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

<p>Abstract</p> <p>Background</p> <p>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 <it>via </it>inhibition of MRE11.</p> <p>Results</p> <p>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 γ-H2AX, NBS1 and phospho-ATM at the sites of DSBs. Furthermore, pentamidine decreased HR activity <it>in vivo</it>. 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.</p> <p>Conclusion</p> <p>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.</p

Induction of Excess Centrosomes in Neural Progenitor Cells during the Development of Radiation-Induced Microcephaly

The embryonic brain is one of the tissues most vulnerable to ionizing radiation. In this study, we showed that ionizing radiation induces apoptosis in the neural progenitors of the mouse cerebral cortex, and that the surviving progenitor cells subsequently develop a considerable amount of supernumerary centrosomes. When mouse embryos at Day 13.5 were exposed to γ-rays, brains sizes were reduced markedly in a dose-dependent manner, and these size reductions persisted until birth. Immunostaining with caspase-3 antibodies showed that apoptosis occurred in 35% and 40% of neural progenitor cells at 4 h after exposure to 1 and 2 Gy, respectively, and this was accompanied by a disruption of the apical layer in which mitotic spindles were positioned in unirradiated mice. At 24 h after 1 Gy irradiation, the apoptotic cells were completely eliminated and proliferation was restored to a level similar to that of unirradiated cells, but numerous spindles were localized outside the apical layer. Similarly, abnormal cytokinesis, which included multipolar division and centrosome clustering, was observed in 19% and 24% of the surviving neural progenitor cells at 48 h after irradiation with 1 and 2 Gy, respectively. Because these cytokinesis aberrations derived from excess centrosomes result in growth delay and mitotic catastrophe-mediated cell elimination, our findings suggest that, in addition to apoptosis at an early stage of radiation exposure, radiation-induced centrosome overduplication could contribute to the depletion of neural progenitors and thereby lead to microcephaly

WRN participates in translesion synthesis pathway through interaction with NBS1.

Werner syndrome (WS), caused by mutation of the WRN gene, is an autosomal recessive disorder associated with premature aging and predisposition to cancer. WRN belongs to the RecQ DNA helicase family, members of which play a role in maintaining genomic stability. Here, we demonstrate that WRN rapidly forms discrete nuclear foci in an NBS1-dependent manner following DNA damage. NBS1 physically interacts with WRN through its FHA domain, which interaction is important for the phosphorylation of WRN. WRN subsequently forms DNA damage-dependent foci during the S phase, but not in the G1 phase. WS cells exhibit an increase in spontaneous focus formation of poleta and Rad18, which are important for translesion synthesis (TLS). WRN also interacts with PCNA in the absence of DNA damage, but DNA damage induces the dissociation of PCNA from WRN, leading to the ubiquitination of PCNA, which is essential for TLS. This dissociation correlates with ATM/NBS1-dependent degradation of WRN. Moreover, WS cells show constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase in the absence of DNA damage. Taken together, these results indicate that WRN participates in the TLS pathway to prevent genomic instability in an ATM/NBS1-dependent manner

ATM activation by a sulfhydryl-reactive inflammatory cyclopentenone prostaglandin

がん研究所がん分子細胞制御ATM (ataxia-telangiectasia mutated) is activated by a variety of noxious agent, including oxidative stress, and ATM deficiency results in an anomalous cellular response to oxidative stress. However, the mechanisms for ATM activation by oxidative stress remain to be established. Furthermore, it is not clear whether ATM responds to oxidative DNA damage or to a change in the intracellular redox state, independent of DNA damage. We found that ATM is activated by N-methyl-N′-nitro-nitrosoguanidine (MNNG) and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), in NBS1- or MSH6-deficient cells. We further found that ATM is activated by treating chromatin-free immunoprecipitated ATM with MNNG or 15d-PGJ2, which modifies free sulfhydryl (SH) groups, and that 15d-PGJ2 binds covalently to ATM. Interestingly, 15d-PGJ2-induced ATM activation leads to p53 activation and apoptosis, but not to Chk2 or H2AX phosphorylation. These results indicate that ATM is activated through the direct modification of its SH groups, independent of DNA damage, and this activation leads, downstream, to apoptosis. © 2006 The Authors Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd

NBS1 and multiple regulations of DNA damage response

DNA damage response is finely tuned, with several pathways including those for DNA repair, chromatin remodeling and cell cycle checkpoint, although most studies to date have focused on single pathways. Genetic diseases characterized by genome instability have provided novel insights into the underlying mechanisms of DNA damage response. NBS1, a protein responsible for the radiation-sensitive autosomal recessive disorder Nijmegen breakage syndrome, is one of the first factors to accumulate at sites of DNA double-strand breaks (DSBs). NBS1 binds to at least five key proteins, including ATM, RPA, MRE11, RAD18 and RNF20, in the conserved regions within a limited span of the C terminus, functioning in the regulation of chromatin remodeling, cell cycle checkpoint and DNA repair in response to DSBs. In this article, we reviewed the functions of these binding proteins and their comprehensive association with NBS1

High LET Radiation Amplifies Centrosome Overduplication Through a Pathway of γ-Tubulin Monoubiquitination.

[Purpose]Radiation induces centrosome overduplication, leading to mitotic catastrophe and tumorigenesis. Because mitotic catastrophe is one of the major tumor cell killing factors in high linear energy transfer (LET) radiation therapy and long-term survivors from such treatment have a potential risk of secondary tumors, we investigated LET dependence of radiation-induced centrosome overduplication and the underlying mechanism. [Methods and Materials]Carbon and iron ion beams (13-200 keV/μm) and γ-rays (0.5 keV/μm) were used as radiation sources. To count centrosomes after IR exposure, human U2OS and mouse NIH3T3 cells were immunostained with antibodies of γ-tubulin and centrin 2. Similarly, Nbs1-, Brca1-, Ku70-, and DNA-PKcs-deficient mouse cells and their counterpart wild-type cells were used for measurement of centrosome overduplication. [Results]The number of excess centrosome-containing cells at interphase and the resulting multipolar spindle at mitosis were amplified with increased LET, reaching a maximum level of 100 keV/μm, followed by sharp decrease in frequency. Interestingly, Ku70 and DNA-PKcs deficiencies marginally affected the induction of centrosome overduplication, whereas the cell killings were significantly enhanced. This was in contrast to observation that high LET radiation significantly enhanced frequencies of centrosome overduplication in Nbs1- and Brca1-deficient cells. Because NBS1/BRCA1 is implicated in monoubiquitination of γ-tubulin, we subsequently tested whether it is affected by high LET radiation. As a result, monoubiquitination of γ-tubulin was abolished in 48 to 72 hours after exposure to high LET radiation, although γ-ray exposure slightly decreased it 48 hours postirradiation and was restored to a normal level at 72 hours. [Conclusions]High LET radiation significantly reduces NBS1/BRCA1-mediated monoubiquitination of γ-tubulin and amplifies centrosome overduplication with a peak at 100 keV/μm. In contrast, Ku70 and DNA-PKcs deficiencies mitigate centrosome overduplication, although deficiencies of both NBS1/BRCA1 and Ku70/DNA-PKcs markedly enhance cell killing

RNF20-SNF2H Pathway of Chromatin Relaxation in DNA Double-Strand Break Repair

Rapid progress in the study on the association of histone modifications with chromatin remodeling factors has broadened our understanding of chromatin dynamics in DNA transactions. In DNA double-strand break (DSB) repair, the well-known mark of histones is the phosphorylation of the H2A variant, H2AX, which has been used as a surrogate marker of DSBs. The ubiquitylation of histone H2B by RNF20 E3 ligase was recently found to be a DNA damage-induced histone modification. This modification is required for DSB repair and regulated by a distinctive pathway from that of histone H2AX phosphorylation. Moreover, the connection between H2B ubiquitylation and the chromatin remodeling activity of SNF2H has been elucidated. In this review, we summarize the current knowledge of RNF20-mediated processes and the molecular link to H2AX-mediated processes during DSB repair