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Selective Inactivation of DNA-dependent Protein Kinase with Antisense Oligodeoxynucleotides

Consequences for the Rejoining of Radiation-induced DNA Double-Strand Breaks and Radiosensitivity of Human Cancer Cell Lines¹

Department of Radiotherapy, University Essen, 45122 Essen [A. S., M. S.], and Department of Radiotherapy, Humboldt-University, Charité, 10117 Berlin [R. W., G. S., B. S., G. W., V. B.], Germany

The inhibition of DNA-dependent protein kinase activity with antisense-oligodeoxynucleotide (As-ODN) and its consequences for the rejoining of DNA-double-strand breaks (Dsbs) and radiation sensitivity was studied in human non-small cell lung cancer (NSCLC) cell lines. Cells were transfected with As-ODNs specific for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). In comparison, cells were treated with Wortmannin, a potent but nonspecific inhibitor of DNA-PK activity. As-ODN efficiently reduced the kinase activity with an IC₅₀ of about 100–200 nM and was attributed to degradation of target mRNA. In comparison, the IC₅₀ of Wortmannin was at 5–10 µM. Treatment of cells with 300 nM As-ODN increased the fraction of residual Dsb at 4 h after irradiation by a factor of 4.4, 2.6, and 1.7 in A549, H460, and H661 cells, respectively. The respective values after treatment with 20 µM Wortmannin were 5.3, 4.3, and 2.2. Inhibition of DNA-PK activity by As-ODN and Wortmannin also decreased the surviving fraction of the NSCLC cell lines. These data show that kinase activity of DNA-PKcs can be specifically inhibited with As-ODN as effective as Wortmannin and results in marked inhibition of DNA-Dsb rejoining and radiosensitization of NSCLC cell lines.

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