Language：English   Publishing type：Research paper (scientific journal)  

Sulforaphane (SFN), an isothiocyanate found in plants of the Brassicaceae family, possesses antioxidant, apoptosis-inducing, and radiosensitizing effects. As one of the mechanisms of cytotoxicity by SFN, SFN has been suggested to be involved in the induction of DNA damage and inhibition of DNA repair. Recently, we reported on the potency of SFN in inducing single-ended double-strand breaks (DSBs) that are caused by the collision of replication forks with single-strand breaks (SSBs). However, the mechanism of SSB accumulation by SFN remains unclear. In this study, we examined the effect of SFN on SSB-inducing factors in HeLa cells. Although the inhibitory effect of SFN on DNA topoisomerase I was not observed, we found that the reduced form of glutathione (GSH; an antioxidant) level was decreased in an SFN concentration-dependent manner. Furthermore, the addition of ascorbic acid partially increased the viability of SFN-treated HeLa cells. We subsequently observed that poly(ADP-ribose) accumulated in SFN-treated HeLa cells, which occurs during early SSB repair. Collectively, these findings suggest that SFN may transiently induce SSBs via reactive oxygen species in HeLa cells.

DOI： 10.1111/gtc.13190

PubMed

researchmap

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2024.150977

researchmap

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Male and female reproductive tracts develop from anterior intermediate mesoderm with similar differentiation processes. The anterior intermediate mesoderm develops into the mesonephros, and the Wolffian duct initiates by epithelialization in the mesonephros. The Müllerian duct invaginates from the coelomic epithelium of the cranial mesonephros for ductal formation and is then regionalized into proximal to caudal female reproductive tracts. In this study, we focused on the epithelialization of the Wolffian duct, initiation of the Müllerian duct, and the regionalization step of the Müllerian ducts as a continuous process. By using intermediate mesodermal cells from mouse pluripotent stem cells, we identified that inhibition of SMAD2/3 signaling might be involved in the differentiation into mesenchymal cells, after which mesonephric cells might be then epithelialized during differentiation of the Wolffian duct. Aggregation of coelomic epithelial cells might be related to initiation of the Müllerian duct. Transcriptomic analysis predicted that consensus sequences of SMAD3/4 were enriched among highly expressed genes in the proximal Müllerian duct. SMAD2/3 signaling to regulate differentiation of the Wolffian duct was continuously activated in the proximal Müllerian duct and was involved in proximal and oviductal regionalization. Therefore, SMAD2/3 signaling may be finely tuned to regulate differentiation from initiation to regionalization steps.

DOI： 10.1002/2211-5463.13729

researchmap

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double‐strand breaks (DSBs) have been implicated in arsenic‐induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As₂O₃) on four different DSB repair pathways using the human pre‐B cell line Nalm‐6. Random integration analysis showed that As₂O₃ does not negatively affect non‐homologous end joining or polymerase theta‐mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As₂O₃ decreases the efficiency of homologous recombination (HR) and, less prominently, single‐strand annealing. Consistent with this finding, As₂O₃ decreased gene‐targeting efficiency, owing to a significant reduction in the frequency of HR‐mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one‐ended DSBs requiring HR for precise repair. Intriguingly, we found that As₂O₃ significantly enhances sensitivity to those anticancer agents in HR‐proficient cells. Our results suggest that arsenic‐induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic‐associated carcinogenesis and therapeutic options.

DOI： 10.1111/febs.16922

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Natural antioxidants derived from plants exert various physiological effects, including antitumor effects. However, the molecular mechanisms of each natural antioxidant have not yet been fully elucidated. Identifying the targets of natural antioxidants with antitumor properties in vitro is costly and time-consuming, and the results thus obtained may not reliably reflect in vivo conditions. Therefore, to enhance understanding regarding the antitumor effects of natural antioxidants, we focused on DNA, one of the targets of anticancer drugs, and evaluated whether antioxidants, e.g., sulforaphane, resveratrol, quercetin, kaempferol, and genistein, which exert antitumor effects, induce DNA damage using gene-knockout cell lines derived from human Nalm-6 and HeLa cells pretreated with the DNA-dependent protein kinase inhibitor NU7026. Our results suggested that sulforaphane induces single-strand breaks or DNA strand crosslinks and that quercetin induces double-strand breaks. In contrast, resveratrol showed the ability to exert cytotoxic effects other than DNA damage. Our results also suggested that kaempferol and genistein induce DNA damage via unknown mechanisms. Taken together, the use of this evaluation system facilitates the analysis of the cytotoxic mechanisms of natural antioxidants.

DOI： 10.3390/genes14020420

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

DNA polymerase ζ (Pol ζ) is a specialized Pol that is involved in translesion DNA synthesis (TLS), in particular, in the extension of primer DNA after bypassing DNA lesions. Previously, we established human cells that express a variant form of Pol ζ with an amino acid change of leucine 2618 to methionine (L2618M) in the catalytic subunit REV3L (DNA Repair, 45, 34-43, 2016). This amino acid change made the cells more sensitive to the mutagenicity of benzo[a]pyrene diol epoxide (BPDE). In this study, we embedded BPDE-N2-guanine at a defined position in the supF gene on the shuttle plasmid and introduced it to REV3 L2618M cells or the wild-type (WT) cells to examine how far Pol ζ L2618M extends the primer DNA after bypassing the lesion. The adduct induced primarily G to T and G to C at the adducted site in both cell lines, but generated additional sequence changes such as base substitutions, deletions and additions in the extension patch much more often in REV3 L2618M cells than in the WT cells. Mutations in the extension patch in REV3 L2618M cells occurred most often within 10 bps from the adducted site. Then, the number of mutations gradually decreased and no mutations were observed between 30 and 40 bps from the lesion. We concluded that human Pol ζ L2618M and perhaps WT Pol ζ extend the primer DNA up to approximately 30 bps from the lesion in vivo. The possibility of involvement of Pol ζ L2618M in the insertion step of TLS is discussed.

DOI： 10.1016/j.dnarep.2021.103052

PubMed

researchmap

Language：English  

[This corrects the article DOI: 10.1371/journal.ppat.1002558.].

DOI： 10.1371/journal.ppat.1008894

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Mammalian cells possess multiple pathways for repairing various types of DNA damage. Although the molecular mechanisms of each DNA repair pathway have been analyzed by biochemical analysis and cell biological analysis, interplay between different pathways has not been fully elucidated. In this study, using human Nalm-6-mutant cell lines, we analyzed the relationship between the base excision repair factor DNA polymerase β (POLβ) and DNA ligase IV (LIG4), which is essential for DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). We found that cells lacking both POLβ and LIG4 grew significantly more slowly than either single mutant, indicating cooperative functions of the two proteins in normal cell growth. To further investigate the genetic interaction between POLβ and LIG4, we examined DNA damage sensitivity of the mutant cell lines. Our results suggested that NHEJ acts as a backup pathway for repairing alkylation damage (when converted into DSBs) in the absence of POLβ. Surprisingly, despite the critical role of POLβ in alkylation damage repair, cells lacking POLβ exhibited increased resistance to camptothecin (a topoisomerase I inhibitor that induces DNA single-strand breaks), irrespective of the presence or absence of LIG4. A LIG4-independent increased resistance associated with POLβ loss was also observed with ionizing radiation; however, cells lacking both POLβ and LIG4 were more radiosensitive than either single mutant. Taken together, our findings provide novel insight into the complex interplay between different DNA repair pathways.

DOI： 10.1111/febs.15012

PubMed

researchmap

DOI： 10.2967/jnumed.118.223461

PubMed

researchmap

DOI： 10.2967/jnumed.118.218321

PubMed

researchmap

DOI： 10.1038/s41598-018-28690-6

PubMed

researchmap

DOI： 10.2967/jnumed.118.210252

PubMed

researchmap

Language：English   Publisher：Society of Nuclear Medicine Inc.  

DOI： 10.2967/jnumed.117.197673

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Gene targeting by homologous recombination provides the definitive tool for analyzing gene function. Promoterless vectors, which do not possess a promoter to drive marker gene expression, confer higher targeting efficiencies than conventional vectors due to the reduced number of drug-resistant clones. We here show that gene-targeting efficiency is typically >= 25% with the use of exon-trapping-type promoterless vectors in a human diploid cell line, Nalm-6. The efficiency of exon-trapping gene targeting was correlated with the level of target gene expression when a 2A peptide sequence was linked to the marker gene. Intriguingly, total arm length was not necessarily a determinant of targeting efficiency, as longer arms tend to enhance both homologous (targeted) and nonhomologous (nontargeted) integration of the vector; rather, the presence of an exon in the 5' arm led to a decreased targeting efficiency. Strikingly, loss of Rad54 did not severely affect the targeting efficiency of exon-trap vectors. Moreover, additional deletion of the Rad54 paralog Rad54B had limited impact on the high-efficiency gene targeting. These results indicate that targeted integration occurs in human cells even when both Rad54 and Rad54B are missing. These studies provide additional important insight into the contribution of various DNA repair factors on the targeting mechanics.

