RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex

SGS1 encodes a DNA helicase whose homologues in human cells include the BLM, WRN, and RECQ4 genes, mutations in which lead to cancer-predisposition syndromes. Clustering of synthetic genetic interactions identified by large-scale genetic network analysis revealed that the genetic interaction profile of the gene RMI1 (RecQ-mediated genome instability, also known as NCE4 and YPL024W) was highly similar to that of SGS1 and TOP3, suggesting a functional relationship between Rmi1 and the Sgs1/Top3 complex. We show that Rmi1 physically interacts with Sgs1 and Top3 and is a third member of this complex. Cells lacking RMI1 activate the Rad53 checkpoint kinase, undergo a mitotic delay, and display increased relocalization of the recombination repair protein Rad52, indicating the presence of spontaneous DNA damage. Consistent with a role for RMI1 in maintaining genome integrity, rmi1Delta cells exhibit increased recombination frequency and increased frequency of gross chromosomal rearrangements. In addition, rmi1Delta strains fail to fully activate Rad53 upon exposure to DNA-damaging agents, suggesting that Rmi1 is also an important part of the Rad53-dependent DNA damage response.     

Mycoplasma hyorhinis Activates the NLRP3 Inflammasome and Promotes Migration and Invasion of Gastric Cancer Cells

Background

Mycoplasma hyorhinis (M.hyorhinis, M.hy) is associated with development of gastric and prostate cancers. The NLRP3 inflammasome, a protein complex controlling maturation of important pro-inflammatory cytokines interleukin (IL)-1β and IL-18, is also involved in tumorigenesis and metastasis of various cancers.

Methodology/Principal Findings

To clarify whether M.hy promoted tumor development via inflammasome activation, we analyzed monocytes for IL-1β and IL-18 production upon M.hy challenge. When exposed to M.hy, human monocytes exhibited rapid and robust IL-1β and IL-18 secretion. We further identified that lipid-associated membrane protein (LAMP) from M.hy was responsible for IL-1β induction. Applying competitive inhibitors, gene specific shRNA and gene targeted mice, we verified that M.hy induced IL-1β secretion was NLRP3-dependent in vitro and in vivo. Cathepsin B activity, K⁺ efflux, Ca²⁺ influx and ROS production were all required for the NLRP3 inflammasome activation by M.hy. Importantly, it is IL-1β but not IL-18 produced from macrophages challenged with M.hy promoted gastric cancer cell migration and invasion.

Conclusions

Our data suggest that activation of the NLRP3 inflammasome by M.hy may be associated with its promotion of gastric cancer metastasis, and anti-M.hy therapy or limiting NLRP3 signaling could be effective approach for control of gastric cancer progress.     

Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells

Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.     

Fhit Deficiency-Induced Global Genome Instability Promotes Mutation and Clonal Expansion

Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome ‘caretaker’ whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit ^-/- MEFs escape senescence to become immortal more rapidly than Fhit ^+/+ MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a ‘mutator’ phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.     

Tumor Suppressor Ras Association Domain Family 5 (RASSF5/NORE1) Mediates Death Receptor Ligand-induced Apoptosis

Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-α and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-α-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-α-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-α- and TNF-related apoptosis-inducing ligand-mediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-α-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.     

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles^1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture^3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue^3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture⁶. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.     

Skp2 promotes adipocyte differentiation via a p27-independent mechanism in primary mouse embryonic fibroblasts

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27^Kip1, a principal target of the SCF^Skp2 complex. Genetic ablation of p27^Kip1 in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27^Kip1 by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2^-/- MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), largely restored lipid accumulation and PPARγ gene expression in Skp2^−/− MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27^Kip1 expression.     

