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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

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 (

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.

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.

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.

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.

ARTS (apoptosis-related protein in the TGF-β signaling pathway) is a mitochondrial protein that binds XIAP (X-linked inhibitor of apoptosis protein) upon entering the cytosol, thus promoting cell death. Expression of ARTS is lost in some malignancies. Here, we show that ARTS binds to XIAP at BIR1, a domain distinct from the caspase-binding sites. Furthermore, ARTS interacts with the E3 ligase Siah-1 (seven in absentia homolog 1) to induce ubiquitination and degradation of XIAP. Cells lacking either Siah or ARTS contain higher steady-state levels of XIAP. Thus, ARTS serves as an adaptor to bridge Siah-1 to XIAP, targeting it for destruction.

Treacher Collins syndrome (TCS) is a congenital disorder of craniofacial development arising from mutations in TCOF1, which encodes the nucleolar phosphoprotein Treacle. Haploinsufficiency of Tcof1 perturbs mature ribosome biogenesis, resulting in stabilization of p53 and the cyclin G1-mediated cell-cycle arrest that underpins the specificity of neuroepithelial apoptosis and neural crest cell hypoplasia characteristic of TCS. Here we show that inhibition of p53 prevents cyclin G1-driven apoptotic elimination of neural crest cells while rescuing the craniofacial abnormalities associated with mutations in Tcof1 and extending life span. These improvements, however, occur independently of the effects on ribosome biogenesis; thus suggesting that it is p53-dependent neuroepithelial apoptosis that is the primary mechanism underlying the pathogenesis of TCS. Our work further implies that neuroepithelial and neural crest cells are particularly sensitive to cellular stress during embryogenesis and that suppression of p53 function provides an attractive avenue for possible clinical prevention of TCS craniofacial birth defects and possibly those of other neurocristopathies.

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.

Baculoviral inhibitor of apoptosis repeat-containing (Birc)6 gene/BIRC6 (Bruce/APOLLON) encodes an inhibitor of apoptosis and a chimeric E2/E3 ubiquitin ligase in mammals. The physiological role of Bruce in antiapoptosis is unknown. Here, we show that deletion of the C-terminal half of Bruce, including the UBC domain, causes activation of caspases and apoptosis in the placenta and yolk sac, leading to embryonic lethality. This apoptosis is associated with up-regulation and nuclear localization of the tumor suppressor p53 and activation of mitochondrial apoptosis, which includes up-regulation of Bax, Bak, and Pidd, translocation of Bax and caspase-2 onto mitochondria, release of cytochrome c and apoptosis-inducing factor, and activation of caspase-9 and caspase-3. Mutant mouse embryonic fibroblasts are sensitive to multiple mitochondrial death stimuli but resistant to TNF. In addition, eliminating p53 by RNA interference rescues cell viability induced by Bruce ablation in human cell line H460. This viability preservation results from reduced expression of proapoptotic factors Bax, Bak, and Pidd and from prevention of activation of caspase-2, -9, and -3. The amount of second mitochondrial-derived activator of caspase and Omi does not change. We conclude that p53 is a downstream effector of Bruce, and, in response to loss of Bruce function, p53 activates Pidd/caspase-2 and Bax/Bak, leading to mitochondrial apoptosis.

The inhibitor of apoptosis (IAP) proteins bind and inhibit caspases via their baculovirus IAP repeat domains. Some of these IAPs are capable of ubiquitinating themselves and their interacting proteins through the ubiquitin-protein isopeptide ligase activity of their RING domain. The Drosophila IAP antagonists Reaper, Hid, and Grim can accelerate the degradation of Drosophila IAP1 and some mammalian IAPs by promoting their ubiquitin-protein isopeptide ligase activity. Here we show that Smac/DIABLO, a mammalian functional homolog of Reaper/Hid/Grim, selectively causes the rapid degradation of c-IAP1 and c-IAP2 but not XIAP and Livin in HeLa cells, although it efficiently promotes the auto-ubiquitination of them all. Smac binding to c-IAP via its N-terminal IAP-binding motif is the prerequisite for this effect, which is further supported by the findings that Smac N-terminal peptide is sufficient to enhance c-IAP1 ubiquitination, and Smac no longer promotes the ubiquitination of mutant c-IAP1 lacking all three baculovirus IAP repeat domains. In addition, different IAPs require the same ubiquitin-conjugating enzymes UbcH5a and UbcH6 for their ubiquitination. Taken together, Smac may serve as a key molecule in vivo to selectively reduce the protein level of c-IAPs through the ubiquitin/proteasome pathway.

The mitochondrial pathway is critical for the efficient execution of death receptor-initiated apoptosis in certain cell types. Questions remain as to why the mitochondria are required in that scenario. We investigated the molecular events that determined the need for the mitochondria by using an in vivo model of anti-Fas-induced hepatocyte apoptosis. In wild-type mice, Fas stimulation resulted in normal activation of caspase-3, with the generation of the active p19-p12 complex. In bid-deficient mice, caspase-3 activation was arrested after the initial cleavage at Asp(175). This allowed the generation of the p12 small subunit, but the p20 large subunit could not be further processed to the p19 subunit. The p20-p12 complex generated by Fas stimulation in bid-deficient hepatocytes was inactive, arresting the death program. Failure of p20/p12 caspase-3 to mature and to exhibit activity was because of the inhibition by the inhibitor-of-apoptosis proteins (IAPs), such as XIAP, and also to a low caspase-8 activity. This block could be overcome in wild-type mice by two mechanisms. Smac was released from mitochondria early following Fas activation and was competitively bound to the IAPs to reverse their effects. XIAP could also be cleaved, and this occurred later and was likely mediated by enhanced caspase activities. Both mechanisms were dependent on Bid and thus were not operative in bid-deficient hepatocytes. In conclusion, mitochondrial activation by Bid is required for reversing the IAP inhibition through Smac release. It is also required for the alternative activation of caspases through cytochrome c release, as demonstrated previously. Together, these events ensure a successful progression of the death program initiated by the death receptor activation in the hepatocyte.

Apoptosis is an essential process in the development and homeostasis of all metazoans. The inhibitor-of-apoptosis (IAP) proteins suppress cell death by inhibiting the activity of caspases; this inhibition is performed by the zinc-binding BIR domains of the IAP proteins. The mitochondrial protein Smac/DIABLO promotes apoptosis by eliminating the inhibitory effect of IAPs through physical interactions. Amino-terminal sequences in Smac/DIABLO are required for this function, as mutation of the very first amino acid leads to loss of interaction with IAPs and concomitant loss of Smac/DIABLO function. Here we report the high-resolution crystal structure of Smac/DIABLO complexed with the third BIR domain (BIR3) of XIAP. Our results show that the N-terminal four residues (Ala-Val-Pro-Ile) in Smac/DIABLO recognize a surface groove on BIR3, with the first residue Ala binding a hydrophobic pocket and making five hydrogen bonds to neighbouring residues on BIR3. These observations provide a structural explanation for the roles of the Smac N terminus as well as the conserved N-terminal sequences in the Drosophila proteins Hid/Grim/Reaper. In conjunction with other observations, our results reveal how Smac may relieve IAP inhibition of caspase-9 activity. In addition to explaining a number of biological observations, our structural analysis identifies potential targets for drug screening.

Apoptosis (programmed cell death), an essential process in the development and homeostasis of metazoans, is carried out by caspases. The mitochondrial protein Smac/DIABLO performs a critical function in apoptosis by eliminating the inhibitory effect of IAPs (inhibitor of apoptosis proteins) on caspases. Here we show that Smac/DIABLO promotes not only the proteolytic activation of procaspase-3 but also the enzymatic activity of mature caspase-3, both of which depend upon its ability to interact physically with IAPs. The crystal structure of Smac/DIABLO at 2.2 A resolution reveals that it homodimerizes through an extensive hydrophobic interface. Missense mutations inactivating this dimeric interface significantly compromise the function of Smac/DIABLO. As in the Drosophila proteins Reaper, Grim and Hid, the amino-terminal amino acids of Smac/DIABLO are indispensable for its function, and a seven-residue peptide derived from the amino terminus promotes procaspase-3 activation in vitro. These results establish an evolutionarily conserved structural and biochemical basis for the activation of apoptosis by Smac/DIABLO.

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication.

The multifunctional caspase-2 protein is involved in apoptosis, NF-κB regulation, and tumor suppression in mice. However, the mechanisms of caspase-2 responsible for tumor suppression remain unclear. Here we identified two sites of caspase-2, the catalytic Cys-320 site and the Ser-139 site, to be important for suppression of cellular transformation and tumorigenesis. Using SV40- and K-Ras-transformed caspase-2 KO mouse embryonic fibroblast cells reconstituted with expression of wild-type, catalytic dead (C320A), or Ser-139 (S139A) mutant caspase-2, we demonstrated that similar to caspase-2 deficiency, when Cys-320 and Ser-139 were mutated, caspase-2 lost its ability to inhibit cellular transformation and tumorigenesis. These mutant cells exhibited enhanced cell proliferation, elevated clonogenic activity, accelerated anchorage-independent growth, and transformation and were highly tumorigenic, rapidly producing large tumors in athymic nude mice. Investigation into the underlying mechanism showed that these two residues are needed for caspase-2 to suppress NF-κB activity, promote apoptosis, and sustain the G(2)/M checkpoint following DNA damage induction. In addition, tumors in nude mice derived from the two mutant cell lines had higher constitutive NF-κB activity and elevated expression of NF-κB targets of antiapoptotic proteins Bcl-xL, XIAP, and cIAP2. A reduction in caspase-2 mRNA was associated with multiple types of cancers in patients. Together, these observations suggest the combined functions of caspase-2 in suppressing NF-κB activation, promoting apoptosis, and sustaining G(2)/M checkpoint contribute to caspase-2 tumor-suppressing function and that caspase-2 may also impact tumor suppression in humans. These findings provide insight into tumor suppression at the cross-roads of apoptosis, cell cycle checkpoint, and NF-κB pathways.

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.

Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

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]

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

Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis to antagonize inhibitors of apoptosis (IAPs) and contribute to caspase-independent cell death. Here, we demonstrate that Omi/HtrA2 directly cleaves various IAPs in vitro, and the cleavage efficiency is determined by its IAP-binding motif, AVPS. Cleavage of IAPs such as c-IAP1 substantially reduces its ability to inhibit and ubiquitylate caspases. In contrast to the stoichiometric anti-IAP activity by Smac/DIABLO, Omi/HtrA2 cleavage of c-IAP1 is catalytic and irreversible, thereby more efficiently inactivating IAPs and promoting caspase activity. Elimination of endogenous Omi by RNA interference abolishes c-IAP1 cleavage and desensitizes cells to apoptosis induced by TRAIL. In addition, overexpression of cleavage-site mutant c-IAP1 makes cells more resistant to TRAIL-induced caspase activation. This IAP cleavage by Omi is independent of caspase. Taken together, these results indicate that unlike Smac/DIABLO, Omi/HtrA2's catalytic cleavage of IAPs is a key mechanism for it to irreversibly inactivate IAPs and promote apoptosis.

Sympathetic neuronal apoptosis after nerve growth factor (NGF) deprivation requires the activation of two events: a protein synthesis-dependent, Bax-dependent release of mitochondrial cytochrome c and a protein synthesis-independent, Bax-independent development of competence. Unlike in most cells, cytosolic cytochrome c is not sufficient to induce cell death in NGF-maintained sympathetic neurons but can do so in neurons that have developed competence. We report that cytosolic cytochrome c-induced apoptosis in competent sympathetic neurons is completely dependent on caspase-9. In addition, the neuroprotective agents KCl and chlorophenylthio-cAMP are potent inhibitors of the development-of-competence pathway in NGF-deprived sympathetic neurons. We also find that the development of competence is reversible. Readdition of NGF reverses competence, and neurons can regain their resistance to cytosolic cytochrome c. Importantly, we examined the mechanism of development of competence and report that the inability of cytochrome c to activate caspases in NGF-maintained sympathetic neurons can be overcome with exogenous Smac that inhibits the inhibitor of apoptosis (IAP) family of proteins. Microinjection of cytochrome c and Smac, but neither alone, induces rapid cell death in NGF-maintained neurons. These data suggest that development of competence may be the result of the loss of the function of one or more members of the IAP family of caspase inhibitors that is needed before cytochrome c can activate caspases and induce cell death in neurons.

The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac(-/-)) mice by using homologous recombination in embryonic stem (ES) cells. Smac(-/-) mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac(-/-) cells, all types of cultured Smac(-/-) cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.