Bisbenzamidine derivative, pentamidine represses DNA damage response through inhibition of histone H2A acetylation - PubMed (original) (raw)

Bisbenzamidine derivative, pentamidine represses DNA damage response through inhibition of histone H2A acetylation

Junya Kobayashi et al. Mol Cancer. 2010.

Abstract

Background: MRE11 is an important nuclease which functions in the end-resection step of homologous recombination (HR) repair of DNA double-strand breaks (DSBs). As MRE11-deficient ATLD cells exhibit hyper radio-sensitivity and impaired DSB repair, MRE11 inhibitors could possibly function as potent radio-sensitizers. Therefore, we investigated whether a bisbenzamidine derivative, pentamidine, which can inhibit endoexonuclease activity, might influence DSB-induced damage responses via inhibition of MRE11.

Results: We first clarified that pentamidine inhibited MRE11 nuclease activity and also reduced ATM kinase activity in vitro. Pentamidine increased the radio-sensitivity of HeLa cells, suggesting that this compound could possibly influence DNA damage response factors in vivo. Indeed, we found that pentamidine reduced the accumulation of gamma-H2AX, NBS1 and phospho-ATM at the sites of DSBs. Furthermore, pentamidine decreased HR activity in vivo. Pentamidine was found to inhibit the acetylation of histone H2A which could contribute both to inhibition of IR-induced focus formation and HR repair. These results suggest that pentamidine might exert its effects by inhibiting histone acetyltransferases. We found that pentamidine repressed the activity of Tip60 acetyltransferase which is known to acetylate histone H2A and that knockdown of Tip60 by siRNA reduced HR activity.

Conclusion: These results indicate that inhibition of Tip60 as well as hMRE11 nuclease by pentamidine underlies the radiosensitizing effects of this compound making it an excellent sensitizer for radiotherapy or chemotherapy.

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Figures

Figure 1

Figure 1

The inhibitory effect of pentamidine on an hMRE11 nuclease and an ATM kinase. (A) Pentamidine inhibited hMRE11 nuclease. hMRE11 nuclease assay was performed using recombinant MRN complex. Indicated amount of pentamidine is added to the reaction mixture. (B) Pentamidine inhibited ATM kinase. Normal lymphoblastoid cells (GM2184) were irradiated by 10 Gy of γ-irradiation and the kinase activity assay was performed using immuno-complex by anti-ATM antibody or control rabbit IgG from these cells. Indicated concentration of pentamidine is added to the reaction mixture. (C) Pentamidine treatment increased radiation sensitivity. HeLa cells were irradiated with indicated dose of γ-ray with or without pentamidine (0.05 mM) and the viability of their cells were analyzed by colony forming assays. (D) Pentamidine treatment induced multinuclear formation. HeLa cells were irradiated with 5 Gy of γ-ray with or without pentamidine (0.05 mM) and cell morphology was observed after 24 hours.

Figure 2

Figure 2

The effect of pentamidine on IR-induced cellular response. (A) Pentamidine repressed γ-H2AX foci formation. MRC5SV cells were irradiated with 5 Gy of γ-ray with or without pre-treatment of pentamidine (indicated concentrations, 30 minutes). After 30 minutes, their cells were fixed and immuno-staining was performed using anti-γ-H2AX. Percentage of γ-H2AX foci-positive cell was shown in (B). (C) Pentamidine repressed focus formation of DNA damage-related factors. MRC5SV cells were irradiated with 5 Gy of γ-ray with or without pre-treatment of pentamidine (0.5 mM, 30 minutes). After 30 minutes, their cells were fixed and immuno-staining was performed using anti-NBS1, anti-phospho-ATM, anti-hMRE11 and anti-MDC1 antibodies.

Figure 3

Figure 3

The effect of pentamidine on ATM/ATR/DNA-PK-dependent phosphorylation. (A) Pentamidine did not disturb ATM-dependent phosphorylation. MRC5SV cells were irradiated by 5 Gy of γ-ray with or without pre-treatment of pentamidine (0.5 mM, 30 minutes). These cells were harvested at 0.5 hour after IR and analyzed by Western blot using indicated antibodies. (B) Pentamidine did not disturb ATR or DNA-PK-dependent phosphorylation. MRC5SV were irradiated by 5 Gy of γ-ray with or without pre-treatment of pentamidine (0.5 mM, 1 hour). These cells were harvested at 0.5 hour after IR and analyzed by Western blot using indicated antibodies.