Fad104, a positive regulator of adipogenesis, negatively regulates osteoblast differentiation

Fad104 (factor for adipocyte differentiation 104) is a novel gene expressed temporarily in the early stages of adipocyte differentiation. Previously, we showed that fad104 promotes adipocyte differentiation in mouse 3T3-L1 cells and mouse embryonic fibroblasts (MEFs). Furthermore, we reported that implanted wild-type MEFs could develop into adipocytes, whereas fad104-deficient MEFs could not. Interestingly, bone-like tissues were only observed in the implants derived from fad104-deficient MEFs. This result implies that fad104 is involved in osteoblast differentiation. However, the functions of fad104 during osteogenesis are unknown. In this paper, we show that fad104 negatively regulates osteoblast differentiation. During the differentiation process, the level of fad104 expression decreased. Deletion of fad104 facilitated osteoblast differentiation in MEFs, and elevated the level of runx2, a master regulator of osteoblast differentiation. Disruption of fad104 suppressed BMP-2-mediated adipocyte differentiation in MEFs. In conclusion, we demonstrate that fad104 reciprocally regulates differentiation of adipocytes and osteoblast; functions as a positive regulator in adipocyte differentiation and as a negative regulator in osteoblast differentiation.     

Binding and activation of DNA topoisomerase III by the Rmi1 subunit

Rmi1 is a conserved oligonucleotide and oligosaccharide binding-fold protein that is associated with RecQ DNA helicase complexes from humans (BLM-TOP3 alpha) and yeast (Sgs1-Top3). Although human RMI1 stimulates the dissolution activity of BLM-TOP3 alpha, its biochemical function is unknown. Here we examined the role of Rmi1 in the yeast complex. Consistent with the similarity of top3Delta and rmi1Delta phenotypes, we find that a stable Top3.Rmi1 complex can be isolated from yeast cells overexpressing these two subunits. Compared with Top3 alone, this complex displays increased superhelical relaxation activity. The isolated Rmi1 subunit also stimulates Top3 activity in reconstitution experiments. In both cases elevated temperatures are required for optimal relaxation unless the substrate contains a single-strand DNA (ssDNA) bubble. Interestingly, Rmi1 binds only weakly to ssDNA on its own, but it stimulates the ssDNA binding activity of Top3 5-fold. Top3 and Rmi1 also cooperate to bind the Sgs1 N terminus and promote its interaction with ssDNA. These results demonstrate that Top3-Rmi1 functions as a complex and suggest that Rmi1 stimulates Top3 by promoting its interaction with ssDNA.     

COP9 Signalosome Subunit Csn8 Is Involved in Maintaining Proper Duration of the G1 Phase

The COP9 signalosome (CSN) is a conserved protein complex known to be involved in developmental processes of eukaryotic organisms. Genetic disruption of a CSN gene causes arrest during early embryonic development in mice. The Csn8 subunit is the smallest and the least conserved subunit, being absent from the CSN complex of several fungal species. Nevertheless, Csn8 is an integral component of the CSN complex in higher eukaryotes, where it is essential for life. By characterizing the mouse embryonic fibroblasts (MEFs) that express Csn8 at a low level, we found that Csn8 plays an important role in maintaining the proper duration of the G₁ phase of the cell cycle. A decreased level of Csn8, either in Csn8 hypomorphic MEFs or following siRNA-mediated knockdown in HeLa cells, accelerated cell growth rate. Csn8 hypomorphic MEFs exhibited a shortened G₁ duration and affected expression of G₁ regulators. In contrast to Csn8, down-regulation of Csn5 impaired cell proliferation. Csn5 proteins were found both as a component of the CSN complex and outside of CSN (Csn5-f), and the amount of Csn5-f relative to CSN was increased in the Csn8 hypomorphic cells. We conclude that CSN harbors both positive and negative regulators of the cell cycle and therefore is poised to influence the fate of a cell at the crossroad of cell division, differentiation, and senescence.     

The Absence of Core Fucose Up-regulates GnT-III and Wnt Target Genes: A POSSIBLE MECHANISM FOR AN ADAPTIVE RESPONSE IN TERMS OF GLYCAN FUNCTION*

