Lentivirus-mediated RNA interference of Ku70 to enhance radiosensitivity of human mammary epithelial cells.
OBJECTIVE
To investigate the radiosensitising effect of Ku autoantigen 70 (Ku70) and Ku autoantigen 80 (Ku80) knockdown by lentivirus-mediated RNA interference (RNAi) in the MCF10A immortalised human mammary epithelial cell line.


METHODS
MCF10A cells were infected with lentiviral vectors for RNAi of Ku70. The Ku70-knockdown cell line (Ku70i) and a mock-infected control cell line (LVTHM) were used to perform radiation experiments. For the in vitro Micronucleus (MN) assay, both cell lines were irradiated with doses of 2 and 4 Gy (60)Co gamma-rays. For cell survival experiments, doses ranging between 0 and 8 Gy were used.


RESULTS
Western blot analysis showed that the Ku70 lentiviral vector was effective in silencing the expression of both Ku70 and Ku80. A significantly higher radiation-induced MN yield was obtained in the Ku70i cell line compared to the control LVTHM cell line. RNAi of Ku70 also resulted in a lower survival yield after irradiation compared to the control cell line. Analysis of cell death mechanisms showed that MCF10A cells (Ku70i and LVTHM) do not undergo apoptosis, but undergo post-irradiation cellular senescence.


CONCLUSIONS
RNAi of Ku70 resulted in increased chromosomal and cellular radiosensitivity in the MCF10A human mammary cell line after irradiation with (60)Co gamma-rays. These results further strengthen the role of the Ku protein in correct DNA double strand break (DSB) repair.
Department of Basic Medical Sciences, Ghent University, B-9000Gent, Belgium.
Veerle
V
Vandersickel
Monica
M
Mancini
Emanuela
E
Marras
Petra
P
Willems
Jacobus
J
Slabbert
Jan
J
Philippé
Ellen
E
Deschepper
Hubert
H
Thierens
Gianpoalo
G
Perletti
Anne
A
Vral
D034961:Antigens, Nuclear / Q000235:genetics; D017209:Apoptosis / Q000528:radiation effects; D001483:Base Sequence; D001940:Breast / Q000166:cytology / Q000276:immunology / Q000378:metabolism / Q000528:radiation effects*; D002453:Cell Cycle / Q000528:radiation effects; D002460:Cell Line; D049109:Cell Proliferation / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D016922:Cellular Senescence / Q000528:radiation effects; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D004307:Dose-Response Relationship, Radiation; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism / Q000528:radiation effects; D005260:Female; D005720:Gamma Rays; D005822:Genetic Vectors; D006801:Humans; D000072200:Ku Autoantigen; D016086:Lentivirus / Q000235:genetics; D015162:Micronucleus Tests; D034622:RNA Interference; D011836:Radiation Tolerance / Q000235:genetics* / Q000276:immunology*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen