Biallelic MAD2L1BP (p31comet) mutation is associated with mosaic aneuploidy and juvenile granulosa cell tumors.

MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.

Externalized histones fuel pulmonary fibrosis via a platelet-macrophage circuit of TGFß1 and IL-27.

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFß and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFß1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFß1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFß1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.

Ginkgo biloba induces different gene expression signatures and oncogenic pathways in malignant and non-malignant cells of the liver.

Ginkgo biloba (EGb761) is a widely used botanical drug. Several reports indicate that EGb761 confers preventive as well as anti-tumorigenic properties in a variety of tumors, including hepatocellular carcinoma (HCC). We here evaluate functional effects and molecular alterations induced by EGb761 in hepatoma cells and non-malignant hepatocytes. Hepatoma cell lines, primary human HCC cells and immortalized human hepatocytes (IH) were exposed to various concentrations (0-1000 µg/ml) of EGb761. Apoptosis and proliferation were evaluated after 72h of EGb761 exposure. Response to oxidative stress, tumorigenic properties and molecular changes were further investigated. While anti-oxidant effects were detected in all cell lines, EGb761 promoted anti-proliferative and pro-apoptotic effects mainly in hepatoma cells. Consistently, EGb761 treatment caused a significant reduction in colony and sphere forming ability in hepatoma cells and no mentionable changes in IH. Transcriptomic changes involved oxidative stress response as well as key oncogenic pathways resembling Nrf2- and mTOR signaling pathway. Taken together, EGb761 induces differential effects in non-transformed and cancer cells. While treatment confers protective effects in non-malignant cells, EGb761 significantly impairs tumorigenic properties in cancer cells by affecting key oncogenic pathways. Results provide the rational for clinical testing of EGb761 in preventive and therapeutic strategies in human liver diseases.

Delivering all in one: Antigen-nanocapsule loaded with dual adjuvant yields superadditive effects by DC-directed T cell stimulation.

Therapeutic vaccination is and remains a major challenge, particularly in cancer treatment. In this process, the effective activation of dendritic cells by a combination of distinctly acting adjuvants and an antigen is crucial for success. While most common vaccine formulations lack the efficiency to trigger sufficient T cell responses in a therapeutic tumor treatment, nanovaccines offer unique properties to tackle that challenge. Here, we report the stepwise development of a nanocapsule for vaccination approaches, comprising a shell consisting of antigen and loaded with a superadditive adjuvant combination. In a first initial step, we identified the combination of resiquimod (R848) and muramyl dipeptide (MDP) to have a superadditive stimulatory potential. Particulated in Spermine-modified dextran-nanoparticles, the dual-adjuvant maintains its superadditive character and stimulates murine dendritic cells (DC) stronger than the soluble equivalents. The second step was to evaluate a protein-based nanocapsule as suitable antigen source for the induction of antigen-specific T cell responses. Therefore, the DC-mediated antigen-specific T cell proliferation upon treatment with nanocapsules, whose shell consists of ovalbumin (OVA), was assessed. At least, the superadditive adjuvant combination was encapsulated into OVA-nanocapsules to create the final nanovaccine. Its immunostimulatory potential for DC was extensively tested by measuring the expression of co-stimulatory surface markers, the secretion of pro-inflammatory cytokines and the capability to mediate OVA-specific T cell responses. The developed nanovaccine triggers strong superadditive dendritic cell stimulation and potent antigen-specific CD4+ and CD8+ T cell proliferation. Combined with a high modifiability, an excellent biocompatibility, low cytotoxicity and an enormous loading capacity, the introduced antigen-nanocapsule provides an enormous potential for the effective delivery of superadditive adjuvant combinations, particularly when they target intracellular receptors.

Inducible knockdown of procollagen I protects mice from liver fibrosis and leads to dysregulated matrix genes and attenuated inflammation.

