技术资料

Autophagy is characterized by the sequestration of bulk cytoplasm,including damaged proteins and organelles,and delivery of the cargo to lysosomes for degradation. Although the autophagic pathway is also linked to tumour suppression activity,the mechanism is not yet clear. Here we report that cytosolic FoxO1,a forkhead O family protein,is a mediator of autophagy. Endogenous FoxO1 was required for autophagy in human cancer cell lines in response to oxidative stress or serum starvation,but this process was independent of the transcriptional activity of FoxO1. In response to stress,FoxO1 was acetylated by dissociation from sirtuin-2 (SIRT2),a NAD(+)-dependent histone deacetylase,and the acetylated FoxO1 bound to Atg7,an E1-like protein,to influence the autophagic process leading to cell death. This FoxO1-modulated cell death is associated with tumour suppressor activity in human colon tumours and a xenograft mouse model. Our finding links the anti-neoplastic activity of FoxO1 and the process of autophagy. View Publication

CD8 + cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide–MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell–cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell–cell contact,promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux,providing the necessary energy through oxidative phosphorylation,and its own protein translation on 55S ribosomes. However,the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here,we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton,mitochondria,and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. View Publication

BACKGROUND: The cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases,which sulfonate proteins,glycoproteins,glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs),which sulfonate various small molecules including hormones,neurotransmitters,and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions,cell signaling,and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism,namely reverse transcription. METHODS: MDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions,MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels,and qPCR was used to measure viral RNA and DNA products. RESULTS: We demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs,and through siRNA knockdown experiments,we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription. CONCLUSIONS: These studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules,raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics. View Publication

The receptor tyrosine kinase c-MET is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-MET axis is often dysregulated in tumors. c-MET activation can be caused by MET gene amplification,activating mutations,and auto- or paracrine mechanisms. Thus,c-MET inhibitors are under development as anticancer drugs. Tivantinib (ARQ 197) was reported as a small-molecule c-MET inhibitor and early clinical studies suggest antitumor activity. To assess whether the antitumor activity of tivantinib was due to inhibition of c-MET,we compared the activity of tivantinib with other c-MET inhibitors in both c-MET-addicted and nonaddicted cancer cells. As expected,other c-MET inhibitors,crizotinib and PHA-665752,suppressed the growth of c-MET-addicted cancers,but not the growth of cancers that are not addicted to c-MET. In contrast,tivantinib inhibited cell viability with similar potency in both c-MET-addicted and nonaddicted cells. These results suggest that tivantinib exhibits its antitumor activity in a manner independent of c-MET status. Tivantinib treatment induced a G(2)-M cell-cycle arrest in EBC1 cells similarly to vincristine treatment,whereas PHA-665752 or crizotinib treatment markedly induced G(0)-G(1) cell-cycle arrest. To identify the additional molecular target of tivantinib,we conducted COMPARE analysis,an in silico screening of a database of drug sensitivities across 39 cancer cell lines (JFCR39),and identified microtubule as a target of tivantinib. Tivantinib-treated cells showed typical microtubule disruption similar to vincristine and inhibited microtubule assembly in vitro. These results suggest that tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET. View Publication

BACKGROUND Emerging evidence of antibody-independent functions,as well as the clinical efficacy of anti-CD20 depleting therapies,helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis. OBJECTIVE To investigate whether CD19+ B cells may share expression of the serine-protease granzyme-B (GzmB),resembling classical cytotoxic CD8+ T lymphocytes,in the peripheral blood from relapsing-remitting MS (RRMS) patients. METHODS In this study,104 RRMS patients during different treatments and 58 healthy donors were included. CD8,CD19,Runx3,and GzmB expression was assessed by flow cytometry analyses. RESULTS RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8+GzmB+ T lymphocytes when compared to healthy volunteers. An increase in circulating CD19+GzmB+ B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA),untreated RRMS patients,and healthy donors but not when compared to interferon-$\beta$ (IFN). Moreover,regarding Runx3,the transcriptional factor classically associated with cytotoxicity in CD8+ T lymphocytes,the expression of GzmB was significantly higher in CD19+Runx3+-expressing B cells when compared to CD19+Runx3- counterparts in RRMS patients. CONCLUSIONS CD19+ B cells may exhibit cytotoxic behavior resembling CD8+ T lymphocytes in MS patients during different treatments. In the future,monitoring cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions." View Publication

The in vitro toxicological evaluation of e-liquid aerosol is an important aspect of consumer protection,but the cell model is of great significance. Due to its water solubility,e-liquid aerosol is deposited in the conducting zone of the respiratory tract. Therefore,primary normal human bronchial epithelial (NHBE) cells are more suitable for e-liquid aerosol testing than the widely used alveolar cell line A549. Due to their prolonged lifespan,immortalized cell lines derived from primary NHBE cells,exhibiting a comparable in vitro differentiation,might be an alternative for acute toxicity testing. In our study,A549 cells freshly isolated NHBE cells and the immortalized cell line CL-1548 were exposed at the air-liquid interface to e-liquid aerosol and cigarette mainstream smoke in a CULTEX(®) RFS compact module. The cell viability was analyzed 24 h post-exposure. In comparison with primary NHBE cells,the CL-1548 cell line showed lower sensitivity to e-liquid aerosol but significantly higher sensitivity compared to A549 cells. Therefore,the immortalized cell line CL-1548 is recommended as a tool for the routine testing of e-liquid aerosol and is preferable to A549 cells. View Publication

The cytotoxicity of paclitaxel against eight human tumour cell lines has been studied with in vitro clonogenic assays. The fraction of surviving cells fell sharply after exposure for 24 h to paclitaxel concentrations ranging from 2 to 20 nM; the paclitaxel IC50 was found to range between 2.5 and 7.5 nM. Increasing the paclitaxel concentration above 50 nM,however,resulted in no additional cytotoxicity after a 24 h drug exposure. Cells incubated in very high concentrations of paclitaxel (10,000 nM) had an increase in survival compared with cells treated with lower concentrations of the drug. Prolonging the time of exposure of cells to paclitaxel from 24 to 72 h increased cytotoxicity from 5 to 200 fold in different cell lines. Exponentially growing cells were more sensitive to paclitaxel than were cells in the plateau phase of growth. Cremophor EL,the diluent in which the clinical preparation of paclitaxel is formulated,antagonised paclitaxel at concentrations of 0.135% (v/v). These data suggest that paclitaxel will be most effective clinically when there is prolonged exposure of tumour to the drug. Further,it appears that modest concentrations (i.e.,50 nM) should be as effective as higher concentrations of paclitaxel. Finally,we have noted that Cremophor EL is a biologically active diluent and,at high concentrations (0.135% v/v),can antagonise paclitaxel cytotoxicity. View Publication

Malignant pleural mesothelioma is an uncommon tumor largely confined to the thoracic cavity,which is resistant to conventional therapies,therefore prompting an intensive search for effective treatment alternatives. This study focuses on dendritic cell (DC) vaccination for malignant pleural mesothelioma and evaluates the in vitro efficacy of antigen-loaded DC-based vaccines for the induction of major histocompatibility complex Class I-restricted antimesothelioma cytotoxic T lymphocyte responses. The source of tumor-associated antigens for HLA-A2(+) DCs from healthy donors was apoptotic HLA-A2(-) mesothelioma cells either lacking or expressing heat shock protein 70 according to whether tumor cells were heat shocked or not before ultraviolet-mediated apoptosis. Our results show that both apoptotic preparations were equivalent regarding the responsiveness of DCs to combined treatment with tumor necrosis factor-alpha and poly(inosinic-cytidylic) acid,as determined by similar increased expression of costimulatory molecules and interleukin-12 production. However,only DCs loaded with apoptotic heat shock protein 70-expressing cells were found to be potent in vitro inducers of cytotoxic T lymphocyte activity against HLA-A2(+) mesothelioma cells. Such elicited cytotoxic T lymphocytes also exhibit cytotoxic activity against an HLA-A2(+) melanoma cell line,suggesting recognition of shared antigens. These findings therefore carry the potential of offering an alternative,promising approach for the therapy of patients with malignant pleural mesothelioma. View Publication

