Effector genes altered in MCF-7 human breast cancer cells after exposure to fractionated ionizing radiation

Understanding the molecular mechanisms involved in the response of tumors t o fractionated exposures to ionizing radiation is important for improving r adiotherapy and/or radiochemotherapy. In the present study, we examined the expression of stress-related genes in an MCF-7 cell population (MCF-IR20) that has been derived through treatment with fractionated irradiation (2 Gy per fraction with a total dose of 40 Gy). MCF-IR20 cells showed a 1.6-fold increase in sensitization with dose at 10% isosurvival in a clonogenic ass ay, and a reduced growth delay (similar to 15 h compared to similar to 27 h ), compared to the parental MCF-7 cells treated with a single dose of 5 Gy. To determine which effector genes were altered in the MCF-IR20 cells, the expression of stress-related effector genes was measured using a filter wit h 588 genes (Clontech) that included major elements involved in cell cycle control, DNA repair, and apoptosis. Compared to MCF-7 cells that were not e xposed to fractionated radiation, 19 genes were upregulated (2.2-5.1-fold) and 4 were down-regulated (2.7-3.4-fold) in the MCF-IR20 cells. In agreemen t with the array results, 6 up-regulated genes tested by RT-PCR showed elev ated expression. Also, activities of the stress-related transcription facto rs NFKB, TP53 and AP1 showed a 1.2-4.5-fold increase after a single dose of 5 Gy in MCF-IR20 cells compared with parental MCF-7 cells. However, when t he radioresistant MCF-IR20 cell were cultured for more than 12 passages aft er fractionated irradiation (MCF-RV), radioresistance was lost, with the ra diosensitivity being the same as the parental MCF-7 cells. Interestingly, e xpression levels of CCNB1, CD9 and CDKN1A in MCF-RV cells returned to level s expressed by the parental cells, whereas the expression levels of three o ther genes, MSH2, MSH6 and RPA remained elevated. To determine if any of th e changes in gene expression could be responsible for the induced radioresi stance, CCNB1 and CDKN1A, both of which were up-regulated in MCF-IR20 cells and down-regulated in MCF-RV cells, were studied further by transfection w ith antisense oligonucleotides. Antisense of CCNB1 significantly reduced th e clonogenic survival of MCF-IR20 cells at doses of 5 and 10 Gy, from 42% t o 26% and from 5.7% to 1.0%, respectively. Antisense of CDKN1A, however, ha d no effect on radiation survival of MCF-IR20 cells. In summary, these resu lts suggest that stress-related effector genes are altered in cells after t reatment with fractionated irradiation, and that up-regulation of CCNB1 is responsible, at least in part, for radioresistance after fractionated irrad iation. (C) 2001 by Radiation Research Society.