Identification of genes whose expression is correlated to radiosensitivity as cell survival

Human cells cultured in vitro are substantially different from each other in their radiosensitivity. Although many experiments using radiosensitive mutants in lower eukaryotes indicated that an alteration in a single gene such as a repair or cell cycle maintenance caused their changing of radiosensitivity, a large number of genes may be involved in determining radiosensitivity in a human cell. To identify genes affecting radiosensitivity in human, gene expression profiles were compared among radiosensitive and radioresistant cell lines. First, a clonogenic assay was used to determine the cellular radiosensitivity of 33-cultured human cell lines derived from cancers such as lung, breast, esophagus, or brain and two lines from normal fibroblast. Estimated values of the survival parameters of X-ray dose-survival responses were determined. Range of D10 value (the dose necessary to reduce survival to 10 %) was between 2.06 and 7.34 Gy in the cells examined. Second, total RNA was extracted from the cells before irradiation and at one, or three hours after X-ray irradiation. Fluorescent complementary RNA was prepared by reverse transcription and RNA synthesis with Cy3- or Cy5-labeled nucleotides. Hybridization was performed with the custom-made oligonucleotide microarray containing 22,500 probes (representing 12,000 unique genes), which was prepared by Agilent Technologies. RNA mixture of totally 11 human tissues (Clontech) was used as a reference of hybridization to compare expression of each gene in different experiments. Finally, expression profiles were analyzed using the Resolver data analysis system (Rosetta BioSoftware). Several gene sets were constructed as follows: genes induced in the radioresistant cells but not in the sensitive cells after irradiation or genes repressed in the radioresistant cells but not in the sensitive cells after irradiation. Expression ratio and measurement accuracy in the microarray experiments were considered for selection of the gene sets. When some gene sets were used for the two-dimensional cluster analysis, the cell lines whose D10 value was more than 6 Gy were clearly separated from the cell lines whose D10 values was relatively low, less than 4 Gy. The cell lines whose D10 value ranged between 4 and 6 Gy were also roughly ordered by the gene sets with the Classification tool of the Resolver. Therefore these data may suggest that detection of the expressed genes selected by these analyses is useful to predict radiosensitive or resistant cells that were clarified by D10 value.1st European Conferen