44. Characterization of Genes in Response to Irradiation though Systematic Transcriptome Analysis

Kumiko Saegusa, Masao Suzuki, Kennichi Ishikawa, Yoshimi Ohtsuka, Tomo Kimura, Atsuko Ishikawa and Takashi Imai

Keywords: microarray, p53, X-rays, D10 value, expression profile, clustering

To predict unusual response to clinical radiations, identifying molecular markers and understanding the variation in radio sensitivity at a molecular level are required. Microarray technology can be used for simultaneously detecting expression of thousands of genes in different cell types in a single hybridization experiment. Microarray technology has also proved to be a powerful tool for comparing gene expression in normal and disease states and/or for analyzing the response of cells exposed to drugs or unknown physiological conditions. We have studied gene expression profiles of two normal fibroblast cell lines (NB1RGB, HFLIII) and 13 cancer cell lines (A549, C32TG, Marcus, U-251MG (KO), SK-MG-1, KNS89, KS-1, A-172, KNS-76, KNS-60, Becker, T98G, SF126) with different radio sensitivities using an oligonucleotide microarray before/after exposure to ionizing radiation.

All cell lines were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum in a 5% C02 incubator at 37°C. The plating efficiencies of cell lines used varied from 20% to 90% in each cell line. Total RNA was purified from the cells at zero, one or three hours after X-ray irradiation. Fluorescent complementary RNA was prepared by reverse transcription and in vitro RNA synthesis with Cy3- or Cy5- labeled nucleotides. Hybridization was performed with the custom-made oligonucleotide microarray containing 22,500 probes (representing 14,000 unique genes). Cells were irradiated with X-rays at room temperature. The dose rate was 0.87Gy/min. We performed mutation analysis spanning p53 exones 5-8 in 15 cell lines by Denaturing High Pressure Liquid Chromatography (DHPLC) using a Wave Fragment Analysis System (Transgenomics). Expression profiles were analyzed using a custom-made oligonucleotide microarray (Agilent Technologies, CA). After hybridization, slides were washed and scanned using a confocal laser scammer (Agilent Technologies, CA). Gene expression analysis was performed using RESOLVER (Rosetta Inpharmatics, WA.)

We compared gene expression profiles of 15 cell lines whose radio sensitivities were quite different. The two-dimensional cluster analysis indicated that the radio-resistant cell lines such as Becker, KNS60, T98G and A549, were clearly separated from the radiosensitive ones. The genes classified by this analysis may be useful to predict radio-resistant cells that were clarified by D10 value.

It has been well documented that p53 is activated in response to DNA-damaging agents including ionizing radiations. We, therefore, examined the relationships between radiation sensitivity and p53 status in the cell lines and found that the D10 values were not correlated with the p53 status. The two-dimensional cluster analysis also indicated that there were at least two groups of genes for classifying the cell lines by D10 values. One group of the genes was useful for clustering the cell lines with wild-type p53 while the other was advantageous for clustering the cell lines with p53 mutants. These results suggest that p53 plays a critical role in the radiation-induced signal transduction in both radiosensitive and resistant cells and that the cells may contain a p53 independent-pathway for determining radiation sensitivity.

Publications:
1) Suzuki M, Kase Y, Yamaguchi H, et al. Relative biological effectiveness for cell-killing effect on various human cell lines irradiated with heavy-ion medical accelerator in Chiba (HIMAC) carbon-ion beams. Int. J. Radiation Oncology Biol. Phys, 2000; 48: 241-250
2) Suzuki M, Kase Y, Kanai T, and Ando K. Correlation between cell killing and residual chromatin breaks measured by PCC in six human cell lines irradiated with different radiation types. Int. J. Radiat. Biol, 2000; 76: 1189-1196