No description available.

Radiation therapy targets cancer cell death by overwhelming cells with harmful DNA damage. Understanding how cells repair radiation damage and in particular how they become resistant to radiation therapy is important for effective cancer treatment. Our lab made the novel discovery that Bcl10, a cytoplasmic protein important for NF-B activation, localizes to endogenous H2AX foci in the nucleus of breast cancer cells. We determined that following radiation treatment Bcl10 is recruited to ionizing radiation-induced foci in a dose-dependent matter and that it is important for the repair of radiation-induced DNA damage. We also observed that breast cancer cells are extremely sensitive to Bcl10 knockdown, causing cellular senescence, while normal breast epithelial cells are insensitive. Our findings identify Bcl10 as potent anti-cancer target. / Experimental Oncology

No description available.

Nucleotide synthesis is a conserved and highly regulated response to DNA damage, required for the efficient repair of DNA double strand breaks (DSB) by homologous recombination (HR). This is essential to prevent loss of heterozygosity (LOH) and maintain genome stability. The aim of this study was to identify new genes important for HR through roles in damage-induced nucleotide synthesis. A screen was performed to identify S. pombe gene deletion strains whose DSB sensitivity was suppressed by deleting the ribonucleotide reductase (RNR) inhibitor spd1⁺ to promote nucleotide synthesis. The screen identified a number of genes including ddb1⁺, cdt2⁺, rad3⁺ and csn1⁺ which have known roles in nucleotide synthesis. Distinct roles were identified for the DNA damage checkpoint in suppressing LOH. rad3⁺, rad26⁺, rad17⁺ and the rad9⁺, rad1⁺ and hus1⁺ genes encoding the 9-1-1 complex were required for DNA damage-induced nucleotide synthesis through Cdt2 induction to promote Spd1 degradation. The HR repair defect of rad3⁺ and rad26⁺ deletion strains was partially suppressed by spd1⁺ deletion. However, the HR repair defect of rad17⁺, rad9⁺, rad1⁺ and hus1⁺ deletion strains was not suppressed. An additional role was confirmed for Rad17 and the 9-1-1 complex in preventing LOH by promoting DSB resection. A role was identified for the Gcn5 histone acetyl transferase (HAT) protein module, consisting of Gcn5, Ngg1, Ada2 and Sgf29, in suppressing DSB sensitivity by promoting nucleotide synthesis. This was independent of Cdt2 or RNR protein levels. The Gcn5 HAT module was also found to regulate DSB repair pathway choice consistent with previous observations. Deletion of gcn5⁺, ngg1⁺ or ada2⁺ decreased HR and increased non-homologous end joining. Surprisingly, deletion of spd1⁺ in a gcn5∆, ngg1∆ or ada2∆ background also promoted HR. This predicts a role for nucleotide pools in regulating DSB repair pathway choice. Eleven other candidates showed repeatable suppression of DSB sensitivity following spd1⁺ deletion. However many of these candidates did not show reduced nucleotide levels. This suggests deleting spd1⁺ may also suppress DSB sensitivity by a different mechanism.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

