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DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1.

Calkins AS, Iglehart JD, Lazaro JB - Nucleic Acids Res. (2013)

Bottom Line: Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis.However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation.Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Department of Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA.

ABSTRACT
RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.

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DNA damage-induced inhibition of rRNA synthesis is dependent on DNA-PK and PARP-1 activity. Representative nuclei stained by EU are shown 22 h after 2 h treatment with 25 µg/ml cisplatin. (A–F) Cells were treated with cisplatin under the following conditions: (A) pretreatment with wortmannin (WRT, W), (B) nonsilencing (NS) or DNA-PKcs (PKcs, PK) shRNA (sh), (C) pretreatment with Nu7026 (Nu7026, 26) or Nu7441 (Nu7441, 41), (D) pretreatment with benzamide (BNZ, B), (E) pretreatment with olaparib (OLP, O) or (F) GFP shRNA (G) or PARP-1 (P) shRNA. (G–I) Cells were treated with IR (2 Gray) under the following conditions: (G) pretreatment with wortmannin or olaparib, (H) pretreatment with Nu7026 or Nu7441 or (I) GFP or PARP-1 shRNA. (J–L) Cells were treated with UV-C (UV) at 5 J/m2 under the following conditions: (J) pretreatment with wortmannin or olaparib, (K) pretreatment with Nu7026 or Nu7441 or (L) GFP or PARP-1 shRNA. Before DNA damage, wortmannin was applied at 100 nM for 30 min, Nu7026 at 10 µM for 1 h, Nu7441 at 1 µM for 1 h, olaparib at 1 µM for 2 h and benzamide at 200 µM for 30 min. Accompanying quantifications of the adjusted nucleolar EU fluorescence are shown. Each condition was normalized to cells treated with DMSO or indicated inhibitors without DNA damaging treatment. Two-way ANOVA was followed by Holm-Sidak’s test for the comparison of cells with and without inhibitors. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively.
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gkt502-F2: DNA damage-induced inhibition of rRNA synthesis is dependent on DNA-PK and PARP-1 activity. Representative nuclei stained by EU are shown 22 h after 2 h treatment with 25 µg/ml cisplatin. (A–F) Cells were treated with cisplatin under the following conditions: (A) pretreatment with wortmannin (WRT, W), (B) nonsilencing (NS) or DNA-PKcs (PKcs, PK) shRNA (sh), (C) pretreatment with Nu7026 (Nu7026, 26) or Nu7441 (Nu7441, 41), (D) pretreatment with benzamide (BNZ, B), (E) pretreatment with olaparib (OLP, O) or (F) GFP shRNA (G) or PARP-1 (P) shRNA. (G–I) Cells were treated with IR (2 Gray) under the following conditions: (G) pretreatment with wortmannin or olaparib, (H) pretreatment with Nu7026 or Nu7441 or (I) GFP or PARP-1 shRNA. (J–L) Cells were treated with UV-C (UV) at 5 J/m2 under the following conditions: (J) pretreatment with wortmannin or olaparib, (K) pretreatment with Nu7026 or Nu7441 or (L) GFP or PARP-1 shRNA. Before DNA damage, wortmannin was applied at 100 nM for 30 min, Nu7026 at 10 µM for 1 h, Nu7441 at 1 µM for 1 h, olaparib at 1 µM for 2 h and benzamide at 200 µM for 30 min. Accompanying quantifications of the adjusted nucleolar EU fluorescence are shown. Each condition was normalized to cells treated with DMSO or indicated inhibitors without DNA damaging treatment. Two-way ANOVA was followed by Holm-Sidak’s test for the comparison of cells with and without inhibitors. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively.

Mentions: We prevented the cisplatin-induced loss of nucleolar SSRP1 using the PI3K-like kinase family inhibitor wortmannin and shRNA against DNA-PKcs [(5), Supplementary Figure S7]. Treatment with wortmannin before addition of cisplatin significantly relieved inhibition of rRNA synthesis (Figure 2A and Supplementary Figure S8). In addition, silencing of DNA-PKcs expression by shRNA prevented cisplatin-induced inhibition of rRNA synthesis (Figure 2B and Supplementary Figure S9). Neither wortmannin nor expression of DNA-PKcs shRNA had visible effect on rRNA synthesis without cisplatin (Supplementary Figures S8A and S9A). As previously described, DNA-PKcs silencing resulted in lower levels of Ataxia Telangiectasia Mutated protein (ATM) [(28), Supplementary Figure S7]. Wortmannin may inhibit ATM or other PI3K-like kinase activities. For these reasons, we performed similar experiments using specific inhibitors of DNA-PK. Treatment with Nu7026 or Nu7441 before cisplatin significantly prevented inhibition of rRNA synthesis (Figure 2C and Supplementary Figure S10). Furthermore, specific inhibitors of the PI3K, ATM and Ataxia Telangiectasia and Rad3-Related protein (ATR) members of the PI3K family as well as C-Abl and p38 MAPK inhibitors did not significantly alter the inhibition of rRNA synthesis in cisplatin-treated cells (Supplementary Figure S11).Figure 2.


DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1.