DOI： 10.1111/febs.14137

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Stress-induced activation of p53 is an essential cellular response to prevent aberrant cell proliferation and cancer development. The ubiquitin ligase MDM2 promotes p53 degradation and limits the duration of p53 activation. It remains unclear, however, how p53 persistently escapes MDM2-mediated negative control for making appropriate cell fate decisions. Here we report that TBP-like protein (TLP), a member of the TBP family, is a new regulatory factor for the p53-MDM2 interplay and thus for p53 activation. We found that TLP acts to stabilize p53 protein to ensure long-lasting p53 activation, leading to potentiation of p53-induced apoptosis and senescence after genotoxic stress. Mechanistically, TLP interferes with MDM2 binding and ubiquitination of p53. Moreover, single cell imaging analysis shows that TLP depletion accelerates MDM2-mediated nuclear export of p53. We further show that a cervical cancer-derived TLP mutant has less p53 binding ability and lacks a proliferation-repressive function. Our findings uncover a role of TLP as a competitive MDM2 blocker, proposing a novel mechanism by which p53 escapes the p53-MDM2 negative feedback loop to modulate cell fate decisions.

DOI： 10.1074/jbc.M116.763318

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioMed Central Ltd.  

Background: Interactions between genes and environment are critical factors for causing cancer in humans. The genotoxicity of environmental chemicals can be enhanced via the modulation of susceptible genes in host human cells. DNA polymerase kappa (Pol κ) is a specialized DNA polymerase that plays an important role in DNA damage tolerance through translesion DNA synthesis. To better understand the protective roles of Pol κ, we previously engineered two human cell lines either deficient in expression of Pol κ (KO) or expressing catalytically dead Pol κ (CD) in Nalm-6-MSH+ cells and examined cytotoxic sensitivity against various genotoxins. In this study, we set up several genotoxicity assays with cell lines possessing altered Pol κ activities and investigated the protective roles of Pol κ in terms of genotoxicity induced by mitomycin C (MMC), a therapeutic agent that induces bulky DNA adduct and crosslinks in DNA. Results: We introduced a frameshift mutation in one allele of the thymidine kinase (TK) gene of the KO, CD, and wild-type Pol κ cells (WT), thereby establishing cell lines for the TK gene mutation assay, namely TK+/- cells. In addition, we formulated experimental conditions to conduct chromosome aberration (CA) and sister chromatid exchange (SCE) assays with cells. By using the WT TK+/- and KO TK+/- cells, we assayed genotoxicity of MMC. In the TK gene mutation assay, the cytotoxic and mutagenic sensitivities of KO TK+/- cells were higher than those of WT TK+/- cells. MMC induced loss of heterozygosity (LOH), base pair substitutions at CpG sites and tandem mutations at GpG sites in both cell lines. However, the frequencies of LOH and base substitutions at CpG sites were significantly higher in KO TK+/-cells than in WT TK+/- cells. MMC also induced CA and SCE in both cell lines. The KO TK+/- cells displayed higher sensitivity than that displayed by WT TK+/- cells in the SCE assay. Conclusions: These results suggest that Pol κ is a modulating factor for the genotoxicity of MMC and also that the established cell lines are useful for evaluating the genotoxicity of chemicals from multiple endpoints in different genetic backgrounds of Pol κ.

DOI： 10.1186/s41021-016-0067-3

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase zeta (Pol zeta)-deficient (KO: knockout) and Pol zeta catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol zeta, i.e., D2781 and D2783, to alanines. Both Pol zeta KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N'-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol zeta plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.mrfmmm.2016.08.004

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase zeta (Pol zeta) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol zeta. The weak-catalytic activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TIC gene to measure the mutant frequencies. Doubling time and spontaneous TIC mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol zeta is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol zeta in genome integrity are discussed. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.dnarep.2016.06.002

Web of Science

PubMed

researchmap

Language：English   Publisher：NATURE PUBLISHING GROUP  

In successive reports from 2014 to 2015, X-ray repair cross-complementing protein 4 (XRCC4) has been identified as a novel causative gene of primordial dwarfism. XRCC4 is indispensable for non-homologous end joining (NHEJ), the major pathway for repairing DNA double-strand breaks. As NHEJ is essential for V(D)J recombination during lymphocyte development, it is generally believed that abnormalities in XRCC4 cause severe combined immunodeficiency. Contrary to expectations, however, no overt immunodeficiency has been observed in patients with primordial dwarfism harboring XRCC4 mutations. Here, we describe the various XRCC4 mutations that lead to disease and discuss their impact on NHEJ and V(D)J recombination.

DOI： 10.1038/jhg.2016.46

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

Gene targeting via homologous recombination, albeit highly inefficient in human cells, is considered a powerful tool for analyzing gene functions. Despite recent progress in the application of artificial nucleases for genome editing, safety issues remain a concern, particularly when genetic modification is used for therapeutic purposes. Therefore, the development of gene-targeting vectors is necessary for safe and sophisticated genetic modification. In this paper, we describe the effect of vector structure on random integration, which is a major obstacle in efficient gene targeting. In addition, we focus on the features of exon-trapping-type gene-targeting vectors, and discuss a novel strategy for negative selection to enhance gene targeting in human cells.

DOI： 10.1248/bpb.b15-00701

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

DNA polymerase (Pol ) is a specialized DNA polymerase involved in translesion DNA synthesis. Although its bypass activities across lesions are well characterized in biochemistry, its cellular protective roles against genotoxic insults are still elusive. To better understand the in vivo protective roles, we have established a human cell line deficient in the expression of Pol (KO) and another expressing catalytically dead Pol (CD), to examine the cytotoxic sensitivity to 11 genotoxins including ultraviolet C light (UV). These cell lines were established in a genetic background of Nalm-6-MSH+, a human lymphoblastic cell line that has high efficiency for gene targeting, and functional p53 and mismatch repair activities. We classified the genotoxins into four groups. Group 1 includes benzo[a]pyrene diolepoxide, mitomycin C, and bleomycin, where the sensitivity was equally higher in KO and CD than in the cell line expressing wild-type Pol (WT). Group 2 includes hydrogen peroxide and menadione, where hypersensitivity was observed only in KO. Group 3 includes methyl methanesulfonate and ethyl methanesulfonate, where hypersensitivity was observed only in CD. Group 4 includes UV and three chemicals, where the chemicals exhibited similar cytotoxicity to all three cell lines. The results suggest that Pol not only protects cells from genotoxic DNA lesions via DNA polymerase activities, but also contributes to genome integrity by acting as a non-catalytic protein against oxidative damage caused by hydrogen peroxide and menadione. The non-catalytic roles of Pol in protection against oxidative damage by hydrogen peroxide are discussed. Environ. Mol. Mutagen. 56:650-662, 2015. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/em.21961

Web of Science

PubMed

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis(-/-), DNA ligase 4(-/-) (LIG4(-/-)), and LIG4(-/-)Artemis(-/-) double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.dnarep.2015.04.004

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioMed Central Ltd.  

Background: Targeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals. In higher eukaryotes, non-homologous integration of targeting vectors occurs several orders of magnitude more frequently than does targeted integration, making the gene-targeting technology highly inefficient. For this reason, negative-selection strategies have been employed to reduce the number of drug-resistant clones associated with non-homologous vector integration, particularly when artificial nucleases to introduce a DNA break at the target site are unavailable or undesirable. As such, an exon-trap strategy using a promoterless drug-resistance marker gene provides an effective way to counterselect non-homologous integrants. However, constructing exon-trapping targeting vectors has been a time-consuming and complicated process. Results: By virtue of highly efficient att-mediated recombination, we successfully developed a simple and rapid method to construct plasmid-based vectors that allow for exon-trapping gene targeting. These exon-trap vectors were useful in obtaining correctly targeted clones in mouse embryonic stem cells and human HT1080 cells. Most importantly, with the use of a conditionally cytotoxic gene, we further developed a novel strategy for negative selection, thereby enhancing the efficiency of counterselection for non-homologous integration of exon-trap vectors. Conclusions: Our methods will greatly facilitate exon-trapping gene-targeting technologies in mammalian cells, particularly when combined with the novel negative selection strategy.

