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A specific inhibitor of protein kinase CK2 delays gamma-H2Ax foci removal and reduces clonogenic survival of irradiated mammalian cells.

Zwicker F, Ebert M, Huber PE, Debus J, Weber KJ - Radiat Oncol (2011)

Bottom Line: 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.The 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, University of Heidelberg, Heidelberg, Germany. f.zwicker@dkfz.de

ABSTRACT

Background: 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 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.

Methods: MRC5 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.

Results: TBB 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.

Conclusions: The 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.

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Related in: MedlinePlus

Cell cycle measurements (FACS) at different times after 5 Gy irradiation of WIDR cells pretreated (2 hours) with 20 μM TBB (lower panels) or DMSO, only (middle panels). The upper panels show histograms after TBB treatment without irradiation which were identical to the respective DMSO controls (not shown).
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Figure 7: Cell cycle measurements (FACS) at different times after 5 Gy irradiation of WIDR cells pretreated (2 hours) with 20 μM TBB (lower panels) or DMSO, only (middle panels). The upper panels show histograms after TBB treatment without irradiation which were identical to the respective DMSO controls (not shown).

Mentions: Sub-G1 flow-cytometry or DAPI-staining of MRC5 or WIDR cells failed to demonstrate apoptosis induction over a period of up to 38 hours following 8 Gy irradiation, incubation with 40 μM TBB, or a combined exposure even when the TBB pretreatment period was extended to 8 hours (data not shown). Cell cycle analysis by flow cytometry was able to detect the well known radiation-induced G2-arrest (WIDR cells after 5 Gy: Figure 7) which was clearly more expressed and prolonged upon 20 μM TBB pretreatment. Radiation inhibition of clonogenic survival was enhanced by the CK2 inhibition with 20 μM TBB compared to 0.2% DMSO controls (normalized data in Figure 8). For the MRC5 cells, this effect was only observed with radiation doses ≥3 Gy.


A specific inhibitor of protein kinase CK2 delays gamma-H2Ax foci removal and reduces clonogenic survival of irradiated mammalian cells.

Zwicker F, Ebert M, Huber PE, Debus J, Weber KJ - Radiat Oncol (2011)

Cell cycle measurements (FACS) at different times after 5 Gy irradiation of WIDR cells pretreated (2 hours) with 20 μM TBB (lower panels) or DMSO, only (middle panels). The upper panels show histograms after TBB treatment without irradiation which were identical to the respective DMSO controls (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045342&req=5

Figure 7: Cell cycle measurements (FACS) at different times after 5 Gy irradiation of WIDR cells pretreated (2 hours) with 20 μM TBB (lower panels) or DMSO, only (middle panels). The upper panels show histograms after TBB treatment without irradiation which were identical to the respective DMSO controls (not shown).
Mentions: Sub-G1 flow-cytometry or DAPI-staining of MRC5 or WIDR cells failed to demonstrate apoptosis induction over a period of up to 38 hours following 8 Gy irradiation, incubation with 40 μM TBB, or a combined exposure even when the TBB pretreatment period was extended to 8 hours (data not shown). Cell cycle analysis by flow cytometry was able to detect the well known radiation-induced G2-arrest (WIDR cells after 5 Gy: Figure 7) which was clearly more expressed and prolonged upon 20 μM TBB pretreatment. Radiation inhibition of clonogenic survival was enhanced by the CK2 inhibition with 20 μM TBB compared to 0.2% DMSO controls (normalized data in Figure 8). For the MRC5 cells, this effect was only observed with radiation doses ≥3 Gy.

Bottom Line: 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.The 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, University of Heidelberg, Heidelberg, Germany. f.zwicker@dkfz.de

ABSTRACT

Background: 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 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.

Methods: MRC5 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.

Results: TBB 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.

Conclusions: The 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.

Show MeSH
Related in: MedlinePlus