Search

Nonsteroidal anti-inflammatory drugs (NSAID) are effective in suppressing the formation of colorectal tumors. However, the mechanisms underlying the antineoplastic effects of NSAIDs remain unclear. The effects of NSAIDs are incomplete, and resistance to NSAIDs is often developed. Growing evidence has indicated that the chemopreventive activity of NSAIDs is mediated by induction of apoptosis. Our previous studies showed that second mitochondria-derived activator of caspase (SMAC)/Diablo, a mitochondrial apoptogenic protein, plays an essential role in NSAID-induced apoptosis in colon cancer cells. In this study, we found that SMAC mediates NSAID-induced apoptosis through a feedback amplification mechanism involving interactions with inhibitor of apoptosis proteins, activation of caspase-3, and induction of cytosolic release of cytochrome c. Small-molecule SMAC mimetics at nanomolar concentrations significantly sensitize colon cancer cells to NSAID-induced apoptosis by promoting caspase-3 activation and cytochrome c release. Furthermore, SMAC mimetics overcome NSAID resistance in Bax-deficient or SMAC-deficient colon cancer cells by restoring caspase-3 activation and cytochrome c release. Together, these results suggest that SMAC is useful as a target for the development of more effective chemopreventive strategies and agents. [Cancer Res 2008;68(1):276–84]

Supplementary Figure 1 from SMAC Mimetics Sensitize Nonsteroidal Anti-inflammatory Drug–Induced Apoptosis by Promoting Caspase-3–Mediated Cytochrome c Release

Supplementary Figure 2 from SMAC Mimetics Sensitize Nonsteroidal Anti-inflammatory Drug–Induced Apoptosis by Promoting Caspase-3–Mediated Cytochrome c Release

Seaweed extracts are widely used as biostimulants in agriculture, with Ascophyllum nodosum (L.) Le Jolis, Ecklonia maxima (Osbeck) Papenfuss, and Macrocystis pyrifera (L.) C. Agardh representing the most commonly used seaweeds. In the present study, the suitability of the abundant and sustainable seaweed Durvillaea antarctica (Chamisso) Hariot as a raw material for biostimulant production was investigated. A thermo-acid method was used to prepare extracts of the brown algae D. antarctica extract (DAE) and A. nodosum extract (ANE) and compared by examining their growth promotion on cucumber seedlings. Germinated cucumber seedlings were grown in soil medium for 7 days and then treated with extracts for another 16 days until the third true leaf was fully extended, and the seedlings were harvested for analysis. The results showed that DAE treatment, at medium (0.12 g·L−1) and high (0.24 g·L−1) concentrations, improved growth parameters such as height and root length of cucumber seedlings. Biochemical analysis revealed that DAE increased the chlorophyll content and levels of indole-3-acetic acid, gibberellin, and nitrate reductase activity in cucumber seedlings, which could be partly responsible for the plant growth promotion effects. Plant height, chlorophyll content, and nitrate reductase activity of DAE-treated cucumber seedlings were significantly enhanced over those of the ANE treatment, suggesting the greater potential applications for D. antarctica in agricultural production when prepared using a thermo-acid extraction process.

Polysaccharides derived from seaweeds can be used as biostimulants to enhance plant resistance to different stressors. In this study, we investigated the effects of applying low molecular weight polysaccharides (LPU) derived from Ulva prolifera with 14.2 kDa on the responses of wheat (Triticum aestivum) to osmotic stress. The results showed that osmotic stress simulated using polyethylene glycol inhibited seedling growth, whereas we observed increases in the fresh weights and shoot lengths of seedlings treated with polysaccharide for 120 h. Furthermore, we observed enhanced activities of antioxidant enzymes, and significant reductions in malondialdehyde content of 23.13%, 19.82%, and 20.04% in response treatment for 120 h with 0.01%, 0.03%, and 0.05% LPU, respectively, relative to those in the group treated with polyethylene glycol alone. In all treatments, expression of the P5CS gene was upregulated to promote proline accumulation. Moreover, after 120 h, exogenously applied LPU induced the expression of stress-related genes, including SnRK2, Wabi5, Wrab18, and Wdhn13. Collectively, these findings indicate that LPU might have the effect of regulating the abscisic acid-dependent pathway in wheat, thereby increasing seedling antioxidant capacity and growth. Application of LPU may accordingly represent an effective approach for enhancing the resistance to osmotic stress in wheat.

Background BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) is a liver tumor suppressor, which is downregulated in a large number of patients with liver diseases. BRUCE facilitates DNA damage repair to protect the mouse liver against the hepatocarcinogen diethylnitrosamine (DEN)-dependent acute liver injury and carcinogenesis. While there exists an established pathologic connection between fibrosis and hepatocellular carcinoma (HCC), DEN exposure alone does not induce robust hepatic fibrosis. Further studies are warranted to identify new suppressive mechanisms contributing to DEN-induced fibrosis and HCC. Aim To investigate the suppressive mechanisms of BRUCE in hepatic fibrosis and HCC development. Methods Male C57/BL6/J control mice [loxp/Loxp; albumin-cre (Alb-cre)-] and BRUCE Alb-Cre KO mice (loxp/Loxp; Alb-Cre+) were injected with a single dose of DEN at postnatal day 15 and sacrificed at different time points to examine liver disease progression. Results By using a liver-specific BRUCE knockout (LKO) mouse model, we found that BRUCE deficiency, in conjunction with DEN exposure, induced hepatic fibrosis in both premalignant as well as malignant stages, thus recapitulating the chronic fibrosis background often observed in HCC patients. Activated in fibrosis and HCC, β-catenin activity depends on its stabilization and subsequent translocation to the nucleus. Interestingly, we observed that livers from BRUCE KO mice demonstrated an increased nuclear accumulation and elevated activity of β-catenin in the three stages of carcinogenesis: Pre-malignancy, tumor initiation, and HCC. This suggests that BRUCE negatively regulates β-catenin activity during liver disease progression. β-catenin can be activated by phosphorylation by protein kinases, such as protein kinase A (PKA), which phosphorylates it at Ser-675 (pSer-675-β-catenin). Mechanistically, BRUCE and PKA were colocalized in the cytoplasm of hepatocytes where PKA activity is maintained at the basal level. However, in BRUCE deficient mouse livers or a human liver cancer cell line, both PKA activity and pSer-675-β-catenin levels were observed to be elevated. Conclusion Our data support a "BRUCE-PKA-β-catenin" signaling axis in the mouse liver. The BRUCE interaction with PKA in hepatocytes suppresses PKA-dependent phosphorylation and activation of β-catenin. This study implicates BRUCE as a novel negative regulator of both PKA and β-catenin in chronic liver disease progression. Furthermore, BRUCE-liver specific KO mice serve as a promising model for understanding hepatic fibrosis and HCC in patients with aberrant activation of PKA and β-catenin.

The purpose of this study was to examine the alleviative effects of selenized polysaccharides from Ulva pertusa (ulvan-Se) on inflammatory bowel disease (IBD) in mice. The dextran sulfate sodium (D...

Multiple genome-wide association studies (GWAS) have identified specific genetic variants in the coiled-coil domain containing 92 (

Obesity-induced lipid metabolism disorders are risk factors for hyperlipidemia, atherosclerosis, and non-alcoholic fatty liver disease. Seaweed oligosaccharides and Zn supplements are potential alternatives to alleviate obesity. Herein, ulvan oligosaccharide (UO) was used as a ligand to prepare a novel Zn supplement (UO-Zn). Subsequently, we explored potential mechanisms underlying UO- and UO-Zn-mediated improvements in lipid metabolism in mice fed a high-fat diet. We found that UO enhanced the abundance of key species (Blautia and Turicibacter) and functions (glycolytic, pentose phosphate, and histidine/lysine biosynthesis pathways) in the gut microbiota, thereby increasing the production of short-chain fatty acids and activating AMPK. Accordingly, UO treatment regulated the transcription of lipid metabolism genes, including ACOX1, ACC, and FASN, thereby reducing blood lipid levels and hepatic lipid accumulation. Zn could act synergistically with UO, enhancing the reversal of cholesterol transport and fatty acid β-oxidation via the MTF1/PPARα pathway, markedly reducing body and adipose tissue weights.

The purpose of this study was to examine the alleviative effects of selenized polysaccharides from

BRUCE is a DNA damage response protein that promotes the activation of ATM and ATR for homologous recombination (HR) repair in somatic cells, making BRUCE a key protector of genomic stability. Preservation of genomic stability in the germline is essential for the maintenance of species. Here, we show that BRUCE is required for the preservation of genomic stability in the male germline of mice, specifically in spermatogonia and spermatocytes. Conditional knockout of Bruce in the male germline leads to profound defects in spermatogenesis, including impaired maintenance of spermatogonia and increased chromosomal anomalies during meiosis. Bruce-deficient pachytene spermatocytes frequently displayed persistent DNA breaks. Homologous synapsis was impaired, and nonhomologous associations and rearrangements were apparent in up to 10% of Bruce-deficient spermatocytes. Genomic instability was apparent in the form of chromosomal fragmentation, translocations, and synapsed quadrivalents and hexavalents. In addition, unsynapsed regions of rearranged autosomes were devoid of ATM and ATR signaling, suggesting an impairment in the ATM- and ATR-dependent DNA damage response of meiotic HR. Taken together, our study unveils crucial functions for BRUCE in the maintenance of spermatogonia and in the regulation of meiotic HR—functions that preserve the genomic stability of the male germline.

Ulva prolifera Muller, an alga rich in nutrients and bioactive compounds, is a natural feed resource for aquatic animals. In this study, instead of extracting polysaccharides (PUs) from U. prolifera, algae powder was used directly as the primary carbon source in a fermentation medium. Five percent (5%) algae powder was more conducive to fermentation. Using submerged liquid fermentation, the soluble components of U. prolifera were released efficiently in a 50-L fermenter. Levels of mannitol acquired using submerged liquid fermentation were 1.8 times higher than those acquired using the hot-water method, and polysaccharides were efficiently hydrolyzed to form oligosaccharides with an average molecular weight of approximately 3300 Da. Furthermore, a 2-month experiment was conducted to evaluate the effects of U. prolifera fermentation extracts on the growth performance of juvenile abalone (Haliotis discus hannai Ino). Diets containing algae fermentation extracts in concentrations ranging from 0.25 to 1% significantly increased linear shell growth rate (LGR), weight growth rate (WGR), and specific growth rate (SGR). Diets supplemented with fermentation extracts in concentrations of 0.5 and 1% significantly increased the degree of green coloration in the shell. Treatment of algae tissue using submerged liquid fermentation has the advantages of high efficiency and convenient process monitoring. Additionally, the technique can be scaled up for use in animal husbandry and aquaculture.

The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N’ terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.

The acidic polysaccharide ulvan extracted from the cell wall of the green algae Ulva is a good ligand for metal ions. Therefore, the adsorption properties of the U. clathrata derived ulvan toward Ca(II), Zn(II), Co(II), Cu(II), and Cr(III) were investigated in this study. The results demonstrate that ulvan exhibited good metal ion adsorption capacity at pH 7 and 50 °C. These adsorption processes can largely be explained by the Freundlich isotherm model and the pseudo-second-order kinetic model. The order of the adsorption capacity and affinity is as follows: Cr(III) > Cu(II) > Zn(II) ≈ Co(II) > Ca(II) and Cr(III) > Zn(II) > Co(II) ≈ Cu(II) > Ca(II). Furthermore, structural characterization revealed that the hydroxyl and carboxyl groups were the main functional groups involved in metal ion binding. Unlike the divalent metal ions, Cr(III) can trigger crosslinking of the ulvan chains, and its adsorption capacity was approximately 4.0 mmol/g.

Polysaccharide from Enteromorpha prolifera (PE) has been reported to have biological activities such as anticancer, antioxidant and immunomodulatory activities, but the structural characteristics of PE remain unclear. In this study, the composition of PE was characterized using chemical methods, IR spectroscopy and HPLC. PE was identified as a sulfated polysaccharide mainly composed of rhamnose (Rha), glucuronic (GlcUA), xylose (Xyl) at a molar ratio of 3.2: 1.1: 1, and the average molecular weight was 620.3kDa. An enzymatic hydrolysis method was used to obtain three carbohydrate products. The MS and NMR results of these residues revealed that the backbone of PE consisted of D-GlcUAp-α-(1→4)-3-sulfate-l-Rha p-β-(1→4)-d-Xyl p-β-(1→4)-3-sulfate-l-Rha p units. In this study, specific carbohydrate residues rich in sulfated rhamnose were prepared from E. prolifera. The structural determination of these sulfated carbohydrates may contribute to future studies on the relationship between structure and bioactivities.

Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.

The ecologically and economically important bivalve species Hyriopsis cumingii is affected by numerous bacterial diseases, but the effects of these harmful bacteria on the antioxidant responses and histological alterations of H. cumingii are unclear. This study evaluated the effects of common pathogenic infections on immune function in H. cumingii. A series of antioxidant enzyme activities, total haemocyte counts, and histopathological changes were measured at 24, 48, and 96 h post infection (hpi) with three pathogens (Aeromonas hydrophila, Streptococcus agalactiae, and Pseudomonas fluorescens). All enzyme activities sharply decreased or remained constant at 24 hpi, except for acid phosphatase and alkaline phosphatase activities in the P. fluorescens-infected group. However, peroxidase activity significantly increased at 24 and 48 hpi in all treatments (p

Ulvan lyase, specifically depolymerizing ulvan to unsaturated sulfated oligosaccharides with diverse biological activities, is becoming more and more popular. To provide an economical fermentative strategy, Catenovulum sp. LP cultured in low-cost medium that directly used Ulva prolifera as the main carbon source was used to produce ulvan lyase. The fermentation conditions were established in a shake flask containing 5% (w/v) U. prolifera powder, 0.2% K2HPO4, 0.4% NH4Cl, 0.5% NaCl, and 0.05% MgSO4. In a 5-L fermentor, series of strategies were used to systematically discuss the fermentation process. The enzyme with the best activity was obtained by the condition of a two-stage temperature (28–32 °C), initial pH 7.0, 1.5 vvm and two-stage agitation (200–400 rpm). The fermentation kinetic model of substrate consumption indicated that a shorter culture time may improve the ulvan lyase activity. Moreover, 10.09% improvement in enzyme activity was observed in a 30-L fermentor by shortening the fermentation time to 20 h. This fermentation method offers a promising prospect to enlarge the production of ulvan lyase and also provides a potential possibility to mildly prepare bioactive unsaturated oligosaccharides.

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

vi CHAPTER L GENERAL INTRODUCTION 1 General background 1 Cisand trans-acdng elements in E. coli DNA replication 3 Cisand trans-acting elements in SV40 viral DNA replication 5 Cisand trans-acting elements in yeast chromosomal DNA replication 7 Cisand trans-acting elements in Tetrakymem iDNA replication 9 Eukaiyotic cellular DNA replicative helicases 13 Dissertation oiganization 14 References 15 CHAPTER IL MODULAR STRUCTURAL ELEMENTS IN THE REPLICATION ORIGIN REGION OF TETRAHYMENA rDNA 23 ABSTRACT 23 INTRODUCnON 24 MATERIALS AND METHODS 27 RESULTS 31 DISCUSSION 36 ACKNOWLEDGMENTS 41 REFERENCES 42

TRIM21 is a RING finger domain-containing ubiquitin E3 ligase whose expression is elevated in autoimmune disease. While TRIM21 plays an important role in immune activation during pathogen infection, little is known about its inherent cellular function. Here we show that TRIM21 plays an essential role in redox regulation by directly interacting with SQSTM1/p62 and ubiquitylating p62 at lysine 7 (K7) via K63-linkage. As p62 oligomerizes and sequesters client proteins in inclusions, the TRIM21-mediated p62 ubiquitylation abrogates p62 oligomerization and sequestration of proteins including Keap1, a negative regulator of antioxidant response. TRIM21-deficient cells display an enhanced antioxidant response and reduced cell death in response to oxidative stress. Genetic ablation of TRIM21 in mice confers protection from oxidative damages caused by arsenic-induced liver insult and pressure overload heart injury. Therefore, TRIM21 plays an essential role in p62-regulated redox homeostasis and may be a viable target for treating pathological conditions resulting from oxidative damage.

A new strain bacteria was isolated and named as Phyllobacterium sp. 921F, due to its high production capacity of exopolysaccharide (EPS). Characterization of physico-chemical properties of the EPS and optimization for high production were conducted to aim at industrial applications. The optimum pH and temperature were 7.0 and 30°C, respectively. The following scale-up fermentation was carried out in 30L bioreactor and amounts of EPS (21.9g/L) were produced. The EPS with a molecular mass of 1082kDa was composed of glucose, galactose, and pyruvate. The EPS solution behaved as Newtonian at low concentrations (≤0.3%) and as shear thinning at higher concentration (e.g, 1%). The moisture retention ability of the EPS was found to be superior to hyaluronic acid. Results suggest that Phyllobacterium sp. 921F is a good candidate for large-scale production of the EPS which might be utilized in food and cosmetics industries.

The DNA damage response (DDR) is crucial for genomic integrity. BRIT1 (breast cancer susceptibility gene C terminus-repeat inhibitor of human telomerase repeat transcriptase expression), a tumor suppressor and early DDR factor, is recruited to DNA double-strand breaks (DSBs) by phosphorylated H2A histone family, member X (γ-H2AX), where it promotes chromatin relaxation by recruiting the switch/sucrose nonfermentable (SWI-SNF) chromatin remodeler to facilitate DDR. However, regulation of BRIT1 recruitment is not fully understood. The baculovirus IAP repeat (BIR)-containing ubiquitin-conjugating enzyme (BRUCE) is an inhibitor of apoptosis protein (IAP). Here, we report a non-IAP function of BRUCE in the regulation of the BRIT1-SWI-SNF DSB-response pathway and genomic stability. We demonstrate that BRIT1 is K63 ubiquitinated in unstimulated cells and that deubiquitination of BRIT1 is a prerequisite for its recruitment to DSB sites by γ-H2AX. We show mechanistically that BRUCE acts as a scaffold, bridging the ubiquitin-specific peptidase 8 (USP8) and BRIT1 in a complex to coordinate USP8-catalyzed deubiquitination of BRIT1. Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with γ-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation. Moreover, BRUCE-depleted cells display reduced homologous recombination repair, and BRUCE-mutant mice exhibit repair defects and genomic instability. These findings identify BRUCE and USP8 as two hitherto uncharacterized critical DDR regulators and uncover a deubiquitination regulation of BRIT1 assembly at damaged chromatin for efficient DDR and genomic stability.

Background and objectives: Management of patients with breast cancer often fails because of inherent or acquired resistance to chemotherapy. BRUCE (BIR repeat containing ubiquitin-conjugating enzyme) is a member of the inhibitor of apoptosis protein (IAP) family. It has various cellular functions including suppression of apoptosis and promotion of cytokinesis. Furthermore, it pays a critical role in promotion of DNA damage repair and preservation of genome stability, a new function recently reported by our group. Although BRUCE is expressed in breast cancer cell lines, its expression in human primary breast tumors and its contribution to chemoresistance in breast cancers has not been explored. Chemotherapeutic drugs are used in the treatment of breast cancer patients. However, they are not effective to all patients and patients often develop resistance. Consequently we explored if BRUCE protein level, as judged by immunohistochemistry (IHC), is higher in primary breast tumors than normal breast tissue. We also examined if depletion of BRUCE, using a lentiviral shRNA approach, enhances cell sensitivity to multiple chemotherapeutic agents, including cisplatin, an agent that induces DNA damage by generating DNA cross-links, and taxol, a microtubule stabilizer and mitotic inhibitor. The reason for including these two chemotherapeutic agents in this study is that they hit two essential cellular processes of DNA repair and cytokinesis in which BRUCE plays critical roles. Results and methods: IHC analysis of BRUCE revealed significantly higher levels of BRUCE in primary breast tumors than normal breast tissue. Knockdown of BRUCE protein expression by lentiviral shRNA resulted in increased sensitivity to cisplatin in the resistant breast cancer MDB-MD-231 cell line. Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone. Conclusions: Our data suggest that elevated protein levels of BRUCE in breast tumors may contribute to chemoresistance in breast cancer patients. In support of this suggestion, our data demonstrate that a reduction in BRUCE expression in breast cancer cell lines increases the toxicity of several chemotherapeutic agents. In all likelihood, the contribution of increased BRUCE levels to chemoresistance are likely due to its roles in suppression of apoptosis, promotion of cytokinesis and facilitation of DNA damage repair. These observations suggest that therapeutic suppression of BRUCE could improve chemosensitivity in chemo-resistant breast cancer patients. Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.

BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.

MCPH1, also known as BRIT1, has recently been identified as a novel key regulatory gene of the DNA damage response pathway. MCPH1 is located on human chromosome 8p23.1, where human cancers frequently show loss of heterozygosity. As such, MCPH1 is aberrantly expressed in many malignancies, including breast and ovarian cancers, and the function of MCPH1 has been implicated in tumor suppression. However, it remains poorly understood whether MCPH1 deficiency leads to tumorigenesis. Here, we generated and studied both Mcph1−/− and Mcph1−/−p53−/− mice; we showed that Mcph1−/− mice developed tumors with long latency, and that primary lymphoma developed significantly earlier in Mcph1−/−p53−/− mice than in Mcph11+/+p53−/− and Mcph1+/−p53−/- mice. The Mcph1−/−p53−/− lymphomas and derived murine embryonic fibroblasts (MEFs) were both more sensitive to irradiation. Mcph1 deficiency resulted in remarkably increased chromosome and chromatid breaks in Mcph1−/−p53−/− lymphomas and MEFs, as determined by metaphase spread assay and spectral karyotyping analysis. Additionally, Mcph1 deficiency significantly enhanced aneuploidy as well as abnormal centrosome multiplication in Mcph1−/−p53−/− cells. Meanwhile, Mcph1 deficiency impaired double strand break (DSB) repair in Mcph1−/−p53−/− MEFs as demonstrated by neutral Comet assay. Compared with Mcph1+/+p53−/− MEFs, homologous recombination and non-homologous end joining activities were significantly decreased in Mcph1−/−p53−/− MEFs. Notably, reconstituted MCPH1 rescued the defects of DSB repair and alleviated chromosomal aberrations in Mcph1−/−p53−/- MEFs. Taken together, our data demonstrate MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. Our study using knockout mouse models provides convincing genetic evidence that MCPH1 is a bona fide tumor suppressor gene. Its deficiency leading to defective DNA repair in tumors can be utilized to develop novel targeted cancer therapies in the future. Citation Format: Yulong Liang, Hong Gao, Shiaw-Yih Lin, John A. Goss, Chunying Du, Kaiyi Li. Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2029. doi:10.1158/1538-7445.AM2015-2029