Organ fibrosis is characterized by a chronic wound-healing response, with excess deposition of extracellular matrix components. Here, collagen type I represents the most abundant scar component and a primary target for antifibrotic therapies. Liver fibrosis can progress to cirrhosis and primary liver cancer, which are the major causes of liver related morbidity and mortality. However, a (pro-)collagen type I specific therapy remains difficult and its therapeutic abrogation may incur unwanted side effects. We therefore designed tetracycline-regulated procollagen alpha1(I) short hairpin (sh)RNA expressing mice that permit a highly efficient inducible knockdown of the procollagen alpha1(I) gene in activated (myo-)fibroblasts, to study the effect of induced procollagen type I deficiency. Transgenic mice were generated using recombinase-mediated integration in embryonic stem cells or zinc-finger nuclease-aided genomic targeting combined with miR30-shRNA technology. Liver fibrosis was induced in transgenic mice by carbon tetrachloride, either without or with doxycycline supplementation. Doxycycline treated mice showed an 80-90% suppression of procollagen alpha1(I) transcription and a 40-50% reduction in hepatic collagen accumulation. Procollagen alpha1(I) knockdown also downregulated procollagens type III, IV and VI and other fibrosis related parameters. Moreover, this was associated with an attenuation of chronic inflammation, suggesting that collagen type I serves not only as major scar component, but also as modulator of other collagens and promoter of chronic inflammation.

Adverse genomic alterations and stemness features are induced by field cancerization in the microenvironment of hepatocellular carcinomas.

Hepatocellular Carcinoma (HCC) commonly develops in chronically damaged liver tissues. The resulting regenerative and inflammatory processes create an adverse milieu that promotes tumor-initiation and progression. A better understanding of the hepatic tumor-microenvironment interaction might infer profound therapeutic implications.Integrative whole genome and transcriptome analyses of different tumor regions, the invasive tumor border and tumor-surrounding liver (SL) were performed to identify associated molecular alterations and integrated with our existing HCC database. Expression levels and localization of established CSC markers were assessed in pre-neoplastic lesions and confirmed in two independent patient cohorts using qRT-PCR, immunohistochemistry and immunofluorescence.Our results indicate that genomic and transcriptomic profiles between SL and different tumor regions are quite distinct. Progressive increase in genetic alterations and activation of pathways related to proliferation as well as apoptosis were observed in the tumor tissue, while activation of stemness markers was present in cirrhotic SL and continuously decreased from pre-neoplastic lesions to HCC. Interestingly, the invasive tumor border was characterized by inflammatory and EMT-related gene sets as well as activation of pro-survival signaling. Consistently, integration of gene expression signatures with two independent HCC databases containing 300 HCCs revealed that border signatures are predictive of HCC patient survival.Prognostic significance of the permissive liver microenvironment might be a consequence of a pro-oncogenic field effect that is caused by chronic regenerative processes. Activation of key oncogenic features and immune-response signaling indicates that the cross-talk between tumor and microenvironment might be a promising therapeutic and/or preventive target.

Glycine-functionalized copper(ii) hydroxide nanoparticles with high intrinsic superoxide dismutase activity.

Superoxide dismutases (SOD) are a group of enzymes that catalyze the dismutation of superoxide (O2-) radicals into molecular oxygen (O2) and H2O2 as a first line of defense against oxidative stress. Here, we show that glycine-functionalized copper(ii) hydroxide nanoparticles (Gly-Cu(OH)2 NPs) are functional SOD mimics, whereas bulk Cu(OH)2 is insoluble in water and catalytically inactive. In contrast, Gly-Cu(OH)2 NPs form water-dispersible mesocrystals with a SOD-like activity that is larger than that of their natural CuZn enzyme counterpart. Based on this finding, we devised an application where Gly-Cu(OH)2 NPs were incorporated into cigarette filters. Cigarette smoke contains high concentrations of toxic reactive oxygen species (ROS, >1016 molecules per puff) including superoxide and reactive nitrogen species which lead to the development of chronic and degenerative diseases via oxidative damage and subsequent cell death. Embedded in cigarette filters Gly-Cu(OH)2 NPs efficiently removed ROS from smoke, thereby protecting lung cancer cell lines from cytotoxic effects. Their stability, ease of production and versatility make them a powerful tool for a wide range of applications in environmental chemistry, biotechnology and medicine.

Activation of silent mating type information regulation 2 homolog 1 by human chorionic gonadotropin exerts a therapeutic effect on hepatic injury and inflammation.

