introduced by the stable isotope labeled amino acids, the individual peptide/protein abundance levels/ ratios can be determined by MS in the precursor mass spectra.…”

Receptor tyrosine kinase MET as potential target of multi‐kinase inhibitor and radiosensitizer sorafenib in HNSCC

“…Although not interacting with cisplatin‐mediated cell inactivation, sorafenib enhances the efficacy of X‐irradiation, leading to radiosensitization and a striking overall cell kill . Sorafenib‐mediated radiosensitization of cancer cells is probably caused by compromised DNA repair and can be observed in various other entities such as colorectal, liver, or breast cancer . It is not, however, a general phenomenon because glioblastoma cells were not sensitized .…”