SILAC and 2D-DIGE Employed in Analysis of Irradiated Endothelial Cells

High doses of ionizing radiation, such as those used in the treatment of breast cancer, can cause endothelial damage. This damage can increase the risk of developing cardiac disease.^1,2 Sriharshan et al.³ investigated the damage of ionizing radiation to the endothelium using two complementary proteomic approaches, SILAC (stable isotope labeling with amino acids in cell culture) and 2D-DIGE (two-dimensional difference-in-gel electrophoresis).

In their publication, Sriharshan et al.³ investigated the effects of radiation in the cell line EA.hy926, which was derived from the PEG-mediated fusion of a thioguanine-resistant clone of human alveolar type II-like epithelial (A549) cells with primary human umbilical vein endothelial cells (HUVECs). Cells were cultured and divided into two parts for SILAC. One portion of cells was grown in SILAC DMEM (Thermo Scientific) supplemented with 0.1 mg/ml heavy ¹³C₆ L-lysine-2HCl, ¹³C₆, and ¹⁵N⁴ L-arginine, while the light labeled cells were supplemented with ¹²C₆ L-lysine-2HCl, ¹²C₆, ¹⁴N₄ L-arginine-HCl, and 10% dialyzed FBS (Thermo Scientific). Cells were exposed to 2.5 Gy gamma radiation and analyzed at 4 and 24 hours.

Harvested cells were lysed and combined together for separation via SDS-PAGE followed by digestion with trypsin. Proteins were separated using liquid phase chromatography and identified using a linear quadrupole ion-trap LTQ Orbitrap LQ mass spectrometer (Thermo Scientific). Proteins were quantified by MALDI-TOF/TOF mass spectrometry. Spectra were analyzed using MaxQuant software 10 (version 1.0.13.13) in combination with Mascot 2.3.02 and the human-specific IPI database (version 3.52, July 29, 2009). A 1% false discovery rate was established.

For 2D-DIGE, cells were harvested by trypsinization 24 hours after irradiation. Cells were lysed in buffer and cleaned and labeled with CyDye DIGE Fluor minimal dyes (300 pmol/50 μg; GE Healthcare). The irradiated samples were labeled with Cy5, the sham-irradiated samples with Cy3, and the internal standard (a 1:1 mixture of both protein extracts) with Cy2.

Results of these experiments revealed that 136 proteins were found to be significantly deregulated (SILAC, 122; 2D-DIGE, 18). The SILAC method was more able to detect deregulated proteins, while only 2D-DIGE could detect protein isoforms, fragments, and modified proteins.

The deregulated proteins were mainly categorized in four key pathways, including glycolysis/gluconeogenesis and synthesis/degradation of ketone bodies, oxidative phosphorylation, Rho-mediated cell motility, and nonhomologous end joining. The Sriharshan group suggests that the changes in these pathways come as a result of the stress caused by irradiation leading to radiation-induced cardio- and cerebrovascular damage. Future directions from this research group involve investigating the effects of ionizing radiation on mouse models and human heart endothelial cells.

References

1. Tukenova, M., et al. (2010) ‘Role of cancer treatment in long-term overall and cardiovascular mortality after childhood cancer‘, Journal of Clinical Oncology, 28 (8), (pp. 1308–1315)

2. Darby, S.C., et al. (2005) ‘Long-term mortality from heart disease and lung cancer after radiotherapy for early breast cancer: prospective cohort study of about 300,000 women in US SEER cancer registries‘, Lancet Oncology, 6 (8), (pp. 557–565)

3. Sriharshan, A., et al. (2012) ‘Proteomic analysis by SILAC and 2D-DIGE reveals radiation-induced endothelial response: four key pathways‘, Journal of Proteomics, 75 (8), (pp. 2319–2330)