Figure 4

Figure 4

The effect of pentamidine on DNA double-strand break repair (A)(B) Pentamidine decreased homologous recombination activity in DR-GFP assay. I-SceI expression plasmids were introduced to M5D (A) or HeLa-DRGFP cells (B) by an electroporation with or without pre-treatment of pentamidine (0.5 mM, 1 hour). After 3 days GFP-positive cells, induced via HR pathway, were analyzed by flowcytometer as described in Materials and Methods. (C) Pentamidine did not disturb NHEJ activity. I-SceI expression plasmids were introduced to MRC5-pEJ cells by electroporation with or without pre-treatment of pentamidine (0.5 mM, 1 hour). After 3 days GFP-positive cells, induced through NHEJ pathway, were analyzed by flowcytometer as described in Materials and Methods.

Figure 5

Figure 5

Pentamidine might affect DNA damage response through acetylation of histone H2A. (A) The effect of pentamidine on histone acetylation. MRC5SV cells were irradiated with 5 Gy of γ-ray with or without pre-treatment of pentamidine (0.5 mM, 30 minutes). These cells were harvested at indicated times after IR and analyzed by Western blot using indicated antibodies. (B) Acetylation of histone H2A is required for IR-induced foci formation. H2A-WT or H2A-K5/9R-expressing cells were generated as described in Materials and Methods. These cells were irradiated with 5 Gy of γ-ray. After 30 minutes, their cells were fixed and immuno-staining was performed using anti-NBS1 and anti-FLAG antibodies. Percentage of NBS1 foci-positive cells was shown in (C). (D) HR activity in H2A (K5/9R)-expressing cells. I-SceI expression plasmids were introduced into H2A-WT or H2A-K5/9R- expressing cells by electroporation. After 2 days GFP-positive cells, induced through HR pathway, were analyzed by flowcytometer.

Figure 6

Figure 6

Tip60-knockdown also repressed both HR activity and acetylation of histone H2A. (A) Tip60-knockdown reduced acetylation of histone H2A. MRC5SV cells were transfected by Tip60 siRNA. After 2 days, these cells were irradiated by 5 Gy of γ-ray and were harvested at indicated times after IR and analyzed by Western blot using indicated antibodies. (B) HR activity in Tip60-knockdowned cells. M5D cells were transfected by Tip60 siRNA. After 2 days, I-SceI expression plasmids were introduced by electroporation. After 3 days, GFP-positive cells were analyzed by flowcytometer. (C) The effect of pentamidine on HAT activity of Tip60. Whole cell extract was prepared from irradiated or un-irradiated HeLa cells. And then, Tip60-dependent HAT activity was measured with or without pentamidine (5 μM) as described in Material and Methods. (D) Pentamidine repressed Tip60-dependent acetylation of p53. MRC5SV cells were added by 0.5 mM adriamycin with or without pre-treatment of pentamidine (0.5 mM, 30 minutes). These cells were harvested after 4 hours and analyzed by Western blot using anti-acetylated p53 antibody.

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References

    1. Kobayashi J, Tauchi H, Sakamoto S, Nakamura A, Morishima K, Matsuura S, Kobayashi T, Tamai K, Tanimoto K, Komatsu K. NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain. Curr Biol. 2002;12:1846–1851. doi: 10.1016/S0960-9822(02)01259-9. - DOI - PubMed
    1. Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, Reina-San-Martin B, Coppola V, Meffre E, Difilippantonio MJ, Redon C, Pilch DR, Olaru A, Eckhaus M, Camerini-Otero RD, Tessarollo L, Livak F, Manova K, Bonner WM, Nussenzweig MC, Nussenzweig A. Genomic instability in mice lacking histone H2AX. Science. 2002;296:922–927. doi: 10.1126/science.1069398. - DOI - PMC - PubMed
    1. Stucki M, Jackson S. gammaH2AX and MDC1: anchoring the DNA-damage-response machinery to broken chromosomes. DNA Repair. 2006;5:534–543. doi: 10.1016/j.dnarep.2006.01.012. - DOI - PubMed
    1. Kobayashi J, Antoccia A, Tauchi H, Matsuura S, Komatsu K. NBS1 and its functional role in the DNA damage response. DNA Repair. 2004;3:855–861. doi: 10.1016/j.dnarep.2004.03.023. - DOI - PubMed
    1. Paull TT, Rogakou E, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol. 2000;10:886–895. doi: 10.1016/S0960-9822(00)00610-2. - DOI - PubMed

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