Checkpoint kinase 2 (CHK2) is an effector kinase of the DNA damage response pathway and although its mechanism of activation has been well studied, the attenuation of its activity following DNA damage has not been explored. Here, we identify the B'α subunit of protein phosphatase 2A (PP2A), a major protein serine/threonine phosphatase of the cell, as a CHK2 binding partner and show that their interaction is modulated by DNA damage. B'α binds to the SQ/TQ cluster domain of CHK2, which is a target of ATM phosphorylation. CHK2 is able to bind to many B' subunits as well as the PP2A C subunit, indicating that it can bind to the active PP2A enzyme. The induction of DNA double-strand breaks by ionizing radiation (IR) as well as treatment with doxorubicin causes dissociation of the B'α and CHK2 proteins, however, it does not have an effect on the binding of B'α to CHK1. IR-induced dissociation is an early event and occurs in a dose-dependent manner. CHK2 and B'α can re-associate hours after DNA damage and this is not dependent upon the repair of the DNA. Dissociation is dependent on ATM activity and correlates with an increase in the ATM-dependent phosphorylation of CHK2 at serines 33 and 35 in the SQ/TQ region. Indeed, mutating these sites to mimic phosphorylation increases the dissociation after IR. CHK2 is able to phosphorylate B'α in vitro; however, in vivo, irradiation has no effect on PP2A activity or localization. Alternatively, PP2A negatively regulates CHK2 phosphorylation at multiple sites, as well as its kinase activity and protein stability. These data reveal a novel mechanism for PP2A to keep CHK2 inactive under normal conditions while also allowing for a rapid release from this regulation immediately following DNA damage. This is followed by a subsequent reconstitution of the PP2A/CHK2 complex in later time points after damage, which may help to attenuate the signal. This mechanism of CHK2 negative regulation by PP2A joins a growing list of negative regulations of DNA damage response proteins by protein serine/threonine phosphatases.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease with primary sites of pathology in the large sensory neurons of the dorsal root ganglia (DRG) and dentate nucleus of the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. FRDA is caused by loss of frataxin (FXN) expression, which is due to GAA repeat expansion in intron 1 of the FXN gene. Frataxin is a mitochondrial protein important in iron-sulphur cluster (ISC) biogenesis and in the electron transport chain (ETC). As a consequence of impaired mitochondrial energy metabolism, FRDA cells show increased levels of and sensitivity to oxidative stress, which is known to be associated with genome instability. In this study, we investigated DNA damage/repair in relation to FXN expression via immunostaining of γ-H2AX, a nuclear protein that is recruited to DNA double strand breaks (DSBs). We found FRDA patient and YG8sR FRDA mouse model fibroblasts to have inherently elevated DNA DSBs (1.8 and 0.9 foci/nucleus) compared to normal fibroblasts (0.6 and 0.2 foci/nucleus, in each case P < 0.001). By delivering the FXN gene to these cells with a lentivirus vector (LV) at a copy number of ~1/cell, FXN mRNA levels reached 48 fold (patient cells) and 42 fold (YG8sR cells) and protein levels reached 20 fold (patient cells) and 3.5 fold (YG8sR cells) that of untreated fibroblasts, without observable cytotoxicity. This resulted in a reduction in DNA DSB foci to 0.7 and 0.43 (in each case P < 0.001) in human and YG8sR fibroblasts, respectively and an increase in cell survival to that found for normal fibroblasts. We next irradiated the FRDA fibroblasts (2Gy) and measured their DSB repair profiles. Both human and mouse FRDA fibroblasts were unable to repair damaged DNA. However, repair returned to near normal levels following LV FXN gene transfer. Our data suggest frataxin may be important for genome stability and cell survival by ensuring ISC for DNA damage repair enzymes or may be required directly for DNA DSB repair.

Abstract Mutations in BRCA1 and BRCA2 explain only about 20% of familial aggregation of breast cancer, suggesting involvement of additional susceptibility genes. In this study five DNA damage response genes, ATM, ATR, MRE11, NBS1 and RAD50, were considered as putative candidates to explain some of the remaining familial breast cancer risk, and were screened for germline mutations in families displaying genetic predisposition. Analysis of ATM indicated that clearly pathogenic mutations seem to be restricted to those reported in ataxia-telangiectasia (A-T). However, a cancer risk modifying effect was suggested for a combination of two ATM polymorphisms, 5557G>A and IVS38-8T>C, as this allele seemed to associate with bilateral breast cancer (OR 10.2, 95% CI 3.1–33.8, p = 0.001). The relevance of ATM mutations, originally identified in Finnish A-T patients, in breast cancer susceptibility was evaluated by a large case-control study. Two such alleles, 6903insA and 7570G>C, in addition to 8734A>G previously associated with breast cancer susceptibility, were observed. The overall mutation frequency in unselected cases (7/1124) was higher than in controls (1/1107), but a significantly elevated frequency was observed only in familial cases (6/541, p = 0.006, OR 12.4, 95% CI 1.5–103.3). These three mutations showed founder effects in their geographical occurrence, and had different functional consequences at protein level. In ATR no disease-related mutations were observed, suggesting that it is not a breast cancer susceptibility gene. The mutation screening of the Mre11 complex genes, MRE11, NBS1 and RAD50, revealed two novel potentially breast cancer associated alleles: NBS1 Leu150Phe and RAD50 687delT were observed in 2.0% (3/151) of the studied families. The subsequent study of newly diagnosed, unselected breast cancer cases indicated that RAD50 687delT is a relatively common low-penetrance susceptibility allele in Northern Finland (cases 8/317 vs. controls 6/1000, OR 4.3, 95% CI 1.5–12.5, p = 0.008). NBS1 Leu150Phe (2/317) together with a novel RAD50 IVS3-1G>A mutation (1/317) was also observed, both being absent from controls. Loss of the wild-type allele was not observed in the tumors of the studied mutation carriers, but they all showed an increase in chromosomal instability of peripheral T-lymphocytes. This suggests an effect for RAD50 and NBS1 haploinsufficiency on genomic integrity and susceptibility to cancer.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19068号 / 人博第721号 / 新制||人||173(附属図書館) / 26||人博||721(吉田南総合図書館) / 32019 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 小松 賢志, 教授 宮下 英明, 准教授 三浦 智行 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM