Abstract
The cellular response to genotoxic stress is a complex cascade of events including altered protein expression, interactions, modifications, and relocalization, leading to cell cycle arrest and DNA repair or to apoptosis. p53 protein has a central role in this process, and p53 status is an important factor in the response of a tumor to genotoxic anticancer therapy. We studied p53-related changes postexposure to ionizing radiation using top-down mass spectrometry. Initially two cell lines were compared, HCT116 p53 wild type (wt) and p53(-/-), in a time course study postirradiation. In the p53 wt cell line a striking increase of a 10.2-kDa protein was detected, and this protein was identified with MS/MS analysis as S100A6. Further MS profiling led to detection of two post-translationally modified variants of S100A6, namely glutathionylated and cysteinylated forms. In p53 wt cells, a specific shift from glutathionylated to cysteinylated S100A6 occurred postirradiation. The p53 dependence of this specific change in protein level and modification pattern of S100A6 postirradiation was confirmed in a panel of four lung cancer cell lines (H23, U1810, H69, and A549) with different p53 status and using small interfering RNA against p53. Interestingly the closely related S100 family protein S100A4 showed the same changes in modification pattern post-ionizing radiation in the p53 wt lung cancer cell line, and S100A4 also showed p53-dependent expression. Using confocal microscopy, relocalization of S100A6 from nucleus to cytosol and a colocalization with tropomyosin in stress fibers was detected in A549 cells postirradiation. This relocalization coincided with the change in S100A6 modification pattern. Based on these results, we suggest that S100A6 and S100A4 are regulated via redox modifications in vivo and that these proteins are involved in the cellular response to genotoxic stress.
Highlights
The cellular response to genotoxic stress is a complex cascade of events including altered protein expression, interactions, modifications, and relocalization, leading to cell cycle arrest and DNA repair or to apoptosis. p53 protein has a central role in this process, and p53 status is an important factor in the response of a tumor to genotoxic anticancer therapy
We used a proteomics approach to further elucidate the complex cellular signaling induced by genotoxic stress
We report here an increase in S100A6 protein level in colon and lung cancer cells postexposure to ionizing radiation
Summary
Cell Culturing, Irradiation, and Protein Extraction—Colon cancer cell lines HCT116 wt and HCT116 p53Ϫ/Ϫ cells [6] were cultured in McCoy’s 5A medium with L-glutamine, 10% calf serum, and 1% penicillin/streptomycin. The protein of interest was located using screening of collected fractions with SELDI-TOF-MS NP20 ProteinChip arrays (normal phase). To improve the sensitivity of analysis, more time-consuming preprocessing parameters were used for peak list generation ( each spectrum was processed using the MaxEnt algorithm of the software) This search resulted in a single significant protein identification, the S100A6 protein (AC: P06703/GI: 116509). Immunocapture Experiments—Anti-S100A6 antibody (Sigma-Aldrich) coupled to an RS100 ProteinChip array (BioRad) was used to capture S100A6 protein from HCT116 cell extract according to the standard protocol recommended by the manufacturer. RS100 ProteinChip arrays were analyzed, and spectra were collected in the 0 –150-kDa range using SELDI-TOF Protein Biology System IIC. The protocol for immunoprecipitation was followed, and the eluate was analyzed using SELDI-TOF-MS and NP20 ProteinChip arrays (normal phase).
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