Widespread dependence of backup NHEJ on growth state: ramifications for the use of DNA-PK inhibitors.
OBJECTIVE
The backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK-dependent pathway of NHEJ (D-NHEJ) is compromised. Our previous results show marked reduction in the activity of B-NHEJ when LIG4(-/-) mouse embryo fibroblasts (MEFs) cease to grow and enter a plateau phase. The dependence of B-NHEJ on growth state is substantially stronger than that of D-NHEJ and points to regulatory mechanisms or processing determinants that require elucidation. Because the different D-NHEJ mutants show phenotypes distinct in their details, it is necessary to characterize the dependence of their DSB repair capacity on growth state and to explore species-specific responses.


METHODS
DSB repair was measured in cells of different genetic background from various species using pulsed-field gel electrophoresis, or the formation of γ-H2AX foci, at different stages of growth.


RESULTS
Using pulsed-field gel electrophoresis, we report a marked reduction of B-NHEJ during the plateau phase of growth in KU and XRCC4, mouse or Chinese hamster, mutants. Notably, this reduction is only marginal in DNA-PKcs-deficient cells. However, reduced B-NHEJ is also observed in repair proficient, plateau-phase cells after treatment with DNA-PK inhibitors. The reduction of B-NHEJ activity in the plateau phase of growth does not derive from the reduced expression of participating proteins, is detectable by γ-H2AX foci analysis, and leads to enhanced cell killing.


CONCLUSIONS
These results further document the marked dependence on growth state of an essential DSB repair pathway and show the general nature of the effect. Molecular characterization of the mechanism underlying this response will help to optimize the administration of DNA repair inhibitors as adjuvants in radiation therapy.
Singh
Satyendra K
SK
Institute of Medical Radiation Biology, University Duisburg-Essen Medical School, Essen, Germany.
Wu
Wenqi
W
Zhang
Lihua
L
Klammer
Holger
H
Wang
Minli
M
Iliakis
George
G
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle / Q000235:genetics*; D048708:Cell Growth Processes / Q000235:genetics* / Q000528:radiation effects; D002470:Cell Survival / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors* / Q000502:physiology; D016521:Electrophoresis, Gel, Pulsed-Field; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000096:biosynthesis; D006801:Humans; D051379:Mice; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D013045:Species Specificity; D009626:Terminology as Topic; D000077191:Wortmannin
D000730:Androstadienes; C468783:H2AX protein, human; D006657:Histones; D047428:Protein Kinase Inhibitors; C475961:gamma-H2AX protein, mouse; D051747:DNA-Activated Protein Kinase; D000077191:Wortmannin