DNA-PKcs deficiency leads to persistence of oxidatively induced clustered DNA lesions in human tumor cells.
DNA-dependent protein kinase (DNA-PK) is a key non-homologous-end-joining (NHEJ) nuclear serine/threonine protein kinase involved in various DNA metabolic and damage signaling pathways contributing to the maintenance of genomic stability and prevention of cancer. To examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three models of DNA-PK deficiency, i.e., chemical inactivation of its kinase activity by the novel inhibitors IC86621 and NU7026, knockdown and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). A compromised DNA-PK repair pathway led to the accumulation of clustered DNA lesions induced by gamma-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidatively induced non-DSB clustered DNA lesions measured using a modified version of pulsed-field gel electrophoresis or single-cell gel electrophoresis (comet assay). In all cases, DNA-PK inactivation led to a higher level of lesion persistence even after 24-72h of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters first by compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions owing to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important for understanding not only cancer etiology in the presence of an NHEJ deficiency but also cancer treatments based on the induction of oxidative stress and inhibition of cluster repair.
Peddi
Prakash
P
Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA.
Loftin
Charles W
CW
Dickey
Jennifer S
JS
Hair
Jessica M
JM
Burns
Kara J
KJ
Aziz
Khaled
K
Francisco
Dave C
DC
Panayiotidis
Mihalis I
MI
Sedelnikova
Olga A
OA
Bonner
William M
WM
Winters
Thomas A
TA
Georgakilas
Alexandros G
AG
D000098:Acetophenones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D020552:Comet Assay; D018736:DNA Adducts / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D049914:DNA Repair-Deficiency Disorders / Q000235:genetics; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D005909:Glioblastoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics
C487214:1-(2-hydroxy-4-morpholin-4-ylphenyl)ethanone; C479235:2-(morpholin-4-yl)benzo(h)chromen-4-one; D000098:Acetophenones; D002867:Chromones; D018736:DNA Adducts; D009025:Morpholines; D034741:RNA, Small Interfering; D051747:DNA-Activated Protein Kinase