DOI： 10.1186/s13104-015-1241-6

Scopus

PubMed

researchmap

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

In aerobically growing cells, in which reactive oxygen species are produced, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine, which induces alterations in gene expression. Here we show that the human Auf1 protein, also called HNRNPD, binds specifically to RNA containing this oxidized base and may be involved in cellular processes associated with managing the problems caused by RNA oxidation. Auf1-deficient cells were constructed from human HeLa and Nalm-6 lines using two different targeting procedures. Both types of Auf1-deficient cells are viable, but exhibit growth retardation. The stability of messenger RNA for four different housekeeping genes was determined in Auf1-deficient and -proficient cells, treated with or without hydrogen peroxide. The level of oxidized messenger RNA was considerably higher in Auf1-deficient cells than in Auf1-proficient cells. Auf1 may play a role in the elimination of oxidized RNA, which is required for the maintenance of proper gene expression under conditions of oxidative stress. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2014.11.018

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4), the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

DOI： 10.1371/journal.pone.0108236

Web of Science

PubMed

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol kappa) bypasses benzo[a]pyrene diolepoxide-N-2-deoxyguanine (BPDE-N-2-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N-2-dG. In the present study, we established human cell lines that express wild-type Pol kappa (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol kappa (POLK-/-) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (-)- or (+)-trans-anti-BPDE-N-2-dG in the supF gene. The frequencies of mutations were in the order of POLK-/- > POLK+/- F171A/- both in (-)- and (+)-trans-anti-BPDE-N-2-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N-2-dG by Pol K and raise the possibility that the cognate substrates for Pol K are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.dnarep.2013.12.008

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2013.08.068

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs)
however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR. © 2013 Kurosawa et al.

DOI： 10.1371/journal.pone.0072253

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20-30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.

DOI： 10.1371/journal.pone.0061189

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54(-/-/)KU70(-/-) DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54(-/-/)LIG4(-/-) Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.

DOI： 10.1371/journal.pone.0060043

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The t(14;18) chromosomal translocation typically involves breakage at the bcl-2 major breakpoint region (MBR) to cause human follicular lymphoma. A theory to explain the striking propensity of the MBR breaks at three CpG clusters within the 175-bp MBR region invoked activation-induced deaminase (AID). In a test of that theory, we used here minichromosomal substrates in human pre-B cell lines. Consistent with the essential elements of the theory, we found that the MBR breakage process is indeed highly dependent on DNA methylation at the CpG sites and highly dependent on the AID enzyme to create lesions at peak locations within the MBR. Interestingly, breakage of the phosphodiester bonds at the AID-initiated MBR lesions is RAG dependent, but, unexpectedly, most are also dependent on Artemis. We found that Artemis is capable of nicking small heteroduplex structures and is even able to nick single-base mismatches. This raises the possibility that activated Artemis, derived from the unjoined D to J(H) DNA ends at the IgH locus on chromosome 14, nicks AID-generated TG mismatches at methyl CpG sites, and this would explain why the breaks at the chromosome 18 MBR occur within the same time window as those on chromosome 14.

DOI： 10.1128/MCB.01436-12

Web of Science

PubMed

researchmap

researchmap

researchmap

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The vertebrate RECQL4 (RECQ4) gene is thought to be the ortholog of budding yeast SLD2. However, RecQL4 contains within its C-terminus a RecQ-like helicase domain, which is absent in Sld2. We established human pre-B lymphocyte Nalm-6 cells, in which the endogenous RECQL4 gene was homozygously targeted such that the entire C-terminus would not be expressed. The RECQL4(Delta C/Delta C) cells behaved like the parental cells during unperturbed DNA replication or after treatment with agents that induce stalling of DNA replication forks, such as hydroxyurea (HIT). However, after exposure to ionizing radiation (IR), the RECQL4(Delta C/Delta C) cells exhibited hypersensitivity, inability to complete S phase and prematurely terminated or paused DNA replication forks. Deletion of BLM, a gene that also encodes a RecQ helicase, had the opposite phenotype; an almost wild-type response to ER, but hypersensitivity to HU. Targeting both RECQL4 and BLM resulted in viable cells, which exhibited mostly additive phenotypes compared with those exhibited by the RECQL4(Delta C/Delta C) and the BLM(-/-) cells. We propose that RecQL4 facilitates DNA replication in cells that have been exposed to ER.

DOI： 10.1093/carcin/bgs149

Web of Science

PubMed

researchmap

Language：English   Publisher：OXFORD UNIV PRESS  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Topoisomerase poisons such as the epipodophyllotoxin etoposide are widely used effective cytotoxic anticancer agents. However, they are associated with the development of therapy-related acute myeloid leukemias (t-AMLs), which display characteristic balanced chromosome translocations, most often involving the mixed lineage leukemia (MLL) locus at 11q23. MLL translocation breakpoints in t-AMLs cluster in a DNase I hypersensitive region, which possesses cryptic promoter activity, implicating transcription as well as topoisomerase II activity in the translocation mechanism. We find that 2-3% of MLL alleles undergoing transcription do so in close proximity to one of its recurrent translocation partner genes, AF9 or AF4, consistent with their sharing transcription factories. We show that most etoposide-induced chromosome breaks in the MLL locus and the overall genotoxicity of etoposide are dependent on topoisomerase II beta, but that topoisomerase II alpha and -beta occupancy and etoposide-induced DNA cleavage data suggest factors other than local topoisomerase II concentration determine specific clustering of MLL translocation breakpoints in t-AML. We propose a model where DNA double-strand breaks (DSBs) introduced by topoisomerase II beta into pairs of genes undergoing transcription within a common transcription factory become stabilized by anti-topoisomerase II drugs such as etoposide, providing the opportunity for illegitimate end joining and translocation.

DOI： 10.1073/pnas.1204406109

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Artemis is an endonuclease that opens coding hairpin ends during V(D)J recombination and has critical roles in postirradiation cell survival. A direct role for the C-terminal region of Artemis in V(D)J recombination has not been defined, despite the presence of immunodeficiency and lymphoma development in patients with deletions in this region. Here, we report that the Artemis C-terminal region directly interacts with the DNA-binding domain of Ligase IV, a DNA Ligase which plays essential roles in DNA repair and V(D)J recombination. The Artemis-Ligase IV interaction is specific and occurs independently of the presence of DNA and DNA-protein kinase catalytic subunit (DNA-PKcs), another protein known to interact with the Artemis C-terminal region. Point mutations in Artemis that disrupt its interaction with Ligase IV or DNA-PKcs reduce V(D)J recombination, and Artemis mutations that affect interactions with Ligase IV and DNA-PKcs show additive detrimental effects on coding joint formation. Signal joint formation remains unaffected. Our data reveal that the C-terminal region of Artemis influences V(D)J recombination through its interaction with both Ligase IV and DNA-PKcs.

DOI： 10.1084/jem.20111437

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASP(-/-) cells showed impaired adhesive capacity and polarization to plate-bound anti-CD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASP(-/-) Nalm-6 cells in a dose-dependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.

DOI： 10.1007/s12185-012-1013-1

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Lens epithelium-derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase (IN) that interacts with IN through its IN binding domain (IBD) and tethers the viral pre-integration complex to the host cell chromatin. Here we report the generation of a human somatic LEDGF/p75 knockout cell line that allows the study of spreading HIV-1 infection in the absence of LEDGF/p75. By homologous recombination the exons encoding the LEDGF/p75 IBD (exons 11 to 14) were knocked out. In the absence of LEDGF/p75 replication of laboratory HIV-1 strains was severely delayed while clinical HIV-1 isolates were replication-defective. The residual replication was predominantly mediated by the Hepatoma-derived growth factor related protein 2 (HRP-2), the only cellular protein besides LEDGF/p75 that contains an IBD. Importantly, the recently described IN-LEDGF/p75 inhibitors (LEDGINs) remained active even in the absence of LEDGF/p75 by blocking the interaction with the IBD of HRP-2. These results further support the potential of LEDGINs as allosteric integrase inhibitors.

DOI： 10.1371/journal.ppat.1002558

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12185-012-1013-1

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Electroporation is a powerful and convenient means for transfection of nonviral vectors into mammalian cells, providing an essential tool for numerous applications including gene targeting via homologous recombination. Recent evidence clearly suggests that high-efficiency gene transfer can be achieved in most cell lines by nucleofection, an electroporation-based transfection method that allows transfected vectors to directly enter the nucleus. In this paper, we analyze the effectiveness of nucleofection for gene targeting using human pre-B cells. For this, we tested 93 different transfection conditions, and found several conditions that gave high (similar to 80%) transfection efficiency with low cytotoxicity (similar to 70% survival rate). Remarkably, under the optimal nucleofection conditions, the gene-targeting efficiency was similar to 2-5-fold higher than that achieved with conventional electroporation methods. We also found that nucleofection conditions with high transfection efficiency and low cytotoxicity tend to provide high gene-targeting efficiency. Our results provide significant implications for gene targeting, and suggest that nucleofection-based nonviral gene transfer is useful for systematic generation of human gene-knockout cell lines. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2011.11.003

Web of Science

PubMed

researchmap

Language：English   Publisher：JAPAN RADIATION RESEARCH SOC  

Artemis is a structure-specific endonuclease when associated with and phosphorylated by DNA-dependent protein kinase catalytic subunit. This structure-specific endonuclease is responsible for the resolution of hairpin coding ends in V(D)J recombination. In DNA double-strand break repair, Artemis is implicated in the end-processing step of the non-homologous end-joining (NHEJ) pathway. Recently, we have demonstrated that the involvement of Artemis in NHEJ depends on the type of DNA damage. Interestingly, recent evidence suggests that the end-processing activity is not the only function of Artemis. Indeed, Artemis is rapidly phosphorylated by ataxia telangiectasia mutated in response to DNA damage, and such phosphorylation of Artemis appears to be involved in the regulation of cell cycle checkpoints. These findings suggest that Artemis is a multifunctional protein participating in the maintenance of genome integrity at two distinct levels; one at the end processing step of NHEJ, and the other at the signaling pathway of cell cycle regulation. Therefore, understanding Artemis function may give us profound insights into the DNA repair network. in this review, we summarize the functions and regulation of Artemis.