Glycans play key roles in a variety of protein functions under normal and pathological conditions, but several glycosyltransferase-deficient mice exhibit no or only mild phenotypes due to redundancy or compensation of glycan functions. However, we have only a limited understanding of the underlying mechanism for these observations. Our previous studies indicated that 70% of Fut8-deficient (Fut8^−/−) mice that lack core fucose structure die within 3 days after birth, but the remainder survive for up to several weeks although they show growth retardation as well as emphysema. In this study, we show that, in mouse embryonic fibroblasts (MEFs) from Fut8^−/− mice, another N-glycan branching structure, bisecting GlcNAc, is specifically up-regulated by enhanced gene expression of the responsible enzyme N-acetylglucosaminyltransferase III (GnT-III). As candidate target glycoproteins for bisecting GlcNAc modification, we confirmed that level of bisecting GlcNAc on β1-integrin and N-cadherin was increased in Fut8^−/− MEFs. Moreover using mass spectrometry, glycan analysis of IgG₁ in Fut8^−/− mouse serum demonstrated that bisecting GlcNAc contents were also increased by Fut8 deficiency in vivo. As an underlying mechanism, we found that in Fut8^−/− MEFs Wnt/β-catenin signaling is up-regulated, and an inhibitor against Wnt signaling was found to abrogate GnT-III expression, indicating that Wnt/β-catenin is involved in GnT-III up-regulation. Furthermore, various oxidative stress-related genes were also increased in Fut8^−/− MEFs. These data suggest that Fut8^−/− mice adapted to oxidative stress, both ex vivo and in vivo, by inducing various genes including GnT-III, which may compensate for the loss of core fucose functions.     

Lack of DNA polymerase μ affects the kinetics of DNA double-strand break repair and impacts on cellular senescence

The specialised DNA polymerase μ (pol μ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol μ intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol μ^?/? mouse embryonic fibroblasts (MEFs) we show that lack of pol μ results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol μ. Here we show pronounced senescence in pol μ^?/? MEFs, associated with high levels of the tumor-suppressor p16^INK4A and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol μ^?/? MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol μ. Pol μ^?/? MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.     

Neuronal and astrocyte dysfunction diverges from embryonic fibroblasts in the Ndufs4fky/fky mouse

Mitochondrial dysfunction causes a range of early-onset neurological diseases and contributes to neurodegenerative conditions. The mechanisms of neurological damage however are poorly understood, as accessing relevant tissue from patients is difficult, and appropriate models are limited. Hence, we assessed mitochondrial function in neurologically relevant primary cell lines from a CI (complex I) deficient Ndufs4 KO (knockout) mouse (Ndufs4^fky/fky) modelling aspects of the mitochondrial disease LS (Leigh syndrome), as well as MEFs (mouse embryonic fibroblasts). Although CI structure and function were compromised in all Ndufs4^fky/fky cell types, the mitochondrial membrane potential was selectively impaired in the MEFs, correlating with decreased CI-dependent ATP synthesis. In addition, increased ROS (reactive oxygen species) generation and altered sensitivity to cell death were only observed in Ndufs4^fky/fky primary MEFs. In contrast, Ndufs4^fky/fky primary isocortical neurons and primary isocortical astrocytes displayed only impaired ATP generation without mitochondrial membrane potential changes. Therefore the neurological dysfunction in the Ndufs4^fky/fky mouse may partly originate from a more severe ATP depletion in neurons and astrocytes, even at the expense of maintaining the mitochondrial membrane potential. This may provide protection from cell death, but would ultimately compromise cell functionality in neurons and astrocytes. Furthermore, RET (reverse electron transfer) from complex II to CI appears more prominent in neurons than MEFs or astrocytes, and is attenuated in Ndufs4^fky/fky cells.     

Exonuclease 1 (Exo1) is required for activating response to SN1 DNA methylating agents

S_N1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O⁶ position of guanine (O⁶meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O⁶meG:T mispairs and is required to activate DNA damage response (DDR). Exonuclease I (EXO1) is a key component of MMR by resecting the damaged strand; however, whether EXO1 is required to activate MMR-dependent DDR remains unknown. Here we show that knockdown of the mouse ortholog (mExo1) in mouse embryonic fibroblasts (MEFs) results in decreased G2/M checkpoint response, limited effects on cell proliferation, and increased cell viability following exposure to the S_N1 methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), establishing a phenotype paralleling MMR deficiency. MNNG treatment induced formation of γ-H2AX foci with which EXO1 co-localized in MEFs, but mExo1-depleted MEFs displayed a significant diminishment of γ-H2AX foci formation. mExo1 depletion also reduced MSH2 association with DNA duplexes containing G:T mismatches in vitro, decreased MSH2 association with alkylated chromatin in vivo, and abrogated MNNG-induced MSH2/CHK1 interaction. To determine if nuclease activity is required to activate DDR we stably overexpressed a nuclease defective form of human EXO1 (hEXO1) in mExo1-depleted MEFs. These experiments indicated that expression of wildtype and catalytically null hEXO1 was able to restore normal response to MNNG. This study indicates that EXO1 is required to activate MMR-dependent DDR in response to S_N1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.     

Knock-in Mutation Reveals an Essential Role for Focal Adhesion Kinase Activity in Blood Vessel Morphogenesis and Cell Motility-Polarity but Not Cell Proliferation

Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAK^R454/R454 mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAK^R454/R454 embryos. In contrast to the inability of embryonic FAK^−/− cells to proliferate ex vivo, primary FAK^R454/R454 mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation, decreased migration, and defects in cell polarity. Within immortalized MEFs, FAK activity was required for fibronectin-stimulated FAK-p190RhoGAP association and p190RhoGAP tyrosine phosphorylation linked to decreased RhoA GTPase activity, focal adhesion turnover, and directional motility. Our results establish that intrinsic FAK activity is essential for developmental processes controlling blood vessel formation and cell motility-polarity but not cell proliferation. This work supports the use of FAK inhibitors to disrupt neovascularization.     

Inactivation of Uaf1 Causes Defective Homologous Recombination and Early Embryonic Lethality in Mice

The deubiquitinating enzyme heterodimeric complex USP1-UAF1 regulates the Fanconi anemia (FA) DNA repair pathway. Absence of this complex leads to increased cellular levels of ubiquitinated FANCD2 (FANCD2-Ub) and ubiquitinated PCNA (PCNA-Ub). Mice deficient in the catalytic subunit of the complex, USP1, exhibit an FA-like phenotype and have a cellular deficiency in homologous-recombination (HR) repair. Here, we have characterized mice deficient in the UAF1 subunit. Uaf1^+/− mice were small at birth and exhibited reduced fertility, thus resembling Usp1^−/− mice. Unexpectedly, homozygous Uaf1^−/− embryos died at embryonic day 7.5 (E7.5). These mutant embryos were small and developmentally retarded. As expected, Uaf1 deficiency in mice led to increased levels of cellular Fancd2-Ub and Pcna-Ub. Uaf1^+/− murine embryonic fibroblasts (MEFs) exhibited profound chromosome instability, genotoxin hypersensitivity, and a significant defect in homologous-recombination repair. Moreover, Uaf1^−/− mouse embryonic stem cells (mESCs) showed chromosome instability, genotoxin hypersensitivity, and impaired Fancd2 focus assembly. Similar to USP1 knockdown, UAF1 knockdown in tumor cells caused suppression of tumor growth in vivo. Taken together, our data demonstrate the important regulatory role of the USP1-UAF1 complex in HR repair through its regulation of the FANCD2-Ub and PCNA-Ub cellular pools.     

Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Background

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.

Methodology/Principal Findings

We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.

Conclusions

We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands.     

Wuho/WDR4 deficiency inhibits cell proliferation and induces apoptosis via DNA damage in mouse embryonic fibroblasts

Wuho known as WDR4 encodes a highly conserved WD40-repeat protein, which has known homologues of WDR4 in human and mouse. Wuho-FEN1 interaction may have a critical role in the growth and development, and in the maintenance of genome stability. However, how Wuho gene deletion contributes to cell growth inhibition and apoptosis is still unknown. We utilized CAGGCre-ER transgenic mice have a tamoxifen-inducible cre-mediated recombination cassette to prepare primary mouse embryonic fibroblasts (MEFs) with Wuho deficiency. We have demonstrated that Wuho deficiency would induces γH2AX protein level elevation, heterochromatin relaxation and DNA damage down-stream sequences, including p53 activation, caspase-mediated apoptotic pathway, and p21-mediated G2/M cell cycle arrest.