Gene Expression Profiling in HeLa Cells After Proton and X-Ray Irradiation with Cisplatin

Abstract

Our previous study demonstrated that cisplatin had more potent radiosensitizing effects and yielded a significantly higher percentage of apoptotic cell death when combined with proton beams than with X-ray irradiation. Although such results may be explained by differences in the DNA repair mechanisms, factors other than cell death may possibly be involved. In this study, we carried out the gene expression profiling in HeLa cells after proton and X-ray irradiation in combination with cisplatin. HeLa cells were provided by the RIKEN BioResource Center. We evaluated differences in the gene expression profile by cDNA microarray activated in response to proton and X-ray irradiation with or without cisplatin in HeLa. The cells were exposed to the medium containing 0 or 2.5 μmol/L cisplatin and were irradiated with single doses of 6 Gy with proton beams or X rays under the same biological conditions. One hour after irradiation, cisplatin was washed out from the medium. Each sample was cryopreserved at 2 h and 24 h after irradiation. Total RNA was further purified using a micro kit and amplified and labeled with Cyanine 3 using Agilent Low Input Quick Amp Labeling Kit (one-color) according to the manufacturer’s instructions. For each hybridization, 0.60 μg of Cyanine 3 labeled cRNA were fragmented, and hybridized at 65 °C for 17 h to an Agilent SurePrint G3 Human GE v3 8x60K Microarray. After washing, microarrays were scanned using an Agilent DNA microarray scanner. Intensity values of each scanned feature were quantified using feature extraction software. The Gene oncology (GO) analysis on microarray data was performed using a data analysis software. The data were then processed with Fisher’s exact test and multiple test correction to identify significant overrepresentation of GO annotations belonging to molecular functions. In addition, the pathway statistical analysis was performed on a pathway collection of WikiPathways database using PathVisio tool to determine pathways that contained the greatest change in expression. Gene expression profiling pathway analyses showed that the activation of different signaling and molecular networks was dependent on the radiation type, time, and presence/absence of cisplatin. In the 24-h samples irradiated with proton beams in combination with cisplatin, particularly, alterations in expression of many genes such as JAK-STAT cascade and MHC class I receptor activity were observed in GO analysis. In pathway analysis, there were significant differences in such as MAPK signaling and NRF2 between protons and X rays combined with cisplatin. In the microarray analysis, proton beam irradiation combined with cisplatin resulted in different gene expression alterations as compared with X rays and cisplatin. These differences suggested the possibility of leading to further development of irradiation methods and new therapeutic strategies using proton therapy.