DOI： 10.1269/jrr.10017

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The 2-oxoglutarate and iron dependent dioxygenase family are crucial for cellular adaptation to changes in oxygen concentration. We found that cells with OGFOD1 gene silencing in this family showed resistance to cell death under ischemia, and cDNA microarray analysis of OGFOD1 knockout human cells revealed downregulation of ATPAF1. Although reintroduction of the OGFOD1 wild-type gene to OGFOD1 KO cells restored ATPAF1 mRNA levels, the catalytically inactive OGFOD1 mutants did not. Furthermore, introduction of ATPAF1 gene to OGFOD1 KO cells induced ischemic cell death. Thus, OGFOD1 plays an important role in ischemic cell survival and an OGFOD1 iron binding residue is required for ATPAF1 gene expression. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2010.06.015

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e. g. embryonic stem cells). Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations.

DOI： 10.1371/journal.pone.0009846

Web of Science

PubMed

researchmap

Language：English  

From in vitro studies, flap endonuclease 1 (FEN1) has been proposed to play a role in the long patch (LP) base excision repair (BER) subpathway. Yet the role of FEN1 in BER in the context of the living vertebrate cell has not been thoroughly explored. In the present study, we cloned a DT40 chicken cell line with a deletion in the FEN1 gene and found that these FEN1-deficient cells exhibited hypersensitivity to H(2)O(2). This oxidant produces genotoxic lesions that are repaired by BER, suggesting that the cells have a deficiency in BER affecting survival. In experiments with extracts from the isogenic FEN1 null and wild-type cell lines, the LP-BER activity of FEN1 null cells was deficient, whereas repair by the single-nucleotide BER subpathway was normal. Other consequences of the FEN1 deficiency were also evaluated. These results illustrate that FEN1 plays a role in LP-BER in higher eukaryotes, presumably by processing the flap-containing intermediates of BER.

DOI： 10.1158/1541-7786.MCR-09-0253

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Bloom syndrome confers strong predisposition to malignancy in multiple tissue types. The Bloom syndrome patient (BLM) protein defective in the disease biochemically functions as a Holliday junction dissolvase and human cells lacking functional BLM show 10-fold elevated rates of sister chromatid exchange. Collectively, these phenomena suggest that dysregulated mitotic recombination drives the genomic instability underpinning the development of cancer in these individuals. Here we use physical analysis of the highly repeated, highly self-similar human ribosomal RNA gene clusters as sentinel biomarkers for dysregulated homologous recombination to demonstrate that loss of BLM protein function causes a striking increase in spontaneous molecular level genomic restructuring. Analysis of single-cell derived sub-clonal populations from wild-type human cell lines shows that gene cluster architecture is ordinarily very faithfully preserved under mitosis, but is so unstable in cell lines derived from BLMs as to make gene cluster architecture in different sub-clonal populations essentially unrecognizable one from another. Human cells defective in a different RecQ helicase, the WRN protein involved in the premature aging Werner syndrome, do not exhibit the gene cluster instability (GCI) phenotype, indicating that the BLM protein specifically, rather than RecQ helicases generally, holds back this recombination-mediated genomic instability. An ataxia-telangiectasia defective cell line also shows elevated rDNA GCI, although not to the extent of BLM defective cells. Genomic restructuring mediated by dysregulated recombination between the abundant low-copy repeats in the human genome may prove to be an important additional mechanism of genomic instability driving the initiation and progression of human cancer.

DOI： 10.1093/hmg/ddp282

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DNA transfection can be too inefficient to establish a desired number of stable transfectants, particularly in lymphocytes; however, this could be circumvented by increasing the absolute frequency of random integration. In this paper, we show that treating cells with topoisomerase II inhibitor following electroporation greatly (similar to 10- to 20-fold) enhances random integration of input DNA in a human pre-B lymphocyte cell line, Nalm-6. With the use of various kinds of topoisomerase II-targeting agents, we also present evidence that topoisomerase II alpha inhibition is critical for the enhancement of random integration, while the contribution of topoisomerase II beta may be negligible. As topoisomerase II alpha is highly expressed in vigorously growing cells, our results show that topoisomerase II alpha inhibition provides a promising way of enhancing random integration in virtually all cultured cell lines. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.03.047

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs-zebrafish ZfL2-2 and human L1-in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

DOI： 10.1371/journal.pgen.1000461

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Oxygen is essential for the life of aerobic organisms, but reactive oxygen species (ROS) derived from oxygen can be a threat for it. Many genes are involved in generation of ROS, but not much attention has been focused on the reactions from which ROS are generated. We therefore screened for mutants that showed an increased sensitivity to oxidative stress in the nematode Caenorhabditis elegans, and isolated a novel mutant, oxy-4(qa5001). This mutant showed an increased sensitivity to a high concentration of oxygen, and decreased longevity at 20 degrees C but not at 26 degrees C. The genetic analysis has revealed that oxy-4 had a causative mutation in an [FeFe]-hydrogenase-like gene (Y54H5A.4). In the yeast Saccharomyces cerevisiae, a deletion of NAR1, a possible homologue of oxy-4, also caused a similar increased sensitivity to oxygen. [FeFe]-hydrogenases are enzymes that catalyze both the formation and the splitting of molecular hydrogen, and function in anaerobic respiration in anaerobes. In contrast, [FeFe]-hydrogenase-like genes identified in aerobic eukaryotes do not generate hydrogen, and its functional roles are less understood. Our results suggested that [FeFe]-hydrogenase-like genes were involved in the regulation of sensitivity to oxygen in S. cerevisiae and C. elegans.

DOI： 10.1111/j.1365-2443.2009.01282.x

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Oxygen is essential for the life of aerobic organisms, but reactive oxygen species (ROS) derived from oxygen can be a threat for it. Many genes are involved in generation of ROS, but not much attention has been focused on the reactions from which ROS are generated. We therefore screened for mutants that showed an increased sensitivity to oxidative stress in the nematode Caenorhabditis elegans, and isolated a novel mutant, oxy-4(qa5001). This mutant showed an increased sensitivity to a high concentration of oxygen, and decreased longevity at 20 degrees C but not at 26 degrees C. The genetic analysis has revealed that oxy-4 had a causative mutation in an [FeFe]-hydrogenase-like gene (Y54H5A.4). In the yeast Saccharomyces cerevisiae, a deletion of NAR1, a possible homologue of oxy-4, also caused a similar increased sensitivity to oxygen. [FeFe]-hydrogenases are enzymes that catalyze both the formation and the splitting of molecular hydrogen, and function in anaerobic respiration in anaerobes. In contrast, [FeFe]-hydrogenase-like genes identified in aerobic eukaryotes do not generate hydrogen, and its functional roles are less understood. Our results suggested that [FeFe]-hydrogenase-like genes were involved in the regulation of sensitivity to oxygen in S. cerevisiae and C. elegans.

DOI： 10.1111/j.1365-2443.2009.01282.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

DNA topoisomerase I (Top1) is a ubiquitous nuclear enzyme that plays essential roles in various cellular processes, such as transcription or replication. Agents that target Top1, involving camptothecin and its derivatives, are among the most effective anticancer drugs used in the clinic. Previous work has suggested that the level of Top1 expression correlates with the cytotoxicity of camptothecin, but no direct evidence has been provided thus far in the context of human cells with a strictly isogenic genetic background. In this study, we perform heterozygous disruption of the Top1 gene (TOP1) by gene targeting in a human pre-B cell line, Nalm-6, which is karyotypically stable and normal for p53 status. We show that the heterozygous loss of the TOP1 gene does confer cellular resistance to camptothecin, to an extent comparable to that observed in the absence of functional p53 protein. Such a tolerance was not observed with other agents that target DNA topoisomerase II. Our results provide direct evidence that human cells with decreased Top1 levels are significantly more resistant to killing by camptothecin than are otherwise isogenic cells.

