Interactive effects of poly(ADP-ribose) polymerase -1 and DNA dependent protein kinase in the cellular responses to DNA damage
DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1) participate in non-homologous end joining and base excision repair respectively, and are key determinants of radio-resistance. The interactive effects of PARP-1 and DNA-PK in the cellular responses to DNA damage were investigated using novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) and cell lines proficient or deficient for DNA-PK or PARP-1. Enzyme deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR), compared to their proficient counterparts. NU7026 potentiated IR cytotoxicity in exponentially growing DNA-PK proficient, but not deficient cells. Similarly AG14361 potentiated IR in PARP-1 +/+ but not PARP-1-/- cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive. Both inhibitors reduced PLDR in the proficient cell lines. Furthermore, inhibitor combination completely abolished PLDR. Both inhibitors prevented IR-induced DNA double strand break repair, but only AG14361 prevented DNA single strand break repair The enzyme activities were investigated using purified enzymes and permeabilised cells. In cell-free assays, PARP- 1 activity was unaffected by the presence of DNA-PK, and vice versa, provided the enzyme substrates were present. DNA-PK inhibited PARP-1 when ATP was absent or NU7026 present. PARP-1 inhibited DNA-PK when NAD+ was absent or AG14361 present. Furthermore, PARP-1 inhibition increased with increasing ratio of DNA-PK to PARP-1, and vice versa. Similar results were obtained using the inhibitors in permeabilised cells. PARP-1 was inactive in the absence of histones, but activity was restored by the addition of DNA-PK. DNA-PK was inactive in the absence of its p53 peptide substrate unless PARP-1 was included. These data suggest reciprocal regulation of PARP-1 and DNA-PK, and co-operation in DSB repair and survival. Thus, individually, or in combination, the DNA-PK and PARP-1 inhibitors act as potent radio-sensitisers.