Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts.
Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10-20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy.
Marangoni
E
E
Unité Propre de l'Enseignement Supérieur Radiosensibilité-Radiocarcinogenèse humaine, Institut Gustave Roussy, Villejuif, France.
Le Romancer
M
M
Foray
N
N
Muller
C
C
Douc-Rasy
S
S
Vaganay
S
S
Abdulkarim
B
B
Barrois
M
M
Calsou
P
P
Bernier
J
J
Salles
B
B
Bourhis
J
J
D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D002588:Cesium Radioisotopes; D002999:Clone Cells / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects; D004265:DNA Helicases; D004260:DNA Repair / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism* / Q000528:radiation effects*; D005720:Gamma Rays; D018014:Gene Transfer Techniques; D006801:Humans; D007700:Kinetics; D000072200:Ku Autoantigen; D015162:Micronucleus Tests; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics* / Q000528:radiation effects; D011836:Radiation Tolerance
D034961:Antigens, Nuclear; D002588:Cesium Radioisotopes; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016372:RNA, Antisense; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen