Abstract: Objective  To investigate the molecular mechanism of radioresistance acquisition after long-term fractionated radiation （FR） in human colon cancer cell line SW480. Methods  Parental SW480 cells and radioresistant subline SW480-R cells were irradiated with single doses of 0，2，5 or 10 Gy.CCND1 mRNA levels were measured using real-time fluorescent quantitative polymerase chain reaction（PCR）.Western blotting was used to measure levels of proteins involved in the DNA-PK/AKT/GSK3β pathway of SW480 cells and SW480-R cells，including cyclin D1，CDK4，Rb，p-Rb-Sebr795，AKT，p-AKT-Ser473，GSK3β， p-GSK3β-Ser9，DNA-PKcs and p-DNA-PKcs. Results  CCND1 mRNA levels were significantly lower in the radioresistant subline SW480-R than in parental SW480 cells after single irradiation doses （P<0.05），with relative expression being 0.31±0.02 in the resistant subline compared to the parental line after 0 Gy，0.32±0.03 after 2 Gy，0.34±0.05 after 5 Gy and 0.44±0.04 after 10 Gy.Conversely levels of all proteins examined were higher in SW480-R cells than in SW480 cells（P<0.05） except the level of GSK3β， which was lower in SW480-R cells（P<0.05）. Conclusions  Acquired radioresistance in the human colon cancer cell line was correlated with alterations in the DNA damage response；these alterations were activated by long-term FR and mediated by cyclin D1 overexpression in the DNA-PK/AKT/GSK3β pathway.

Key words: Colon neoplasms, Cyclin D1, Fractionated-radiation；Radioresistance, Real-time fluorescent quantitative polymerase chain reaction, Western blot