Incidence and prevalence of inflammatory liver diseases has increased over the last years, but therapeutic options are limited. Pregnancy induces a state of immune tolerance, which can result in spontaneous improvement of clinical symptoms of certain autoimmune diseases including autoimmune hepatitis (AIH). We investigated the immune-suppressive mechanisms of the human pregnancy hormone, chorionic gonadotropin (hCG), in the liver. hCG signaling activates silent mating type information regulation 2 homolog 1 (SIRT1), which deacetylates forkhead box o3 (FOXO3a), leading to repression of proapoptotic gene expression, because the immunosuppressive consequence attributed to the absence of caspase-3 activity of hepatocellular interleukin 16 (IL-16) is no longer processed and released. Thus, serum levels of IL-16, a key chemotactic factor for CD4+ lymphocytes, were reduced and migration to injured hepatocytes prevented. Furthermore, elevated IL-16 levels are found in the sera from patients with AIH, hepatitis B virus, hepatitis C virus, and nonalcoholic steatohepatitis. CONCLUSION: Here, we report that hCG regulates the SIRT1/FOXO3a axis in hepatocytes, resulting in immune suppression by attenuating caspase-3-dependent IL-16 processing and release, which concomitantly prevents autoaggressive T-cell infiltration of the liver. Considering the low toxicity profile of hCG in humans, interrupting the inflammatory cycle by hCG opens new perspectives for therapeutic intervention of inflammatory liver diseases. (Hepatology 2017;65:2074-2089).

Hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal NADPH oxidase.

OBJECTIVES: Hydroxychloroquine (HCQ) has been used for decades to treat patients with rheumatic diseases, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis or the antiphospholipid syndrome (APS). We hypothesise that HCQ might target endosomal NADPH oxidase (NOX), which is involved in the signal transduction of cytokines as well as antiphospholipid antibodies (aPL). METHODS: For in vitro experiments, monocytic cells were stimulated with tumour necrosis factor α (TNFα), interleukin-1ß (IL-1ß) or a human monoclonal aPL and the activity of NOX was determined by flow cytometry. The expression of genes known to be induced by these stimuli was quantified by quantitative reverse transcription PCR. Live cell imaging was performed by confocal laser scanning microscopy. Finally, the effects of HCQ on NOX-induced signal transduction were analysed in an in vivo model of venous thrombosis. RESULTS: HCQ strongly reduces or completely prevents the induction of endosomal NOX by TNFα, IL-1ß and aPL in human monocytes and MonoMac1 cells. As a consequence, induction of downstream genes by these stimuli is reduced or abrogated. This effect of HCQ is not mediated by direct interference with the agonists but by inhibiting the translocation of the catalytic subunit of NOX2 (gp91phox) into the endosome. In vivo, HCQ protects mice from aPL-induced and NOX2-mediated thrombus formation. CONCLUSIONS: We describe here a novel mechanism of action of HCQ, that is, interference with the assembly of endosomal NOX2. Since endosomal NOX2 is involved in many inflammatory and prothrombotic signalling pathways, this activity of HCQ might explain many of its beneficial effects in rheumatic diseases including the APS.

Identification of a classic nuclear localization signal at the N terminus that regulates the subcellular localization of Rbfox2 isoforms during differentiation of NMuMG and P19 cells.

Nuclear localization of the alternative splicing factor Rbfox2 is achieved by a C-terminal nuclear localization signal (NLS) which can be excluded from some Rbfox2 isoforms by alternative splicing. While this predicts nuclear and cytoplasmic localization, Rbfox2 is exclusively nuclear in some cell types. Here, we identify a second NLS in the N terminus of Rbfox2 isoform 1A that is not included in Rbfox2 isoform 1F. Rbfox2 1A isoforms lacking the C-terminal NLS are nuclear, whereas equivalent 1F isoforms are cytoplasmic. A shift in Rbfox2 expression toward cytoplasmic 1F isoforms occurs during epithelial to mesenchymal transition (EMT) and could be important in regulating the activity and function of Rbfox2.

Dextran-based therapeutic nanoparticles for hepatic drug delivery.

AIM: Evaluation of dextran-based nanoparticles (DNP) as a drug delivery system to target myeloid cells of the liver. MATERIALS & METHODS: DNP were synthesized and optionally PEGylated. Their toxicity and cellular uptake were studied in vitro. Empty and siRNA-carrying DNP were tested in vivo with regard to biodistribution and cellular uptake. RESULTS: In vitro, DNP were taken up by cells of the myeloid lineage without compromising their viability. In vivo, empty and siRNA-carrying DNP distributed to the liver where a single treatment addressed approximately 70% of macrophages and dendritic cells. Serum parameters indicated no in vivo toxicity. CONCLUSION: DNP are multifunctional liver-specific drug carriers which lack toxic side effects and may be utilized in clinical applications targeting liver macrophages.