DOI： 10.1248/bpb.32.724

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Conditional gene knockout by homologous recombination combined with an inducible gene expression system is a powerful approach for studying gene function, although homologous recombination in human cells occurs infrequently. The tetracycline-regulated gene expression (Tet-Off) system is a convenient method for achieving conditional gene knockout, but it is not always promising in Nalm-6. a rare human cell line highly effective for gene targeting. Here we modified the Tet-Off system and applied it to the Nalm-6 cell line successfully by using an internal ribosome entry site to drive a selectable marker from the same tetracycline-responsive promoter for the transgene. We also inserted the gene for the tetracycline-controlled transactivator under the control of a potent CAG promoter. These modifications enabled LIS to easily obtain rare clones that express optimal amounts of tetracycline-regulated transgenes. We thereby generated a 'tetracycline-inducible conditional gene knockout' for the proliferation-associated SNF2-like gene (PASG) in a Nalm-6 cell line, in which the expression of PASG can be depleted in a tetracycline-dependent manner on a knockout background. This method is applicable to any human genes, making this gene-targeting system using the Nalm-6 cell line a promising tool for analyzing gene function. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2008.12.010

Web of Science

PubMed

researchmap

Language：Japanese  

PubMed

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2009184530

Language：English   Publisher：JAPAN RADIATION RESEARCH SOC  

Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) in mammalian species. Upon DSB induction, a living cell quickly activates the NHEJ pathway comprising of multiple molecular events. However, it has been difficult to analyze the initial phase of DSB responses in living cells, primarily due to technical limitations. Recent advances in real-time imaging and site-directed DSB induction using laser microbeam allow us to monitor the spatiotemporal dynamics of NHEJ factors in the immediate-early phase after DSB induction. These new approaches, together with the use of cell lines deficient in each essential NHEJ factor, provide novel mechanistic insights into DSB recognition and protein assembly on DSBs in the NHEJ pathway. In this review, we provide an overview of recent progresses in the imaging analyses of the NHEJ core factors. These studies strongly suggest that the NHEJ core factors are pre-assembled into a large complex on DSBs prior to the progression of the biochemical reactions in the NHEJ pathway. Instead of the traditional step-by-step assembly model from the static view of NHEJ, a novel model for dynamic protein assembly in the NHEJ pathway is proposed. This new model provides important mechanistic insights into the protein assembly at DSBs and the regulation of DSB repair.

DOI： 10.1269/jrr.08119

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G(1) cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated historic foci (gamma-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase II alpha (top2 alpha) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2 alpha knockout cells (top2 alpha(+/-)) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2 alpha(+/+) or top2 beta(-/-) cells. Specificity for top2 alpha was confirmed using top2 alpha and top2 beta small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2 alpha. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2 alpha activity. [Cancer Res 2009;69(3):948-57]

DOI： 10.1158/0008-5472.CAN-08-1437

Web of Science

PubMed

researchmap

Language：English   Publisher：IRCA-BSSA  

Tightly controlled expression of transgenes in mammalian cells is an important tool for biological research, drug discovery, and future genetic therapies. The tetracycline-regulated gene depletion (Tet-Off) system has been widely used to control gene activities in mammalian cells, because it allows strict regulation of transgenes but no pleiotropic effects of prokaryotic regulatory proteins. However, the Tet-Off system is not compatible with every cell type and this is the main remaining obstacle left for this system. Recently, we overcame this problem by inserting an internal ribosome entry site (IRES) to drive a selectable marker from the same tetracycline-responsive promoter for the transgene. We also employed a CMV immediate early enhancer/beta-actin (CAG) promoter to express a Tet-controlled transactivator. Indeed, the Tet-Off system with these technical modifications was applied successfully to the human pre-B Nalm-6 cell line in which conventional Tet-Off systems had not worked efficiently. These methodological improvements should be applicable for many other mammalian proliferating cells. In this review we give an overview and introduce a new method for the improved application of the Tet-Off system.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

KU70(-/-) and DNA-PKcs(-/-/-)chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. in the presence of 100 mu M etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70(-/-) and DNA-PKcs(-/-/-) cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis(-/-) cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4(-/-), XRCC4(-), and XLF-/- cells were capable to induce apoptosis. When cells Were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cellsig.2008.07.006

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In higher animal cells, the principal limitation of gene-targeting technology is the extremely low efficiency of targeted integration, which occurs three to four orders of magnitude less frequently than random integration. Assuming that random integration mechanistically involves non-homologous end-joining (NHEJ), inactivation of this pathway should reduce random integration and may enhance gene targeting. To test this possibility, we examined the frequencies of random and targeted integration in NHEJ-deficient chicken DT40 and human Nalm-6 cell lines. As expected, loss of NHEJ resulted in drastically reduced random integration in DT40 cells. Unexpectedly, however, this was not the case for Nalm-6 cells, indicating that NHEJ is not the sole mechanism of random integration. Nevertheless, we present evidence that NHEJ inactivation can lead to enhanced gene targeting through a reduction of random integration and/or an increase in targeted integration by homologous recombination. Most intriguingly, our results show that, in the absence of functional NHEJ, random integration of targeting vectors occurs more frequently than non-targeting vectors (harboring no or little homology to the host genome), implying that suppression of NHEJ-independent random integration events is needed to greatly enhance gene targeting in animal cells.

DOI： 10.1093/nar/gkn649

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The repair of DNA double-strand breaks by nonhomologous end-joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Central to the molecular mechanism of NHEJ is DNA ligase IV/XRCC4/XLF complex, which rejoins the DNA. During adenovirus (Ad5) infection, ligase IV is targeted for degradation in a process that requires expression of the viral E1B 55k and E4 34k proteins while XRCC4 and XLF protein levels remain unchanged. We show that in Ad5-infected cells, loss of ligase IV is accompanied by loss of DNA binding by XRCC4. Expression of E1B 55k and E4 34k was sufficient to cause loss of ligase IV and loss of XRCC4 DNA binding. Using ligase IV mutant human cell lines, we determined that the absence of ligase IV, and not expression of viral proteins, coincided with inhibition of DNA binding by XRCC4. In ligase IV mutant human cell lines, DNA binding by XLF was also inhibited. Expression of both wild-type and adenylation-mutant ligase IV in ligase IV-deficient cells restored DNA binding by XRCC4. These data suggest that the intrinsic DNA-binding activities of XRCC4 and XLF may be subject to regulation and are down regulated in human cells that lack ligase IV.

DOI： 10.1093/nar/gkn552

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Non-homologous end joining (NHEJ) plays a major role in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs), especially during the G1-phase of the cell cycle. Using a flow cytometric cell sorter, we fractionated G1- and S/G2-phase cells based on size to assess the DSB-repair activity in NHEJ factor-deficient DT40 and Nalm-6 cell lines. Colony formation assays revealed that the X-ray sensitivities of the G1-enriched populations correctly reflected the DSB-repair activities of both the DT40 and Nalm-6 cell lines. Furthermore, as assessed by gamma-H2AX foci formation, the sorted cells exhibited less DNA damage than chemically synchronized cells. Given that it does not use fluorescent labeling or chemical agents, this method of cell sorting is simpler and less toxic than other methods, making it applicable to a variety of cell lines, including those that cannot be synchronized by standard chemical treatments. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.05.147

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Topoisomerase II ( Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2 beta is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2 beta is nonessential for growth and has recently been implicated in treatment- associated secondary malignancies, highlighting the validity of a Top2 beta-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c] phenanthridine alkaloid, targets Top2 beta and not Top2 beta in vivo. Unlike other Top2 inhibitors, NK314 induces Top2- DNA complexes and double- strand breaks ( DSBs) in an beta isoform- specific manner. Heterozygous disruption of the humanTOP2 beta gene confers increased NK314 resistance, whereas TOP2 beta homozygous knock- out cells display increased NK314 sensitivity, indicating that the beta isoform is the cellular target. Wefurther show that the absence of Top2 beta does not alleviate NK314 hypersensitivity of cells deficient in non- homologous end- joining, a critical pathway for repairing Top2- mediated DSBs. Our results indicate that NK314 acts as a Top2 beta- specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock- out cell lines are useful in assessingDNAdamage and repair induced by potential topoisomerase- targeting agents.

DOI： 10.1074/jbc.M803936200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC  

Artemis is a recently identified factor involved in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Here, we performed targeted disruption of the Artemis gene (ARTEMIS) in the human pre-B cell line Nalm-6. Unexpectedly, we found that cells lacking Artemis exhibit increased sensitivity to low doses, but not high doses, of ionizing radiation. We also show that ARTEMIS-deficient cells are hypersensitive to the topoisomerase II inhibitor etoposide, but to a much lesser extent than cells lacking DNA ligase IV, a critical component of NHEJ. Unlike DNA ligase IV-deficient cells, ARTEMIS-deficient cells were not hypersensitive to ICRF-193, a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA cleavable complexes. Collectively, our results suggest that Artemis only partially participates in the NHEJ pathway to repair DSBs in human somatic cells.

DOI： 10.1089/dna.2007.0649

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press  

Gene targeting provides a powerful means for studying gene function by a reverse genetic approach. Despite recent rapid progress in gene knockdown technologies, gene knockout studies using human somatic cells will be of greater importance for analyzing the functions of human genes in greater detail. Although the frequency of gene targeting is typically very low in human cultured cells, we have recently shown that a human precursor B cell line, Nalm-6, exceptionally allows for high-efficiency gene targeting by homologous recombination. In addition, we have developed a quick and simplified method to construct gene-targeting vectors, which is applicable to all sequenced organisms as well as embryonic stem cells. The combination of the simplified vector construction technology and the highly efficient gene-knockout system using Nalm-6 cells has enabled us to disrupt virtually any locus of the human genome within one month. Our system will greatly facilitate gene-knockout studies in human cells. © 2008 Humana Press Inc.

