Abstract
BackgroundThe protein kinase CK2 sustains multiple pro-survival functions in cellular DNA damage response and its level is tightly regulated in normal cells but elevated in cancers. Because CK2 is thus considered as potential therapeutic target, DNA double-strand break (DSB) formation and rejoining, apoptosis induction and clonogenic survival was assessed in irradiated mammalian cells upon chemical inhibition of CK2.MethodsMRC5 human fibroblasts and WIDR human colon carcinoma cells were incubated with highly specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB), or mock-treated, 2 hours prior to irradiation. DSB was measured by pulsed-field electrophoresis (PFGE) as well as gamma-H2AX foci formation and removal. Apoptosis induction was tested by DAPI staining and sub-G1 flow cytometry, survival was quantified by clonogenic assay.ResultsTBB treatment did not affect initial DNA fragmention (PFGE; up to 80 Gy) or foci formation (1 Gy). While DNA fragment rejoining (PFGE) was not inhibited by the drug, TBB clearly delayed gamma-H2AX foci disappearence during postirradiation incubation. No apoptosis induction could be detected for up to 38 hours for both cell lines and exposure conditions (monotherapies or combination), but TBB treatment at this moderately toxic concentration of 20 μM (about 40% survival) enhanced radiation-induced cell killing in the clonogenic assay.ConclusionsThe data imply a role of CK2 in gamma-H2AX dephosporylation, most likely through its known ability to stimulate PP2A phosphatase, rather than DSB rejoining. The slight but definite clonogenic radiosensitization by TBB does apparently not result from interference with an apoptosis suppression function of CK2 in these cells but could reflect inhibitor-induced uncoupling of DNA damage response decay from break ligation.
Highlights
Protein kinase CK2 is a ubiquitous and highly conserved protein serine/threonine kinase with a broad spectrum of target proteins the majority of which play a role in signal transduction and gene expression promoting cell survival upon phosphorylation [1,2,3]
CK2 predominantly exists as heteroterameric holoenzyme and, in mammalian cells, basal activity of CK2 is conferred by intramolecular interaction of either catalytic isoform CK2a or CK2a’ and may be regulated via the association with a dimer of the regulatory CK2b subunit [2,4]
The DNA damage response (DDR) invokes chromatin structure changes extending over megabasepair regions flanking a double-strand break (DSB), phosphorylations of the histone variant H2AX [9], as well as the concomitant accumulation of the diverse factors mediating DNA damage signaling [10]
Summary
Protein kinase CK2 is a ubiquitous and highly conserved protein serine/threonine kinase with a broad spectrum of target proteins the majority of which play a role in signal transduction and gene expression promoting cell survival upon phosphorylation [1,2,3]. The DNA damage response (DDR) invokes chromatin structure changes extending over megabasepair regions flanking a DSB, phosphorylations of the histone variant H2AX [9], as well as the concomitant accumulation of the diverse factors mediating DNA damage signaling [10]. Their visualization by means of immunostaining and fluorescence microscopy (the socalled “focus assay”). The protein kinase CK2 sustains multiple pro-survival functions in cellular DNA damage response and its level is tightly regulated in normal cells but elevated in cancers. Because CK2 is considered as potential therapeutic target, DNA double-strand break (DSB) formation and rejoining, apoptosis induction and clonogenic survival was assessed in irradiated mammalian cells upon chemical inhibition of CK2
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