AbstractThe effect of targeted therapeutics on anticancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2,and the therapeutic effect has been partially attributed to GCN2 activation. Because ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function,we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T-cell activity,which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration,and attenuated transcriptional programs required for PMN development. Moreover,we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways,and increased activity of transcriptional regulons driven by Atf5,Mafg,and Zbtb7a. This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest,skewing immature MDSC development toward monocytic lineage cells. In vivo,we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally,in the tumor immune infiltrate. Thus,our data reveal transcriptional networks that govern MDSC developmental programs,and the impact of GCN2 stress signaling on the innate immune landscape in tumors,providing novel insight into potentially beneficial off-target effects of dabrafenib.Significance:An important,but poorly understood,aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities. View Publication

The purpose of this study is to review the development of BRAF inhibitors,with emphasis on the trials conducted with dabrafenib (GSK2118436) and the evolving role of dabrafenib in treatment for melanoma patients. Fifty percent of cutaneous melanomas have mutations in BRAF,resulting in elevated activity of the mitogen-activated protein kinase signaling pathway. Dabrafenib inhibits the mutant BRAF (BRAF(mut)) protein in melanomas with BRAF(V600E) and BRAF(V600K) genotypes. BRAF(V600E) metastatic melanoma patients who receive dabrafenib treatment exhibit high clinical response rates and compared with dacarbazine chemotherapy,progression-free survival. Efficacy has also been demonstrated in BRAF(V600K) patients and in those with brain metastases. Dabrafenib has a generally mild and manageable toxicity profile. Cutaneous squamous cell carcinomas and pyrexia are the most significant adverse effects. Dabrafenib appears similar to vemurafenib with regard to efficacy but it is associated with less toxicity. It is expected that new combinations of targeted drugs,such as the combination of dabrafenib and trametinib (GSK1120212,a MEK inhibitor),will provide higher response rates and more durable clinical benefit than dabrafenib monotherapy. View Publication

The effect of resiquimod (R-848),an immune-response modifier that is similar to imiquimod,on recurrent herpes simplex virus (HSV) was evaluated using the guinea pig model of genital herpes. Guinea pigs were intravaginally infected with HSV-2 and then were randomized on day 14 to receive nothing or 0.1 mL/kg per dose of subcutaneous resiquimod,given either daily,every other day,or weekly from days 15-35. During a 3-week course of therapy,recurrences in all 3 treated groups were reduced by textgreater80%,compared with the control group. After therapy,recurrences remained significantly (Ptextless.05) decreased in all 3 groups for the next 3 weeks. The group treated weekly developed the fewest recurrences. Significant increases in interleukin-2 levels,produced by incubation of mononuclear cells with HSV-2 antigens,but not in circulating antibody also were detected in the treated groups. Resiquimod treatment may offer significant advantages to present antiviral therapies for the control of recurrent genital herpes. View Publication

Myeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD),donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer,resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen-induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival,indicating in vivo biologic effects,no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1$\beta$ (IL-1$\beta$),we expected that IL-1$\beta$ KO donor MDSCs would be superior in subverting GVHD,but such MDSCs proved inferior relative to WT. IL-1$\beta$ release and IL-1 receptor expression was required for optimal MDSC function,and exogenous IL-1$\beta$ added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1$\beta$ could diminish survival in GVHD. However,loss of inflammasome activation and IL-1$\beta$ release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1$\beta$ signaling,improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum,the ATP release site,synergized with WT MDSCs,as did regulatory T-cell infusion,which we showed reduced but did not eliminate MDSC inflammasome activation,as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments. View Publication