DOI： 10.1007/978-1-59745-232-8_2

Scopus

PubMed

researchmap

Language：Japanese  

Scopus

PubMed

researchmap

Language：English   Publisher：IRCA-BSSA  

Gene targeting by homologous recombination is a powerful tool to precisely manipulate the genome in order to study the function of a gene of interest (GOI). Indeed, it has become a routine methodology in yeasts, murine embryonic stem cells, and a chicken DT40 cell line. However, gene targeting has not been used often in human somatic cells to date since the relative efficiency of gene targeting (the ratio of homologous integrations to random integrations) is remarkably low. In this review, we introduce a fundamental strategy and a protocol to generate a null allele and/or 'tetracycline-inducible conditional gene knockout' for the GOI by gene targeting in the human Nalm-6 pre-B cell line. The Nalm-6 is a rare cell line in which gene targeting by homologous recombination takes place efficiently, and it carries a stable near-diploid karyotype with a doubling time of around 20 h. In addition, the tetracycline-regulated gene depletion (Tet-Off) system is steadily applicable to this cell line. Therefore, gene targeting systems using the Nalm-6 cell are used increasingly and offer promise in the study of human gene functions. This review should prove useful to researchers in a wide rage of fields.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

5- Bromodeoxyuridine (BrdU) is known to modulate expression of particular genes, and eventually arrest cell division in mammalian and yeast cells. To study a molecular basis for these phenomena, we adopted a genetic approach with a yeast cell system. We screened multicopy suppressor genes that confer resistance to BrdU with a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae. One of such genes was found to encode Ham1 protein, which was originally identified as a possible triphosphatase for N-6- hydroxylaminopurine triphosphate. Consistent with this, overexpression of the HAM1 gene reversed growth arrest caused by BrdU, and blocked incorporation of BrdU into genomic DNA. On the contrary, disruption of the gene sensitized cells to BrdU. A crude extract from Ham1- overproducing cells showed a high activity to hydrolyze BrdUTP to BrdUMP and pyrophosphate in addition to abnormal purine nucleotides. Purified recombinant Ham1 protein showed the same activity. These results demonstrate that Ham1 protein detoxifies abnormal pyrimidine as well as purine nucleotides.

DOI： 10.1007/s00294-007-0152-z

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

5- Bromodeoxyuridine (BrdU) is known to modulate expression of particular genes, and eventually arrest cell division in mammalian and yeast cells. To study a molecular basis for these phenomena, we adopted a genetic approach with a yeast cell system. We screened multicopy suppressor genes that confer resistance to BrdU with a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae. One of such genes was found to encode Ham1 protein, which was originally identified as a possible triphosphatase for N-6- hydroxylaminopurine triphosphate. Consistent with this, overexpression of the HAM1 gene reversed growth arrest caused by BrdU, and blocked incorporation of BrdU into genomic DNA. On the contrary, disruption of the gene sensitized cells to BrdU. A crude extract from Ham1- overproducing cells showed a high activity to hydrolyze BrdUTP to BrdUMP and pyrophosphate in addition to abnormal purine nucleotides. Purified recombinant Ham1 protein showed the same activity. These results demonstrate that Ham1 protein detoxifies abnormal pyrimidine as well as purine nucleotides.

DOI： 10.1007/s00294-007-0152-z

Web of Science

researchmap

Language：English   Publisher：日本癌学会  

researchmap

Language：English   Publisher：日本癌学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC  

The Bloom syndrome helicase BLM and the tumor-suppressor protein p53 play important roles in preserving genome integrity. Here, we knock out the genes for BLM and p53 in a human pre-B-cell line, Nalm-6. We show that p53 plays an important role in cell proliferation, but not apoptosis, when BLM is absent. Intriguingly, despite the apoptotic function of p53, BLM(-/-)TP53(-/-) cells were more sensitive than either single mutant to etoposide, an anticancer agent that poisons DNA topoisomerase II. Our results suggest a direct, BLM-independent role for p53 in etoposide-induced, topoisomerase II-mediated DNA damage in human cells.

DOI： 10.1089/dna.2007.0578

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：放射線生物研究会  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2008119203

Language：English   Publishing type：Research paper (scientific journal)   Publisher：EATON PUBLISHING CO  

Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway (R) technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human preB cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest call be created within 2-3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian-particularly human-genes.

DOI： 10.2144/000112233

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Ionizing radiation (IR) induces a variety of DNA lesions. The most significant lesion is a DNA double-strand break (DSB), which is repaired by homologous recombination or nonhomologous end joining (NHEJ) pathway. Since we previously demonstrated that IR-responsive protein 53BP1 specifically enhances activity of DNA ligase IV, a DNA ligase required for NHEJ, we investigated responses of 53BP1-deficient chicken DT40 cells to IR. 53BP1-deficient cells showed increased sensitivity to X-rays during G1 phase. Although intra-S and G2/M checkpoints were intact, the frequency of isochromatid-type chromosomal aberrations was elevated after irradiation in 53BP1-deficient cells. Furthermore, the disappearance of X-ray-induced gamma-H2AX foci, a marker of DNA DSBs, was prolonged in 53BP1-deficient cells. Thus, the elevated X-ray sensitivity in G1 phase cells was attributable to repair defect for IR-induced DNA-damage. Epistasis analysis revealed that 53BP1 plays a role in a pathway distinct from the Ku-dependent and Artemis-dependent NHEJ pathways, but requires DNA ligase IV. Strikingly, disruption of the 53BP1 gene together with inhibition of phosphatidylinositol 3-kinase family by wortmannin completely abolished colony formation by cells irradiated during G1 phase. These results demonstrate that the 53BP1-dependent repair pathway is important for survival of cells irradiated with IR during the G1 phase of the cell cycle.

DOI： 10.1111/j.1365-2443.2006.00989.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Gene targeting via homologous recombination is a powerful tool for studying gene function, but the targeting efficiency in human cell lines is too low for generating knockout mutants. Several cell lines null for the gene responsible for Bloom syndrome, BLM, have shown elevated targeting efficiencies. Therefore, we reasoned that gene targeting would be enhanced by transient suppression of BLM expression by RNA interference. To test this, we constructed a gene correction assay system to measure gene targeting frequencies using a disrupted hypoxanthine phosphoribosyltransferase (HPRT) locus in the human HT1080 cell line, and examined the effect of small interfering RNA (siRNA) for BLM on gene targeting. When HPRT-null cells pretreated with BLM siRNA were co-transfected with the siRNA and a gene correction vector, the gene targeting frequency was elevated three-fold, while the random integration frequency was marginally affected. Remarkably, in BLM heterozygous (+/-) cells derived from HPRT-null cells, the BLM siRNA treatment gave more than five-fold higher targeting frequencies, even with one-tenth the amount of BLM siRNA used for BLM+/+ cells. Furthermore, in the human pre-B cell line Nalm-6, the siRNA treatment enhanced gene targeting 6.3-fold and > 5.8-fold at the HPRT and adenine phosphoribosyltransferase (APRT) loci, respectively. These results indicate that transient suppression of BLM expression by siRNA stimulates gene targeting in human cells, facilitating a further improvement of gene targeting protocols for human cell lines.

DOI： 10.1111/j.1365-2443.2006.00944.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1(-/-) DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB-binding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1(-/-) cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.

DOI： 10.1038/sj.emboj.7601015

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Ku, the heterodimer of Ku70 and Ku86, plays crucial roles in non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. It has recently been reported that heterozygous disruption of the human KU86 locus results in haploinsufficient phenotypes, including retarded growth, increased radiosensitivity, elevated p53 levels and shortened telomeres. in this paper, however, we show that heterozygous inactivation of either the KU70 or KU86 gene does not cause any defects in cell proliferation or DSB repair in human somatic cells. Moreover, although these heterozygous cell lines express reduced levels of both Ku70 and Ku86, they appear to maintain overall genome integrity with no elevated p53 levels or telomere shortening. These results clearly indicate that Ku haploinsufficiency is not a commonly observed phenomenon in human cells. Our data also suggest that the impact of KU70/KU86 mutations on telomere metabolism varies between cell types in humans. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.dnarep.2005.10.008

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC  

Gene targeting provides a powerful means for analyzing gene function, as exemplified by knockout mouse studies and recent work with the highly recombinogenic chicken DT40 B-lymphocyte line. In human cultured cells, however, the low frequency of gene targeting is a serious barrier to efficiently generate knockout clones. Moreover, commonly used human cell lines are karyotypically abnormal or unstable. Here, we show using promoterless targeting constructs that Nalm-6, a human pre-B ALL cell line, is highly proficient for gene targeting by homologous recombination. Indeed, the efficiency of TP53 gene targeting in Nalm-6 appears nearly two orders of magnitude higher than that in HCT116, a colon cancer cell line popularly used for gene targeting. Expression analysis revealed a lack of MSH2 expression in this cell line. As Nalm-6 has a stable neardiploid karyotype with normal p53 status, our results underscore the usefulness of Nalm-6 for gene knockout studies in humans.

