Quantitative proteomics reveals proteins associated with radiotherapy resistance in breast cancer cells
© BioMed Central Ltd 2008
Published: 13 May 2008
Resistance to radiotherapy may be a significant factor in the development of local recurrence following surgical resection and radiotherapy. We aimed to develop a novel in vitro model of radioresistance using a breast cancer cell line and to subsequently identify molecular biomarkers that may be associated with the radioresistant phenotype. We utilised a quantitative proteomics technique (iTRAQ; Applied Biosystems, Warrington, UK) based on matrix-assisted laser desorption/ionisation (MALDI) time-of-flight (TOF)/TOF mass spectrometry to identify differentially expressed proteins.
We established three novel breast cancer cell sublines that were significantly resistant to radiotherapy when compared with the parental cells. The radioresistant sublines were created by irradiating cells in fractionated doses of 2 Gy up to a total dose of 40 Gy. Sufficient time was allowed for the cells to recover between subsequent irradiations. A dose–response curve was assessed at the end of treatment to demonstrate a statistically significant increase in radioresistance for each novel cell subline when compared with parental cells. One radioresistant/parental cell pair was first analysed using in-solution digestion and liquid chromatographic separation with protein identification by MALDI-TOF/TOF (LC-MALDI analysis) on an Applied Biosystems 4800 Plus instrument (Applied Biosystems, Warrington, UK). Quantitative iTRAQ was then performed on the same instrument for all three radioresistant/parental cell pairs.
A total of 586 and 652 proteins were identified in T47D and T47DRR cells, respectively, by LC-MALDI. Those proteins identified in both cell lines and any redundant entries were removed to reveal those proteins that were unique to each cell line. In total, 244 unique proteins were identified in T47D cells and 311 unique proteins were identified in T47DRR cells. Comparison of the three pairs of radioresistant/parental cell samples by iTRAQ revealed a number of differentially expressed proteins. Using a standard ≥ 2-fold change in expression, these iTRAQ analyses revealed significant changes in the expression of 51 proteins in one or more of the radio-resistant derivatives. Further confirmation by immunoblotting is underway. Currently the decrease in expression of 26S proteasome associated subunits has been confirmed by this method.
LC-MALDI and iTRAQ analysis has revealed a large number of candidate proteins that may be associated with a radioresistant phenotype. These now require further confirmatory studies. These mass spectrometry-based techniques offer a powerful proteomic approach to identify candidate biomarkers that may be involved in radioresistance.