WORTMANNIN INHIBITS REPAIR OF DNA DOUBLE-STRAND BREAKS IN IRRADIATED NORMAL HUMAN-CELLS

Wortmannin, a specific inhibitor of PI-3 kinase, was recently found to be an effective radiosensitizer in cells of various human and murine cell lines. Another study indicated that wortmannin inhibited repair o f DNA double-strand breaks (DSBs) in irradiated Chinese hamster ovary cells using the neutral elution assay. To further clarify the mechanis m behind radiosensitization by wortmannin, we have studied DSB repair in gamma-irradiated normal human fibroblasts using pulsed-field gel el ectrophoresis. The rejoining of DSBs in irradiated cells was significa ntly inhibited when 20 mu M or more of wortmannin was added to the cel ls. The colony formation assay in cultures treated with wortmannin sho wed that the radiosensitization occurred in a manner that was dependen t on the drug concentration. However, significant sensitization was ob served only with a concentration of wortmannin of 20 mu M or higher, r eflecting the results of DSB rejoining studies. No marked reduction in plating efficiencies was observed for cells treated with wortmannin a lone. The studies of the levels of expression of DNA-dependent protein kinase (DNA-PK) indicated that, while there were no significant chang es in expression of Ku protein, the expression of the DNA-PK catalytic subunit (DNA-PKcs) was reduced markedly in cultures treated with wort mannin using an antibody against the C-terminus region of DNA-PKcs. In addition, no reduction in the levels of expression of DNA-PKcs was ob served in cells treated with wortmannin using an antibody which recogn izes a mid-region of this large protein. These results together with t hose of related studies suggest that wortmannin radiosensitizes normal human cells by inhibiting DSB repair and that this inhibition is a co nsequence of an inactivation of kinase activity and/or a structural ch ange caused by binding of wortmannin to the C-terminus region of DNA-P Kcs. (C) 1998 by Radiation Research Soeiety.