DOI： 10.1089/dna.2006.25.19

Web of Science

PubMed

researchmap

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Homologous recombination (HR) requires nuclease activities at multiple steps, but the contribution of individual nucleases to the processing of double-strand DNA ends at different stages of HR has not been clearly defined. We used chicken DT40 cells to investigate the role of flap endonuclease 1 (Fen-1) in HR. FEN-1-deficient cells exhibited a significant decrease in the efficiency of immunoglobulin gene conversion while being proficient in recombination between sister chromatids, suggesting that Fen-1 may play a role in HR between sequences of considerable divergence. To clarify whether sequence divergence at DNA ends is truly the reason for the observed HR defect in FEN-1(-/-) cells we inserted a unique I-SceI restriction site in the genome and tested various donor and recipient HR substrates. We found that the efficiency of HR-mediated DNA repair was indeed greatly diminished when divergent sequences were present at the DNA break site. We conclude that Fen-1 eliminates heterologous sequences at DNA damage site and facilitates DNA repair by HR.

DOI： 10.1128/MCB.25.16.6948-6955.2005

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC  

DNA topoisomerase II (Top2) inhibitors are useful as anticancer agents, mostly by virtue of their ability to induce DNA double-strand breaks (DSBs). These DSBs are repaired almost exclusively by Rad52-dependent homologous recombination (HR) in yeast. However, we have recently shown that in vertebrate cells such lesions are primarily repaired by nonhomologous end-joining, but not HR. This finding, taken together with previous observations that disruption of RAD52 does not severely affect HR in vertebrate cells, makes it highly unlikely that Rad52 contributes to the repair of Top2-mediated DNA damage. However, in this paper we show that chicken cells lacking Rad52 do exhibit increased sensitivity to the Top2 inhibitor VP-16. Remarkably, the level of hypersensitivity of RAD52-null cells was comparable to that of RAD54-null cells, albeit only at high doses. Our data thus provide the first demonstration of a major repair defect associated with loss of Rad52 in vertebrate cells.

DOI： 10.1089/dna.2005.24.388

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Cytogenetic analysis was performed on peripheral lymphocytes collected from 20 cattle clones (19 showed no overt phenotypic abnormalities except for high birth weight while 1 exhibited left forelimb contracture), the donor cell cultures from which they were derived and lymphocytes from six insemination produced control cattle. All animals and cell cultures had a modal chromosome number of 60. The frequency of abnormal cells for donor cell cultures, clones, and controls was 6.68 +/- 0.30%, 5.30 +/- 5.49%, and 5.08 +/- 1.04%, respectively, and did not differ significantly among the groups. There were, however, two clones derived from different donor cell cultures with high incidences of 21.29% and 20.13%, of abnormal cells consisting of pseudodiploid (near-diploid), near-triploid and neartetraploid, and tetraploid cells. Among these two clones, one had only a few encloreduplicated nuclei although further studies are necessary to precisely define the cytological origin and nature of the abnormal cells. The clones were evaluated at multiple time points for up to 20 months of age and the incidence of abnormal lymphocytes remained stable indicating that the chromosomally abnormal nuclei found in cloned animals was not a transient event. These results show that the majority of phenotypically normal clones have normal chromosomal make up but that instability of chromosome number can occur in clones that are phenotypically normal. Therefore, cytogenetical evaluation of peripheral lymphocytes and other tissues with follow up of the phenotypical consequences of these abnormalities is warranted even in phenotypically normal clones. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/mrd.20283

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Endonuclease G (endo G) is one of the most abundant nucleases in eukaryotic cells. It is encoded in the nucleus and imported to the mitochondrial intermembrane space. This nuclease is active on single- and double-stranded DNA. We genetically disrupted the endo G gene in mice without disturbing a conserved, overlapping gene of unknown function that is oriented tail to tail with the endo G gene. In these mice, the production of endo G protein is not detected, and the disruption abolishes the nuclease activity of endo G. The absence of endo G has no effect on mitochondrial DNA copy number, structure, or mutation rate over the first five generations. There is also no obvious effect on nuclear DNA degradation in standard apoptosis assays. The endo G null mice are viable and show no age-related or generational abnormalities anatomically or histologically. We infer that this highly conserved protein has no mitochondrial or apoptosis function that can discerned by the assays described here and that it may have a function yet to be determined. The early embryonic lethality of endo G null mice recently reported by others may be due to the disruption of the gene that overlaps the endo G gene.

DOI： 10.1128/MCB.25.1.294-302.2005

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure. To explore this, we generated BLM-/- and BLM(-/-)LIG4(-/-) cells from the human pre-B cell line Nalm-6. BLM-/- cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression. Interestingly, these phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. We propose that the absence of BLM leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by NHEJ. In addition, the NHEJ pathway per se was marginally affected by BLM deficiency, as evidenced by x-ray sensitivity and I-SceI-based DSB repair assays. More intriguingly, however, these experiments revealed the presence of an alternative, DNA ligase IV-independent end-joining pathway, which was significantly affected by the loss of BLM. Collectively, our results provide the first evidence for genetic interactions between BLM and NHEJ in human cells.

DOI： 10.1074/jbc.M409827200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

DNA topoisomerase I (Top1) generates transient DNA single-strand breaks via the formation of cleavage complexes in which the enzyme is linked to the 3'-phosphate of the cleavage strand. The anticancer drug camptothecin (CPT) poisons Top1 by trapping cleavage complexes, thereby inducing Top1-linked single-strand breaks. Such DNA lesions are converted into DNA double-strand breaks (DSBs) upon collision with replication forks, implying that DSB repair pathways could be involved in the processing/repair of Top1-mediated DNA damage. Here we report that Top1-mediated DNA damage is repaired primarily by homologous recombination, a major pathway of DSB repair. Unexpectedly, however, we found that nonhomologous end joining (NHEJ), another DSB repair pathway, has no positive role in the relevant repair; notably, DT40 cell mutants lacking either of the NHEJ factors ( namely, Ku70, DNA-dependent protein kinase catalytic subunit, and DNA ligase IV) were resistant to killing by CPT. In addition, we showed that the absence of NHEJ alleviates the requirement of homologous recombination in the repair of CPT-induced DNA damage. Our results indicate that NHEJ can be a cytotoxic pathway in the presence of CPT, shedding new light on the molecular mechanisms for the formation and repair of Top1-mediated DNA damage in vertebrates. Thus, our data have significant implications for cancer chemotherapy involving Top1 inhibitors.

DOI： 10.1074/jbc.M313910200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

In vertebrate cells, DNA double-strand breaks are efficiently repaired by homologous recombination or nonhomologous end-joining (NHEJ). The latter pathway relies on Ku (the Ku70/Ku86 heterodimer), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (Lig4). Here, we show that a human pre-B cell line nullizygous for Lig4 exhibits hypersensitivity to topoisomerase II (Top2) inhibitors, demonstrating a crucial role for the NHEJ pathway in repair of Top2-induced DNA damage in vertebrates. We also show that in the chicken DT40 cell line, all NHEJ mutants (i.e., Ku70-, Lig4-, and DNA-PKcs-null cells) are equally hypersensitive to the Top2 inhibitor ICRF-193, indicating that the drug-induced damage is repaired by NHEJ involving DNA-PKcs. Intriguingly, however, DNA-PKcs-null cells display considerably less severe phenotype than other NHEJ mutants in terms of hypersensitivity to VP-16, a Top2 poison that stabilizes cleavable complexes. The results indicate that two distinct NHEJ pathways, involving or not involving DNA-PKcs, are important for the repair of VP-16-induced DNA damage, providing additional evidence for the biological relevance of DNA-PKcs-independent NHEJ. Our results provide significant insights into the mechanisms of repair of Top2-mediated DNA damage, with implications for chemotherapy involving Top2 inhibitors. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2004.04.099

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase ( topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA double-strand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 cell line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4(-/-) and KU70(-/-) cells, which are defective in nonhomologous DNA end-joining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54(-/-) cells ( defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.