Vaccination with trifunctional nanoparticles that address CD8+ dendritic cells inhibits growth of established melanoma.

AIM: We wanted to assess the potency of a trifunctional nanoparticle (NP) that targeted and activated CD8+ dendritic cells (DC) and delivered an antigen to induce antitumor responses. MATERIALS & METHODS: The DC targeting and activating properties of ferrous NPs conjugated with immunostimulatory CpG-oligonucleotides, anti-DEC205 antibody and ovalbumin (OVA) as a model antigen to induce antigen-specific T-cell responses and antitumor responses were analyzed. RESULTS: OVA-loaded NP conjugated with immunostimulatory CpG-oligonucleotides and anti-DEC205 antibody efficiently targeted and activated CD8+ DC in vivo, and induced strong OVA-specific T-cell activation. Vaccination of B16/OVA tumor-burdened mice with this NP formulation resulted in tumor growth arrest. CONCLUSION: CD8+ DC-targeting trifunctional nanocarriers bear significant potential for antitumor immunotherapy.

CAF-like state in primary skin fibroblasts with constitutional BRCA1 epimutation sheds new light on tumor suppressor deficiency-related changes in healthy tissue.

Constitutive epimutations of tumor suppressor genes are increasingly considered as cancer predisposing factors equally to sequence mutations. In light of the emerging role of the microenvironment for cancer predisposition, initiation, and progression, we aimed to characterize the consequences of a BRCA1 epimutation in cells of mesenchymal origin. We performed a comprehensive molecular and cellular comparison of primary dermal fibroblasts taken from a monozygous twin pair discordant for recurrent cancers and BRCA1 epimutation, whose exceptional clinical case we previously reported in this journal. Comparative transcriptome analysis identified differential expression of extracellular matrix-related genes and pro-tumorigenic growth factors, such as collagens and CXC chemokines. Moreover, genes known to be key markers of so called cancer-associated fibroblasts (CAFs), such as ACTA2, FAP, PDPN, and TNC, were upregulated in fibroblasts of the affected twin (BRCA1(mosMe)) in comparison to those of the healthy twin (BRCA1(wt)). Further analyses detected CAF-typical cellular features, including an elevated growth rate, enhanced migration, altered actin architecture and increased production of ketone bodies in BRCA1(mosMe) fibroblasts compared to BRCA1(wt) fibroblasts. In addition, conditioned medium of BRCA1(mosMe) fibroblasts was more potent than conditioned medium of BRCA1(wt) fibroblasts to promote cell proliferation in an epithelial and a cancer cell line. Our data demonstrate, that a CAF-like state is not an exclusive feature of tumor-associated tissue but also exists in healthy tissue with tumor suppressor deficiency. The naturally occurring phenomenon of twin fibroblasts differing in their BRCA1 methylation status revealed to be a unique powerful tool for exploring tumor suppressor deficiency-related changes in healthy tissue, reinforcing their significance for cancer predisposition.

SIRT1 prevents genotoxic stress-induced p53 activation in acute myeloid leukemia.

SIRT1 is an important regulator of cellular stress response and genomic integrity. Its role in tumorigenesis is controversial. Whereas sirtuin 1 (SIRT1) can act as a tumor suppressor in some solid tumors, increased expression has been demonstrated in many cancers, including hematologic malignancies. In chronic myeloid leukemia, SIRT1 promoted leukemia development, and targeting SIRT1 sensitized chronic myeloid leukemia progenitors to tyrosine kinase inhibitor treatment. In this study, we investigated the role of SIRT1 in acute myeloid leukemia (AML). We show that SIRT1 protein, but not RNA levels, is overexpressed in AML samples harboring activating mutations in signaling pathways. In FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)(+)-cells protein, expression of SIRT1 is regulated by FLT3 kinase activity. In addition, SIRT1 function is modulated via the ATM-DBC1-SIRT1 axis in a FLT3-ITD-dependent manner. In murine leukemia models driven by MLL-AF9 or AML1-ETO coexpressing FLT3-ITD, SIRT1 acts as a safeguard to counteract oncogene-induced stress, and leukemic blasts become dependent on SIRT1 activity. Pharmacologic targeting or RNAi-mediated knockdown of SIRT1 inhibited cell growth and sensitized AML cells to tyrosine kinase inhibitor treatment and chemotherapy. This effect was a result of the restoration of p53 activity. Our data suggest that targeting SIRT1 represents an attractive therapeutic strategy to overcome primary resistance in defined subsets of patients with AML.

Molybdenum trioxide nanoparticles with intrinsic sulfite oxidase activity.

Sulfite oxidase is a mitochondria-located molybdenum-containing enzyme catalyzing the oxidation of sulfite to sulfate in the amino acid and lipid metabolism. Therefore, it plays a major role in detoxification processes, where defects in the enzyme cause a severe infant disease leading to early death with no efficient or cost-effective therapy in sight. Here we report that molybdenum trioxide (MoO3) nanoparticles display an intrinsic biomimetic sulfite oxidase activity under physiological conditions, and, functionalized with a customized bifunctional ligand containing dopamine as anchor group and triphenylphosphonium ion as targeting agent, they selectively target the mitochondria while being highly dispersible in aqueous solutions. Chemically induced sulfite oxidase knockdown cells treated with MoO3 nanoparticles recovered their sulfite oxidase activity in vitro, which makes MoO3 nanoparticles a potential therapeutic for sulfite oxidase deficiency and opens new avenues for cost-effective therapies for gene-induced deficiencies.

Multifunctional two-photon active silica-coated Au@MnO Janus particles for selective dual functionalization and imaging.

Monodisperse multifunctional and nontoxic Au@MnO Janus particles with different sizes and morphologies were prepared by a seed-mediated nucleation and growth technique with precise control over domain sizes, surface functionalization, and dye labeling. The metal oxide domain could be coated selectively with a thin silica layer, leaving the metal domain untouched. In particular, size and morphology of the individual (metal and metal oxide) domains could be controlled by adjustment of the synthetic parameters. The SiO2 coating of the oxide domain allows biomolecule conjugation (e.g., antibodies, proteins) in a single step for converting the photoluminescent and superparamagnetic Janus nanoparticles into multifunctional efficient vehicles for theranostics. The Au@MnO@SiO2 Janus particles were characterized using high-resolution transmission electron microscopy (HR-)TEM, powder X-ray diffraction (PXRD), optical (UV-vis) spectroscopy, confocal laser fluorescence scanning microscopy (CLSM), and dynamic light scattering (DLS). The functionalized nanoparticles were stable in buffer solution or serum, showing no indication of aggregation. Biocompatibility and potential biomedical applications of the Au@MnO@SiO2 Janus particles were assayed by a cell viability analysis by coincubating the Au@MnO@SiO2 Janus particles with Caki 1 and HeLa cells. Time-resolved fluorescence spectroscopy in combination with CLSM revealed the silica-coated Au@MnO@SiO2 Janus particles to be highly two-photon active; no indication for an electronic interaction between the dye molecules incorporated in the silica shell surrounding the MnO domains and the attached Au domains was found; fluorescence quenching was observed when dye molecules were bound directly to the Au domains.

Sirtuin-6-dependent genetic and epigenetic alterations are associated with poor clinical outcome in hepatocellular carcinoma patients.

UNLABELLED: Sirtuin 6 (SIRT6) is a member of the sirtuin family of NAD+-dependent deacetylases. Genetic deletion of Sirt6 in mice results in a severe degenerative phenotype with impaired liver function and premature death. The role of SIRT6 in development and progression of hepatocellular carcinoma is currently unknown. We first investigated SIRT6 expression in 153 primary human liver cancers and in normal and cirrhotic livers using microarray analysis. SIRT6 was significantly down-regulated in both cirrhotic livers and cancer. A Sirt6 knockout (KO) gene expression signature was generated from primary hepatoctyes isolated from 3-week-old Sirt6-deficient animals. Sirt6-deficient hepatocytes showed up-regulation of established hepatocellular carcinoma (HCC) biomarkers alpha-fetoprotein (Afp), insulin-like growth factor 2 (Igf2), H19, and glypican-3. Furthermore, decreased SIRT6 expression was observed in hepatoma cell lines that are known to be apoptosis-insensitive. Re-expression of SIRT6 in HepG2 cells increased apoptosis sensitivity to CD95-stimulation or chemotherapy treatment. Loss of Sirt6 was characterized by oncogenic changes, such as global hypomethylation, as well as metabolic changes, such as hypoglycemia and increased fat deposition. The hepatocyte-specific Sirt6-KO signature had a prognostic impact and was enriched in patients with poorly differentiated tumors with high AFP levels as well as recurrent disease. Finally, we demonstrated that the Sirt6-KO signature possessed a predictive value for tumors other than HCC (e.g., breast and lung cancer). CONCLUSION: Loss of SIRT6 induces epigenetic changes that may be relevant to chronic liver disease and HCC development. Down-regulation of SIRT6 and genes dysregulated by loss of SIRT6 possess oncogenic effects in hepatocarcinogenesis. Our data demonstrate that deficiency in one epigenetic regulator predisposes a tumorigenic phenotype that ultimately has relevance for outcome of HCC and other cancer patients.

Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells.

The multidrug resistance-associated protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel "Screen and Insert" (ScIn) technology to achieve stable and highly reproducible expression of 13 ABCC2 variants in HT1080 cells. Western blotting revealed lower (30-65%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Variants N1244K and R1174H decreased transport of glutathione-methylfluorescein (GS-MF) and glutathione-monochlorobimane (GS-MCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GS-MCB transport by 50% as compared with WT. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for GS-MF and GS-MCB by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation.

A variant of Smurf2 protects mice against colitis-associated colon cancer by inducing transforming growth factor ß signaling.

BACKGROUND & AIMS: Transforming growth factor (TGF)-ß signaling, which is down-regulated by the E3 ubiquitin ligase Smad ubiquitin regulating factor 2 (Smurf2), promotes development of cancer. We identified a splice variant of Smurf2 (ΔE2Smurf2) and investigated its role in colon carcinogenesis in mice. METHODS: Colitis-associated colon cancer was induced in mice by administration of azoxymethane, followed by 3 cycles of oral administration of dextran sodium sulfate. Messenger RNA levels of Smurf2 in colon tumors and control tissue were measured by quantitative polymerase chain reaction; lymphocyte and cytokine levels were measured in tumor and tissue samples. RESULTS: Tumor-infiltrating CD4(+) cells expressed higher levels of ΔE2Smurf2 than CD4(+) cells from nontumor tissues of wild-type mice. T cell-specific overexpression of ΔE2Smurf2 increased TGF-ß signaling by suppressing protein levels of Smurf2, accompanied by an increase in levels of TGF-ß receptor type II. Transgenic mice that overexpress ΔE2Smurf2 were protected against development of colitis-associated tumors and down-regulated proinflammatory cytokines such as interleukin-6. Patients with chronic inflammatory bowel disease had a significantly lower ratio of Smurf2/ΔE2Smurf2 than control individuals. CONCLUSIONS: T cell-specific ΔE2Smurf2 degrades wild-type Smurf2 and controls intestinal tumor growth in mice by up-regulating TGF-ß receptor type II, reducing proliferation and production of proinflammatory cytokines.

Antiphospholipid antibodies induce translocation of TLR7 and TLR8 to the endosome in human monocytes and plasmacytoid dendritic cells.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by thromboembolic events and/or fetal loss in the presence of antiphospholipid antibodies (aPLs). The mechanisms underlying the pathogenicity of aPLs are still poorly understood. Here we show that 3 human monoclonal aPLs as well as IgG fractions from patients with the APS increase mRNA expression of the intracellular toll-like receptor (TLR) 7 in plasmacytoid dendritic cells and TLR8 in monocytes. Simultaneously they induce the translocation of TLR7 or TLR8 from the endoplasmic reticulum to the endosome. These effects depend on the uptake of aPLs into the endosome, subsequent activation of endosomal NADPH oxidase, and generation of superoxide. As a consequence cells are dramatically sensitized to ligands for TLR7 and TLR8. This observation delineates a novel signal transduction pathway in innate immunity originating from the endosome. Because the overexpression of TLR7 can also be detected in plasmacytoid dendritic cells from patients with the APS ex vivo, our results provide an explanation for proinflammatory and procoagulant effects of aPLs. Because inappropriate expression of TLR7 has been implicated in the development of systemic autoimmunity, these findings may also be relevant for the understanding of autoimmunity.