Calkins AS, Iglehart JD, Lazaro JB - Nucleic Acids Res. (2013)

DNA damage-induced inhibition of rRNA synthesis is dependent on DNA-PK and PARP-1 activity. Representative nuclei stained by EU are shown 22 h after 2 h treatment with 25 µg/ml cisplatin. (A–F) Cells were treated with cisplatin under the following conditions: (A) pretreatment with wortmannin (WRT, W), (B) nonsilencing (NS) or DNA-PKcs (PKcs, PK) shRNA (sh), (C) pretreatment with Nu7026 (Nu7026, 26) or Nu7441 (Nu7441, 41), (D) pretreatment with benzamide (BNZ, B), (E) pretreatment with olaparib (OLP, O) or (F) GFP shRNA (G) or PARP-1 (P) shRNA. (G–I) Cells were treated with IR (2 Gray) under the following conditions: (G) pretreatment with wortmannin or olaparib, (H) pretreatment with Nu7026 or Nu7441 or (I) GFP or PARP-1 shRNA. (J–L) Cells were treated with UV-C (UV) at 5 J/m2 under the following conditions: (J) pretreatment with wortmannin or olaparib, (K) pretreatment with Nu7026 or Nu7441 or (L) GFP or PARP-1 shRNA. Before DNA damage, wortmannin was applied at 100 nM for 30 min, Nu7026 at 10 µM for 1 h, Nu7441 at 1 µM for 1 h, olaparib at 1 µM for 2 h and benzamide at 200 µM for 30 min. Accompanying quantifications of the adjusted nucleolar EU fluorescence are shown. Each condition was normalized to cells treated with DMSO or indicated inhibitors without DNA damaging treatment. Two-way ANOVA was followed by Holm-Sidak’s test for the comparison of cells with and without inhibitors. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively.
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gkt502-F2: DNA damage-induced inhibition of rRNA synthesis is dependent on DNA-PK and PARP-1 activity. Representative nuclei stained by EU are shown 22 h after 2 h treatment with 25 µg/ml cisplatin. (A–F) Cells were treated with cisplatin under the following conditions: (A) pretreatment with wortmannin (WRT, W), (B) nonsilencing (NS) or DNA-PKcs (PKcs, PK) shRNA (sh), (C) pretreatment with Nu7026 (Nu7026, 26) or Nu7441 (Nu7441, 41), (D) pretreatment with benzamide (BNZ, B), (E) pretreatment with olaparib (OLP, O) or (F) GFP shRNA (G) or PARP-1 (P) shRNA. (G–I) Cells were treated with IR (2 Gray) under the following conditions: (G) pretreatment with wortmannin or olaparib, (H) pretreatment with Nu7026 or Nu7441 or (I) GFP or PARP-1 shRNA. (J–L) Cells were treated with UV-C (UV) at 5 J/m2 under the following conditions: (J) pretreatment with wortmannin or olaparib, (K) pretreatment with Nu7026 or Nu7441 or (L) GFP or PARP-1 shRNA. Before DNA damage, wortmannin was applied at 100 nM for 30 min, Nu7026 at 10 µM for 1 h, Nu7441 at 1 µM for 1 h, olaparib at 1 µM for 2 h and benzamide at 200 µM for 30 min. Accompanying quantifications of the adjusted nucleolar EU fluorescence are shown. Each condition was normalized to cells treated with DMSO or indicated inhibitors without DNA damaging treatment. Two-way ANOVA was followed by Holm-Sidak’s test for the comparison of cells with and without inhibitors. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively.
Mentions: We prevented the cisplatin-induced loss of nucleolar SSRP1 using the PI3K-like kinase family inhibitor wortmannin and shRNA against DNA-PKcs [(5), Supplementary Figure S7]. Treatment with wortmannin before addition of cisplatin significantly relieved inhibition of rRNA synthesis (Figure 2A and Supplementary Figure S8). In addition, silencing of DNA-PKcs expression by shRNA prevented cisplatin-induced inhibition of rRNA synthesis (Figure 2B and Supplementary Figure S9). Neither wortmannin nor expression of DNA-PKcs shRNA had visible effect on rRNA synthesis without cisplatin (Supplementary Figures S8A and S9A). As previously described, DNA-PKcs silencing resulted in lower levels of Ataxia Telangiectasia Mutated protein (ATM) [(28), Supplementary Figure S7]. Wortmannin may inhibit ATM or other PI3K-like kinase activities. For these reasons, we performed similar experiments using specific inhibitors of DNA-PK. Treatment with Nu7026 or Nu7441 before cisplatin significantly prevented inhibition of rRNA synthesis (Figure 2C and Supplementary Figure S10). Furthermore, specific inhibitors of the PI3K, ATM and Ataxia Telangiectasia and Rad3-Related protein (ATR) members of the PI3K family as well as C-Abl and p38 MAPK inhibitors did not significantly alter the inhibition of rRNA synthesis in cisplatin-treated cells (Supplementary Figure S11).Figure 2.

Bottom Line: Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis.However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation.Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Department of Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA.

ABSTRACT
RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.

Show MeSH
Related in: MedlinePlus