Effects of novel inhibitors of poly(ADP-ribose) polymerase-1 and the DNA-dependent protein kinase on enzyme activities and DNA repair.
DNA-dependent protein kinase (DNA-PK) and poly (ADP-ribose) polymerase-1 (PARP-1) participate in nonhomologous end joining and base excision repair, respectively, and are key determinants of radio- and chemo-resistance. Both PARP-1 and DNA-PK have been identified as therapeutic targets for anticancer drug development. Here we investigate the effects of specific inhibitors on enzyme activities and DNA double-strand break (DSB) repair. The enzyme activities were investigated using purified enzymes and in permeabilized cells. Inhibition, or loss of activity, was compared using potent inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361), and cell lines proficient or deficient for DNA-PK or PARP-1. Inactive DNA-PK suppressed the activity of PARP-1 and vice versa. This was not the consequence of simple substrate competition, since DNA ends were provided in excess. The inhibitory effect of DNA-PK on PARP activity was confirmed in permeabilized cells. Both inhibitors prevented ionizing radiation-induced DSB repair, but only AG14361 prevented single-strand break repair. An increase in DSB levels caused by inhibition of PARP-1 was shown to be caused by a decrease in DSB repair, and not by the formation of additional DSBs. These data point to combined inhibition of PARP-1 and DNA-PK as a powerful strategy for tumor radiosensitization.
Northern Institute for Cancer Research, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK.
Stephany J
SJ
Veuger
Nicola J
NJ
Curtin
Graeme C M
GC
Smith
Barbara W
BW
Durkacz
D000818:Animals; D052176:Azulenes; D001569:Benzodiazepines / Q000494:pharmacology; D001667:Binding, Competitive; D002460:Cell Line; D045744:Cell Line, Tumor; D002867:Chromones / Q000138:chemical synthesis / Q000494:pharmacology; D004260:DNA Repair / Q000187:drug effects*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009025:Morpholines / Q000138:chemical synthesis / Q000494:pharmacology; D009687:Nuclear Proteins; D010766:Phosphorylation; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*
C479234:1-(4-dimethylaminomethylphenyl)-8,9-dihydro-7H-2,7,9a-benzo(cd)azulen-6-one; C479235:2-(morpholin-4-yl)benzo(h)chromen-4-one; D052176:Azulenes; D002867:Chromones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009025:Morpholines; D009687:Nuclear Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D001569:Benzodiazepines; C483041:PARP2 protein, human; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases