Gene Expression Profiling Pathway Analyses Research Articles

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

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.

Gene expression profiling of breast cancer cell lines treated with proton and electron radiations.

Technological advances in radiation therapy are evolving with the use of hadrons, such as protons, indicated for tumors where conventional radiotherapy does not give significant advantages or for tumors located in sensitive regions, which need the maximum of dose-saving of the surrounding healthy tissues. The genomic response to conventional and non-conventional linear energy transfer exposure is a poor investigated topic and became an issue of radiobiological interest. The aim of this work was to analyze and compare molecular responses in term of gene expression profiles, induced by electron and proton irradiation in breast cancer cell lines. We studied the gene expression profiling differences by cDNA microarray activated in response to electron and proton irradiation with different linear energy transfer values, among three breast cell lines (the tumorigenic MCF7 and MDA-MB-231 and the non-tumorigenic MCF10A), exposed to the same sublethal dose of 9 Gy. Gene expression profiling pathway analyses showed the activation of different signaling and molecular networks in a cell line and radiation type-dependent manner. MCF10A and MDA-MB-231 cell lines were found to induce factors and pathways involved in the immunological process control. Here, we describe in a detailed way the gene expression profiling and pathways activated after electron and proton irradiation in breast cancer cells. Summarizing, although specific pathways are activated in a radiation type-dependent manner, each cell line activates overall similar molecular networks in response to both these two types of ionizing radiation. Advances in knowledge: In the era of personalized medicine and breast cancer target-directed intervention, we trust that this study could drive radiation therapy towards personalized treatments, evaluating possible combined treatments, based on the molecular characterization.