Hypersensitivity of Ku-Deficient Cells toward the DNA Topoisomerase II Inhibitor ICRF-193 Suggests a Novel Role for Ku Antigen during the G₂ and M Phases of the Cell Cycle

ABSTRACT

Ku antigen is a heterodimer, comprised of 86- and 70-kDa subunits, which binds preferentially to free DNA ends. Ku is associated with a catalytic subunit of 450 kDa in the DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double-strand break (DSB) repair and V(D)J recombination of immunoglobulin and T-cell receptor genes. We now demonstrate that Ku86 (86-kDa subunit)-deficient Chinese hamster cell lines are hypersensitive to ICRF-193, a DNA topoisomerase II inhibitor that does not produce DSB in DNA. Mutant cells were blocked in G₂ at drug doses which had no effect on wild-type cells. Moreover, bypass of this G₂ block by caffeine revealed defective chromosome condensation in Ku86-deficient cells. The hypersensitivity of Ku86-deficient cells toward ICRF-193 was not due to impaired in vitro decatenation activity or altered levels of DNA topoisomerase IIα or -β. Rather, wild-type sensitivity was restored by transfection of a Ku86 expression plasmid into mutant cells. In contrast to cells deficient in the Ku86 subunit of DNA-PK, cells deficient in the catalytic subunit of the enzyme neither accumulated in G₂/M nor displayed defective chromosome condensation at lower doses of ICRF-193 compared to wild-type cells. Our data suggests a novel role for Ku antigen in the G₂ and M phases of the cell cycle, a role that is not related to its role in DNA-PK-dependent DNA repair.

ACKNOWLEDGMENTS

We thank A. M. Creighton for the generous gift of ICRF-193, G. F. Whitmore for V-3 cells, A. Kikuchi for monoclonal antibody 3H10 against DNA topoisomerase IIβ, and G. Chu for plasmid pBJ5-Ku86. The expertise of M. Chatenet and F. Clerc for PFGE analysis and V. Marechal for metaphase spreads was invaluable. The comments of N. Taylor and Y. Robbins on the manuscript were particularly helpful. Particular thanks go to E. Moustacchi for hospitality and advice and to U. Hibner and M. Olivier for discussion.

This work was supported by the ACC/SV8 from the Ministère de la Recherche et de l’Enseignement Supérieur and a grant from the Association pour la Recherche contre le Cancer. P.M. was supported by a postdoctoral fellowship from the Spanish Ministery and the European Community.