Adenovirus-Mediated Expression of a Dominant Negative Ku70 Fragment Radiosensitizes Human Tumor Cells under Aerobic and Hypoxic Conditions

Abstract

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 endbinding 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 ;-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. [Cancer Res 2007;67(2):634-42]

Non-invasive molecular and functional imaging of cytosine deaminase and uracil phosphoribosyltransferase fused with red fluorescence protein

“…To measure the protein expression of CD, UPRT and mDsRed, protein samples (20 mg) from transiently transfected Cos7 cells, stably transfected R3327-CD/mDsRed and R3327-CD/UPRT/mDs Red cells were analyzed by Western blotting [20].…”

“…Cells were treated with 5-FC or 5-FU (InvivoGen, San Diego, CA) at various concentrations for 24 h. After the drug treatment, cells were trypsinized, counted, serially diluted, and plated into 60 mm Petri dishes. After incubation for 10Á14 days, colonies were fixed, stained with crystal violet, and counted [20]. Surviving fractions were normalized to the plating efficiency of untreated controls.…”

Low expression of Ku70/80, but high expression of DNA-PKcs, predict good response to radiotherapy in early breast cancer

“…Similarly, mice with mutated Ku70 also display defect DNA repair and hypersensitivity to ionising radiation (15). Furthermore, experimental studies have shown that inhibition of DNA-PK cause radiosensitisation in vitro and in vivo (16)(17)(18)(19). This is in line with the central role of the DNA-PK complex in DNA repair, and forms the basis of pharmacological inhibitors that might become useful in cancer therapy in the future.…”

Selective radiosensitization of hypoxic cells using BCCA621C: a novel hypoxia activated prodrug targeting DNA-dependent protein kinase

“…It is well established that genetic deficiency or inhibition of DNA-PK results in radiosensitivity of oxygenated cells [18,19,[36][37][38][39][40].…”