47. Radiation Sensitivity of 49 Cultured Human Cancer Cell Lines
Sadayuki Ban, Hitomi Sudo, Masashi Sagara, Yuichi Michikawa, Kumiko Saegusa, Yoshinobu Harada, Yoshiya Shimada, Masami Arai, Yutaka Shimada, Jhoji Inazawa and Takashi Imai
Keywords: radiation sensitivity, clonogenic assay, human cancer cells
Classification of cells with different radiosensitivities provides the base for a genome-wide search for radiation-responsible genes by using cDNA microarray technology.
A clonogenic assay was used to determine the cellular radiosensitivity of 49 cultured human cancer cell lines, of which five were established from breast cancer, 3, from pancreas cancer, 31, from esophageal cancer, 4, from cervical cancer, and 6, from colon/rectum cancer. One breast cancer cell line had a homologous mutation in BRCA11, one pancreas cancer cell line, in BRCA2 (1), 3 colon/rectum cancer cell lines, in APC and 3 colon/rectum cancer cell lines, in mismatch-repair genes. Alpha MEM supplemented with 10 % FCS was used for maintenance and subculture for all experiments. Actively growing cells were harvested and suspended in growth medium, after which cells were put into plastic test tubes and exposed to various doses of X rays with a dose rate of 1 Gy/min. Immediately after irradiation, appropriate known numbers of cells were seeded into plastic culture dishes and cultured for 10 - 14 days in 95 % air plus 5 % CO2 at 37 °C. The dose responses were analyzed using a multitarget model, S/S0 = 100[1-(1-e-D/Do)n] (2,3). Chromosomal radiosensitivity of six colon/rectum cancer cell lines was also determined with a micronucleus assay (1,4,5).
There was a wide variation in each of the three dose-survival parameters among 49 cell lines (D0 = 0.77 - 2.37 Gy, D10 = 2.06 - 7.34 Gy, Dq = 0.16 - 2.71 Gy). Dq was not correlated with D10, or D0, although there was a weak correlation between D0 and D10. These results suggested that the repair pathway in the sub-lethal dose range was different from that in the lethal dose range. The BRCA1-defective cell line had the highest radiosensitivity (D0 = 0.77 Gy, D10 = 2.06 Gy, Dq = 0.29 Gy) among cell lines tested. BRCA2-defective cells also showed unusual radiation sensitivity (D0 = 1.43 Gy, D10 = 3.92 Gy, Dq = 0.62 Gy). Cells with mutation in mismatch repair genes had a slightly higher radiosensitivity (D0 = 0.80 - 0.81 Gy, D10 = 2.23 - 4.31 Gy, Dq = 0.35 - 2.45 Gy) than APC-defective cells (D0 = 1.23 - 1.59 Gy, D10 = 5.00 - 5.86 Gy, Dq = 2.15 - 2.19 Gy), but the chromosomal radiosensitivity of APC-defective cells was higher than that of the others. For other cell lines, genes involved in the heterogeneity in radiation sensitivity remained unknown. Our cancer cell collection is characteristic of the number of esophageal cancer cell lines (31 among 49 lines) (6). One of the worst diagnoses is given to esophageal cancer, because most patients do not feel any symptoms in an early stage and they are not diagnosed till the disease has progressed to an advanced stage (7). A wide variation was also observed in each of three dose-survival parameters of esophageal cancer cell lines. D0 values were 0.96 - 2.37 Gy, D10, 2.81 - 7.34 Gy, and Dq, 0.16 - 2.71 Gy. Preliminary study demonstrated that some cell lines lost the G1- or G2/M- checkpoint, others did not.
Detection and identification of radiation-related genes in various types of cells are urgently needed to deliver radiation effectively to tumor tissues, and to minimize the side effects on normal tissues. We are planning a genome-wide search of the genes involved in heterogeneous radiation susceptibility and the cell-cycle checkpoint in various types of cultured human cells. Our studies are extremely useful to design radiation-therapy protocols for advanced cancers with heterogeneous radiation sensitivities.
Publications:
1) Ban, S., Shinohara, T., Hirai, Y., Moritaku, Y., Cologne, J.B., MacPhee, D.G.: Mutation Research 474, 15-23, 2001.
2) Ban, S., Setlow, R.B., Bender, M.A., Ezaki, H., Hiraoka, T., Yamane, M., Nishiki, M., Dohi, K., Awa, A.A., Miller, R.C., Parry, Mulvihill, D.M., J.J., Beebe, J.W.: Cancer Res. 50, 4050-4055, 1990.
3) Ban, S., Setlow, R.B., Ezaki, H., Hiraoka, T., Yamane, M., Nishiki, M., Dohi, K.: J. Radiat. Res. (Suppl.) 32, 330-338, 1991.
4) Ban, S., Cologne, J.B., Radiat. Res. 131, 60-65, 1992.
5) Ban, S., Cologne, J.B., Fujita, S., Awa, A.A.: Radiat. Res. 134, 170-178, 1993.
6) Shimada, Y., Imamura, M., Wagata, T.,Yamaguchi, N., Tobe, T.: Cancer 69, 277-284, 1992.
7) Kan, T., Shimada, Y., Sato, F., Maeda, M., Kawabe, A., Kaganoi, J., Itami, A., Yamasaki, S., Imamura, M.: Biochem. Biophys. Res. Commun. 286, 792-801, 2001.