Radioresistance in a tumour cell line correlates with radiation inducible Ku 70/80 end-binding activity.
OBJECTIVE
The aims of the present study were to better understand the role of Ku 80, which is involved in double-strand break repair in mammalian cells in the mechanism of radiation resistance and to verify the possibility of increasing cell radiosensitivity by targeted inhibition of Ku autoantigen 80 (Ku 80).


METHODS
Western blot and electrophoretic mobility shift assay (EMSA) were performed on the human bladder carcinoma cell line RT112 (radioresistant) and on the human colorectal carcinoma cell line SW48 (radiosensitive) to assess the expression levels of DNA-dependent protein kinase (DNA-PK) components and the DNA-binding activity of the Ku 70/80 heterodimer after exposure to radiation, respectively. Ku 80 silencing was carried out with the use of small interfering RNA (siRNA).


RESULTS
Greater differences in the DNA-binding activity of Ku 70/80 and Ku 80 phosphorylation level were observed in RT112 as compared to SW48 after X-ray treatment. There is no correlation between Ku expression and DNA-binding activity at lower doses. A significant increase in nuclear Ku 80 expression was observed one hour after the exposure, only at the higher doses, while the DNA-PK catalytic subunits (DNA-PKcs) and Ku 70 levels did not change significantly. Inhibition of Ku 80 expression by siRNA induced radiosensitivity in the RT112 cell line.


CONCLUSIONS
Our data demonstrate that in a bladder tumour cell line up-regulation of Ku end-binding activity without any marked change in Ku expression underlie radiation resistance.
Negroni
Anna
A
Section of Toxicology and Biomedical Sciences, ENEA-National Agency for New Technology, Energy and Environment, Rome, Italy. Anna.negroni@casaccia.enea.it
Stronati
Laura
L
Grollino
Maria G
MG
Barattini
Paola
P
Gumiero
Daniela
D
Danesi
Donatella Tirindelli
DT
D034961:Antigens, Nuclear / Q000378:metabolism*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D006801:Humans; D000072200:Ku Autoantigen; D011485:Protein Binding / Q000528:radiation effects; D011829:Radiation Dosage; D011836:Radiation Tolerance; D013223:Statistics as Topic; D001749:Urinary Bladder Neoplasms / Q000378:metabolism*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D051747:DNA-Activated Protein Kinase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen