Inhibition of B-NHEJ in plateau-phase cells is not a direct consequence of suppressed growth factor signaling.
OBJECTIVE
It has long been known that the proliferation status of a cell is a determinant of radiation response, and the available evidence implicates repair of DNA double-strand breaks (DSBs) in the underlying mechanism. Recent results have shown that a novel, highly error-prone pathway of nonhomologous end joining (NHEJ) operating as backup (B-NHEJ) processes DSBs in irradiated cells when the canonical, DNA-PK (DNA-dependent protein kinase)-dependent pathway of NHEJ (D-NHEJ) is compromised. Notably, B-NHEJ shows marked reduction in efficiency when D-NHEJ-deficient cells cease to grow and enter a plateau phase. This phenomenon is widespread and observed in cells of different species with defects in core components of D-NHEJ, with the notable exception of DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Using new, standardized serum-deprivation protocols, we re-examine the growth requirements of B-NHEJ and test the role of epidermal growth factor receptor (EGFR) signaling in its regulation.


METHODS
DSB repair was measured by pulsed-field gel electrophoresis in cells maintained under different conditions of growth.


RESULTS
Serum deprivation in D-NHEJ-deficient cells causes a rapid reduction in B-NHEJ similar to that measured in normally growing cells that enter the plateau phase of growth. Upon serum deprivation, reduction in B-NHEJ activity is evident at 4 h and reaches a plateau reflecting maximum inhibition at 12-16 h. The inhibition is reversible, and B-NHEJ quickly recovers to the levels of actively growing cells upon supply of serum to serum-deprived cells. Chemical inhibition of EGFR in proliferating cells inhibits only marginally B-NHEJ and addition of EGFR in serum-deprived cells increases only a marginally B-NHEJ.


CONCLUSIONS
The results document a rapid and fully reversible adaptation of B-NHEJ to growth activity and point to factors beyond EGFR in its regulation. They show notable differences in the regulation of error-prone DSB repair pathways between proliferating and non proliferating cells that may present new treatment design opportunities in radiation therapy.
Institute of Medical Radiation Biology, University Duisburg-Essen Medical School, Essen, Germany.
Satyendra K
SK
Singh
Theresa
T
Bednar
Lihua
L
Zhang
Wenqi
W
Wu
Emil
E
Mladenov
George
G
Iliakis
D000818:Animals; D048708:Cell Growth Processes / Q000235:genetics*; D049109:Cell Proliferation; D016895:Culture Media, Serum-Free; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000502:physiology*; D051379:Mice; D013997:Time Factors
D016895:Culture Media, Serum-Free; D066246:ErbB Receptors; D051747:DNA-Activated Protein Kinase