DNA-PKcs deficiency leads to persistence of oxidatively induced clustered DNA lesions in human tumor cells

Abstract

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. In order to examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three different models of DNA-PK deficiency i.e. chemical inactivation of its kinase activity by novel inhibitors IC86621 and NU7026, knock-down and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). Compromised DNA-PK repair pathway has lead to accumulation of clustered DNA lesions induced by γ-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidativelyinduced 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 lead to a higher level of lesion persistence even after 24-72 hrs of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters by first compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions due to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important not only for understating cancer etiology in the presence of a NHEJ deficiency but also lead to a better understanding of cancer treatments based on the induction of oxidative stress and inhibition of cluster repair.

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

“…The inaccurate repair of such "hybrid clusters", consisting of DSBs and non-DSBs, can be attributed to the error-prone DNA repair pathways implicated in this case like NHEJ and its backup subpathways alt-EJ (or B-NHEJ) and BER, respectively [7,8,37]. Previous studies by Peddi et al [57] have shown, using an adaptation of PFGE and different types and levels of DNA-PK deficiency, that these deficient cells not only exhibit as expected DSB repair problems, but also the non-DSB oxidative clustered DNA lesions processing is significantly impeded. Therefore, these repair resistant DSB sites (foci) will be expected to carry a heavy load of non-DSB damage increasing the possibility for misrepair and chromosomal breaks [50].…”

Phosphorylation of H2AX at Ser139 and a New Phosphorylation Site Ser16 by RSK2 Decreases H2AX Ubiquitination and Inhibits Cell Transformation

“…1B). The peptides were each incubated with active RSK2 and [g- 32 P]ATP in an in vitro kinase assay. The results showed that Ser16 was consistently and strongly phosphorylated by RSK2 (Fig.…”

“…Members of the phosphatidylinositol-3 (PI-3) kinase family, including ATM, ATR, and DNA-PK, also regulate histone H2AX phosphorylation and are known to be involved in the DNA damage response induced by replication stress (9, 12, 14, 15). DNA-PK responds to oxidative stress as well in human tumor cell lines (32), and phosphorylation of H2AX at Ser139 is induced in lymphocytes after oxidative stress (33). To determine the role of these kinases in EGF-induced H2AX phosphorylation, we treated cells with the PI-3 kinase family inhibitor, wortmannin (5, 34), for 1 hour before EGF treatment.…”

DNA-PKcs deficiency sensitizes the human hepatoma HepG2 cells to cisplatin and 5-fluorouracil through suppression of the PI3K/Akt/NF-κB pathway

“…Depletion of DNA-PKcs through shRNA transfection resulted in inhibition of cell proliferation and increased apoptosis in human cervical cancer HeLa cell line exposed to cisplatin [33]. Moreover, besides the capability of the non-homologous-end-joining repair for DNA double strands breaks (DSBs), DNA-dependent protein kinase (DNA-PK) is involved in processing the oxidatively induced non-DSB clustered DNA lesions, which is important for the therapeutic efficiency of cancer treatments based on the induction of oxidative stress [34]. Mutant forms of DNA-PKcs at serine/threonine residues in the 2,609-2,647 cluster or specific inhibitor KU57788 could completely block or markedly reduce the efficiency of end joining [35].…”