DOI： 10.1074/jbc.M306500200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

Background: There are two distinct DNA topoisomerase II (topo II) isoforms, designated topo IIalpha and topo IIbeta, in mammalian cells. The function of topo IIalpha in the development of mammalian cells has not been elucidated because of a lack of topo IIalpha mutants.
Results: We generated mice with a targeted disruption of the topo IIalpha gene. The development of topo IIalpha(-/-) embryos was terminated at the 4- or 8-cell stage. When wild-type embryos at the 2- or 4-cell stage were treated with ICRF-193, a catalytic inhibitor of topo II, nuclear division occurred followed by cytokinesis to form 4 or 8 cells, respectively, then development was terminated. Microscope analysis of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei of both topo IIalpha(-/-) and ICFR-193-treated embryonic cells revealed a droplet-like structure connecting the terminals of two adjacent nuclei forming a bridge-like structure. Phosphorylated histone H3, a marker for the M phases, disappeared from the nuclei of the topo IIalpha-depleted embryonic cells. Laser scanning cytometry of the topo IIalpha-depleted cells revealed the presence of 2N DNA cells.
Conclusions: Our results indicate that topo IIalpha has an essential role in the early stages of mouse development and that depletion of topo IIalpha from the embryonic cells causes incomplete nuclear division followed by enforced cytokinesis.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

There are two general pathways by which multicellular eukaryotes repair double-strand DNA breaks (DSB): homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). All mammalian mutants in the NHEJ pathway demonstrate a lack of B and T lymphocytes and ionizing radiation sensitivity. Among these NHEJ mutants, the DNA-PKcs and Artemis mutants are the least severe, having no obvious phenotype other than the general defects described above. Ku mutants have an intermediate severity with accelerated senescence. The XRCC4 and DNA ligase IV mutants are the most severe, resulting in embryonic lethality. Here we show that the lethality of DNA ligase IV-deficiency in the mouse can be rescued when Ku86 is also absent. To explain the fact that simultaneous gene mutations in the NHEJ pathway can lead to viability when a single mutant is not viable, we propose a nuclease/ligase model. In this model, disrupted NHEJ is more severe if the Artemis:DNA-PKcs nuclease is present in the absence of a ligase, and Ku mutants are of intermediate severity, because the nuclease is less efficient. This model is also consistent with the order of severity in organismal phenotypes; consistent with chromosomal breakage observations reported here; and consistent with the NHEJ mutation identified in radiation sensitive human SCID patients. (C) 2002 Elsevier Science B.V. All rights reserved.

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The structure-specific FEN-1 endonuclease has been implicated in various cellular processes, including DNA replication, repair and recombination. In vertebrate cells, however, no in vivo evidence has been provided so far. Here, we knocked out the FEN-1 gene (FEN1) in the chicken DT40 cell line. Surprisingly, homozygous mutant (FEN1(-/-)) cells were viable, indicating that FEN-1 is not essential for cell proliferation and thus for Okazaki fragment processing during DNA replication. However, compared with wild-type cells, FEN1(-/-) cells exhibited a slow growth phenotype, probably due to a high rate of cell death. The mutant cells were hypersensitive to methylmethane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and H2O2, but not to UV light, X-rays and etoposide, suggesting that FEN-1 functions in base excision repair in vertebrate cells.

DOI： 10.1093/nar/gkf440

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We established a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae to examine biological effects of thymidine analogues. 5-Bromo-2'-deoxyuridine efficiently suppressed the division potential of yeast showing morphology similar to senescent cells.

DOI： 10.1271/bbb.66.906

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Flap endonuclease 1 (FEN-1) is a nuclear enzyme involved in DNA metabolism, such as replication, repair, and recombination. Here, we report the comparative genomic organization of the chicken, mouse, and human FEN1 genes as well as the comparative organization of a small gene (C11orf10) located immediately upstream of the FEN1 gene in reverse orientation. Immunostaining revealed that the C11orf10 protein, unlike FEN-1, is located in the cytoplasm, suggesting that these two proteins do not form a physical complex. Importantly, in the human genome, the two mRNAs are overlapping (14 bp) in their 5′ ends. Thus, the FEN1/C11orf10 locus is a new example of two overlapping, divergent transcription units in the human genome.

DOI： 10.1089/15362310260256927

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

To elucidate the relationship between topoisomerase (topo) II expression and sensitivity to anti-topo II drugs in mammalian cells, we generated mouse embryonic stem cell mutants heterozygous for the topo IIα gene by gene targeting. The level of topo IIα in the heterozygous cells reduced to one-half of that found in wild-type cells, while topo IIβ levels were similar in both cell types. Importantly, the heterozygous cells exhibited an increased resistance to ICRF-193 as well as VP-16, suggesting that ICRF-193, like VP-16, exerts its cytotoxicity through converting topo II to a poison. © 2001 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0304-3835(01)00447-5

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

In mammalian cells, nonhomologous (illegitimate) recombination is a predominant pathway to repair DNA double-strand breaks. We have shown that DNA topoisomerase II inhibitors are capable of enhancing random integration of foreign DNA via nonhomologous recombination. Since this enhancement is likely due to stabilized DNA strand breaks, we examined the effect of a radiomimetic antitumor drug, bleomycin (BLM), on nonhomologous recombination. We found that BLM greatly enhances the random integration of transfected plasmids into human cells. Importantly, this enhancement was independent of the molecular form of the plasmid, the cell type or the transfection method, suggesting that the BLM effect is intrinsically general. Transient expression analysis revealed no stimulation of reporter gene expression by the drug, suggesting that the effect is not attributable to increased uptake and/or accumulation of transfected DNA in the drug-treated cell nuclei. In addition, the comet assay and flow cytometric analyses revealed the occurrence of low but significant strand breaks in cells treated with the BLM concentration which maximally enhanced the integration. These results strongly suggest that BLM acts directly at a nonhomologous recombination reaction that is initiated through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes. Our findings will facilitate the understanding of DNA integration events through nonhomologous recombination and the development of transfection protocols with higher efficiencies. Copyright (C) 1998 Elsevier Science B.V.

DOI： 10.1016/S0921-8777(98)00036-6

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Expression of DNA topoisomerase IIα protein varies through the cell cycle with its peak in G2/M. This cell-cycle-dependent expression depends on changes in topoisomerase IIα mRNA stability as well as promoter activity. We isolated the 3' genomic region of the mouse topoisomerase IIα gene and investigated whether or not the 3' untranslated region (UTR) of the topoisomerase IIα mRNA participates in the cell-cycle-dependent mRNA stability. Interestingly, genomic- and RT-PCR analyses revealed that the topoisomerase IIα 3' UTR is formed via splicing in mouse, but not in human and hamster. Comparison of the mouse 3' region with the human and hamster regions suggests that this mouse-specific splicing has resulted from an accidental acquisition of the consensus 5' splice site. The minority of the non-spliced topoisomerase IIα 3' UTR in mouse was confirmed by Northern blot analysis. We performed transient expression assays using luciferase constructs with the mouse topoisomerase IIα 3' genomic region, or the major spliced form of the 3' UTR. However, neither construct affected the cell-cycle-dependent expression of the reporter gene driven by the topoisomerase IIα promoter. Our results strongly suggest that the mouse topoisomerase IIα 3' UTR by itself is not involved in the cell-cycle-dependent mRNA stability.

DOI： 10.1016/S0378-1119(98)00283-2

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

Expression of DNA topoisomerase (topo) IIα varies through the cell cycle with its peak in G2/M. To investigate the mechanism controlling the topo IIα gene expression, we cloned the 5' upstream region of the mouse topo IIα gene. Although there was no TATA-like sequence, two GC and seven CCAAT boxes were found in the upstream region 5' distal to the major transcription start sites, which were located 137, 124, and 105 bp upstream from the ATG start codon. Luciferase vectors with the upstream sequences were constructed and transfected into HeLa cells, followed by cell cycle arrest either in G1 by treatment with mimosine, in S with thymidine, or in G2/M with colcemid. We found that the topo IIα gene promoter has the cell cycle-dependent activity, which is low in G1, rises in S, and peaks in G2/M. We suggest that the level of topo IIα mRNA is determined by the cell cycle-regulated promoter.

DOI： 10.1006/bbrc.1996.5893

Scopus

PubMed

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：(公財)上原記念生命科学財団  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(株)ニュー・サイエンス社  

ヒト細胞においてたった2つの遺伝子を欠損させるだけで、導入したベクターDNAの非特異的な組込みを完全に抑制できることが明らかとなった。これにより、CRISPR/Cas9などの人工ヌクレアーゼを使用せずとも効率100%で相同組換えによる遺伝子ターゲティングが可能であることが実証された。今後はこうした表現型を遺伝子破壊に依らず再現させることが課題となる。(著者抄録)

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

J-GLOBAL

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

J-GLOBAL

researchmap

Language：English   Publisher：生命科学系学会合同年次大会運営事務局  

researchmap

Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：Japanese   Publisher：(公社)日本薬学会  

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

Web of Science

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：Japanese  

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

Web of Science

researchmap

J-GLOBAL

researchmap

Language：English   Publisher：(一社)日本放射線影響学会  

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：日本環境変異原学会  

CiNii Books

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BIOMED CENTRAL LTD  

DOI： 10.1186/gb-2010-11-S1-P1

Web of Science

researchmap

Language：Japanese   Publisher：日本環境変異原学会  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese   Publisher：放射線生物研究会  

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2004319827

Language：Japanese  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese   Publisher：秀潤社  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese   Publisher：The Pharmaceutical Society of Japan  

CiNii Books

researchmap