Adenovirus-mediated expression of a dominant negative Ku70 fragment radiosensitizes human tumor cells under aerobic and hypoxic conditions.
Ku70 is one component of a protein complex, the Ku70/Ku80 heterodimer, which binds to DNA double-strand breaks and activates DNA-dependent protein kinase (DNA-PK), leading to DNA damage repair. Our previous work has confirmed that Ku70 is important for DNA damage repair in that Ku70 deficiency compromises the ability of cells to repair DNA double-strand breaks, increases the radiosensitivity of cells, and enhances radiation-induced apoptosis. Because of the radioresistance of some human cancers, particularly glioblastoma, we examined the use of a radio-gene therapy paradigm to sensitize cells to ionizing radiation. Based on the analysis of the structure-function of Ku70 and the crystal structure of Ku70/Ku80 heterodimer, we designed and identified a candidate dominant negative fragment involving an NH(2)-terminal deletion, and designated it as DNKu70. We generated this mutant construct, stably overexpressed it in Rat-1 cells, and showed that it has a dominant negative effect (i.e., DNKu70 overexpression results in decreased Ku-DNA end-binding activity, and increases radiosensitivity). We then constructed and generated recombinant replication-defective adenovirus, with DNKu70 controlled by the cytomegalovirus promoter, and infected human glioma U-87 MG cells and human colorectal tumor HCT-8 cells. We show that the infected cells significantly express DNKu70 and are greatly radiosensitized under both aerobic and hypoxic conditions. The functional ramification of DNKu70 was further shown in vivo: expression of DNKu70 inhibits radiation-induced DNA-PK catalytic subunit autophosphorylation and prolongs the persistence of gamma-H2AX foci. If radiation-resistant tumor cells could be sensitized by down-regulating the cellular level/activity of Ku/DNA-PK, this approach could be evaluated as an adjuvant to radiation therapy.
He
Fuqiu
F
Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Li
Ligeng
L
Kim
Dooha
D
Wen
Bixiu
B
Deng
Xuelong
X
Gutin
Philip H
PH
Ling
Clifton C
CC
Li
Gloria C
GC
D000256:Adenoviridae / Q000235:genetics; D000332:Aerobiosis; D000818:Animals; D034961:Antigens, Nuclear / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000532:radiotherapy*; D003131:Combined Modality Therapy; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015316:Genetic Therapy / Q000379:methods; D005910:Glioma / Q000235:genetics / Q000378:metabolism / Q000532:radiotherapy*; D006657:Histones / Q000096:biosynthesis; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D010766:Phosphorylation / Q000528:radiation effects; D011836:Radiation Tolerance / Q000235:genetics; D051381:Rats; D014162:Transfection
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D051747:DNA-Activated Protein Kinase; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen