Rad9, Rad1 and Hus1 are essential genes conserved from yeast to humans. They form a heterotrimer complex (9-1-1 complex) that participates in the cell cycle checkpoint activation and DNA damage repair in eukaryotic cells. Rad9, Rad1 and Hus1 deficient cells are hypersensitive to ionizing radiation and mouse cells deleted for anyone of the three genes are highly sensitive to the killing by gamma rays. We propose that ionizing radiation-induced transcription of these genes is a mechanism by which cells respond to radiation-induced damage. In this study we used quantitative real-time RT-PCR(qPCR) to analyze the mRNA levels of Rad9, Rad1 and Hus1 in various tissues isolated from mice that were either mock irradiated or exposed to 10 Gy gamma radiation. Our results indicated that the mRNA levels of Rad9, Rad1 and Hus1 genes were very different among these tissues, and we found high natural levels of mRNA in the spleen, lung, ovary and testis of mice before exposure to radiation. The mRNA levels of the three genes were well correlated across these tissues, being high, medium or low in each of the tissues simultaneously. The mRNA levels of the three genes were analyzed at 2, 6, 12, 24 and 48 h after irradiation. In most tissues Rad9 was strongly induced at 2 and 12 h time points and Hus1 was strongly induced at 2, 12 and 48 h time points, but Rad1 was minimally induced in most of the tissues with the exception of slightly higher levels in the heart and lung tissues at the 48 h time point. These results suggest that the regulation mechanisms for the mRNA levels of the three genes in response to ionizing radiation are complex and not well orchestrated. We also detected the induction of Rad9 and Hus1 proteins in the heart and liver of the animals after irradiation, and found that Rad9 protein levels were highly induced in both the heart and liver, while the Hus1 protein levels were significantly induced only in the liver, suggesting that Rad9 and Hus1 protein levels are not regulated in a coordinated manner in response to irradiation. We then went on to measure the mRNA levels of the three genes and the Rad9 and Hus1 protein levels in the mouse liver cell line (NCTC 1469) in response to irradiation in vitro. All three genes in the cultured cells were minimally induced at mRNA level, obviously different from the highly dynamic induction in liver. Rad9 and Hus1 were significantly induced at the protein level, but the induced Rad9 protein levels were higher than the Hus1 levels. Taken together, the good correlation of the mRNA levels of Rad9, Hus1 and Rad1 genes across different tissues isolated from the animals that were mock irradiated and the lack of correlation in mRNA as well as protein levels after irradiation suggest that the 9-1-1 complex has evolved to play various physiological roles in tissues rather than dealing with high doses of gamma radiation or other genotoxic agents.
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Vol. 183 • No. 1