技术资料

Receptor tyrosine kinases (RTK) are therapeutic targets for the treatment of malignancy. However,tumor cells develop resistance to targeted therapies through the activation of parallel signaling cascades. Recent evidence has shown that redundant or compensatory survival signals responsible for resistance are initiated by nontargeted glycoprotein RTKs coexpressed by the cell. We hypothesized that disrupting specific functions of the posttranslational machinery of the secretory pathway would be an effective strategy to target both primary and redundant RTK signaling. Using the N-linked glycosylation inhibitor,tunicamycin,we show that expression levels of several RTKS (EGFR,ErbB2,ErbB3,and IGF-IR) are exquisitely sensitive to inhibition of N-linked glycosylation. Disrupting this synthetic process reduces both cellular protein levels and receptor activity in tumor cells through retention of the receptors in the endoplasmic reticulum/Golgi compartments. Using U251 glioma and BXPC3 pancreatic adenocarcinoma cell lines,two cell lines resistant to epidermal growth factor receptor-targeted therapies,we show that inhibiting N-linked glycosylation markedly reduces RTK signaling through Akt and radiosensitizes tumor cells. In comparison,experiments in nontransformed cells showed neither a reduction in RTK-dependent signaling nor an enhancement in radiosensitivity,suggesting the potential for a therapeutic ratio between tumors and normal tissues. This study provides evidence that enzymatic steps regulating N-linked glycosylation are novel targets for developing approaches to sensitize tumor cells to cytotoxic therapies. View Publication

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs),and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo,with a consequent enhancement of MCC. To this end,camostat,a trypsin-like protease inhibitor,provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells,a potency order of placental bikunin {\textgreater} camostat {\textgreater} 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester {\textgreater} aprotinin {\textgreater} soybean trypsin inhibitor = alpha1-antitrypsin,was largely consistent with that observed for inhibition of prostasin,a molecular candidate for the airway CAP. In vivo,topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep,camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary,camostat attenuates ENaC function and enhances MCC,providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically. View Publication

In human cytomegalovirus (HCMV)-seropositive patients,CD34+ hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34+ HPCs to investigate mechanisms of viral latency. However,analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34+ HPCs from different donors,availability of cells,and low frequency of reactivation. In addition,multiple progenitor cell types express surface CD34,and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro In this study,we generated CD34+ progenitor cells from two different embryonic stem cell (ESC) lines,WA01 and WA09,to circumvent limitations associated with primary CD34+ HPCs. HCMV infection of CD34+ HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34+ HPCs. Analysis of HCMV-infected primary or ESC-derived CD34+ HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38+ CD90+ and CD38+/low CD90- HPCs but persistently infected CD38- CD90+ cells to produce infectious virus. These results indicate that ESC-derived CD34+ HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34+ cells.IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34+ hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however,these cells are a heterogeneous pool with donor-to-donor variation in functional,genetic,and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states,which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation. View Publication

BACKGROUND While essential to the normal differentiation of ciliated airway epithelial cells,upregulated Wnt signaling in chronic rhinosinusitis with nasal polyps (CRSwNP) has been proposed to result in abnormal epithelial morphology and dysfunctional mucociliary clearance. The mechanism of epithelial Wnt signaling dysregulation in CRSwNP is unknown,and importantly cellular sources of Wnt ligands in CRSwNP have not yet been investigated. METHODS Human sinonasal epithelial cells (hSNECs) and human sinonasal fibroblasts (hSNFs) were collected from 34 human subjects (25 control and 9 CRSwNP) and differentiated as primary air-liquid interface (ALI) and organoid co-cultures. hSNECs were isolated to the apical compartment of the transwell and hSNFs were isolated to the basolateral compartment. After 21 days of ALI culture,ciliary expression and sinonasal epithelial morphology were examined by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). An organoid model was used to evaluate proliferation of basal cells in presence of hSNFs. RESULTS Epithelial cells co-cultured with CRSwNP-hSNFs revealed significantly decreased ciliated cells,altered epithelial cell morphology,and increased colony forming efficiency compared to epithelial cells co-cultured with control-hSNFs. CRSwNP-hSNFs showed significantly higher messenger RNA (mRNA) expression of canonical WNT3A. A Wnt agonist,CHIR99021,replicated CRSwNP-hSNF co-cultures,and treatment with the Wnt inhibitor IWP2 prevented abnormal morphologies. CONCLUSION These results suggest that abnormal interactions between epithelial cells and fibroblasts may contribute to CRSwNP pathogenesis and supports the concept that dysregulated Wnt signaling contributes impairment to epithelial function in CRSwNP. View Publication

We used synthetic lethal high-throughput screening to interrogate 23,550 compounds for their ability to kill engineered tumorigenic cells but not their isogenic normal cell counterparts. We identified known and novel compounds with genotype-selective activity,including doxorubicin,daunorubicin,mitoxantrone,camptothecin,sangivamycin,echinomycin,bouvardin,NSC146109,and a novel compound that we named erastin. These compounds have increased activity in the presence of hTERT,the SV40 large and small T oncoproteins,the human papillomavirus type 16 (HPV) E6 and E7 oncoproteins,and oncogenic HRAS. We found that overexpressing hTERT and either E7 or LT increased expression of topoisomerase 2alpha and that overexpressing RAS(V12) and ST both increased expression of topoisomerase 1 and sensitized cells to a nonapoptotic cell death process initiated by erastin. View Publication

PURPOSE A major clinical problem in the treatment of breast cancer is the inherent and acquired resistance to antiestrogen therapy. In this study,we sought to determine whether antiprogestin treatment,used as a monotherapy or in combination with antiestrogen therapy,induced growth arrest and active cell death in antiestrogen-resistant breast cancer cells. EXPERIMENTAL DESIGN MCF-7 sublines were established from independent clonal isolations performed in the absence of drug selection and tested for their response to the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (fulvestrant),and the antiprogestin mifepristone (MIF). The cytostatic (growth arrest) effects of the hormones were assessed with proliferation assays,cell counting,flow cytometry,and a determination of the phosphorylation status of the retinoblastoma protein. The cytotoxic (apoptotic) effects were analyzed by assessing increases in caspase activity and cleavage of poly(ADP-ribose) polymerase. RESULTS All of the clonally derived MCF-7 sublines expressed estrogen receptor and progesterone receptor but showed a wide range of antiestrogen sensitivity,including resistance to physiological levels of 4-OHT. Importantly,all of the clones were sensitive to the antiprogestin MIF,whether used as a monotherapy or in combination with 4-OHT. MIF induced retinoblastoma activation,G(1) arrest,and apoptosis preceded by caspase activation. CONCLUSIONS We demonstrate that: (a) estrogen receptor(+)progesterone receptor(+),4-OHT-resistant clonal variants can be isolated from an MCF-7 cell line in the absence of antiestrogen selection; and (b) MIF and MIF plus 4-OHT combination therapy induces growth arrest and active cell death of the antiestrogen-resistant breast cancer cells. These preclinical findings show potential for a combined hormonal regimen of an antiestrogen and an antiprogestin to combat the emergence of antiestrogen-resistant breast cancer cells and,ultimately,improve the therapeutic index of antiestrogen therapy. View Publication

BACKGROUND Taking into consideration a recent surge of a lung injury condition associated with electronic cigarette use,we devised an in vitro model of sub-chronic exposure of human bronchial epithelial cells (HBECs) in air-liquid interface,to determine deterioration of epithelial cell barrier from sub-chronic exposure to cigarette smoke (CS),e-cigarette aerosol (EC),and tobacco waterpipe exposures (TW). METHODS Products analyzed include commercially available e-liquid,with 0{\%} or 1.2{\%} concentration of nicotine,tobacco blend (shisha),and reference-grade cigarette (3R4F). In one set of experiments,HBECs were exposed to EC (0 and 1.2{\%}),CS or control air for 10 days using 1 cigarette/day. In the second set of experiments,exposure of pseudostratified primary epithelial tissue to TW or control air exposure was performed 1-h/day,every other day,until 3 exposures were performed. After 16-18 h of last exposure,we investigated barrier function/structural integrity of the epithelial monolayer with fluorescein isothiocyanate-dextran flux assay (FITC-Dextran),measurements of trans-electrical epithelial resistance (TEER),assessment of the percentage of moving cilia,cilia beat frequency (CBF),cell motion,and quantification of E-cadherin gene expression by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS When compared to air control,CS increased fluorescence (FITC-Dextran assay) by 5.6 times,whereby CS and EC (1.2{\%}) reduced TEER to 49 and 60{\%} respectively. CS and EC (1.2{\%}) exposure reduced CBF to 62 and 59{\%},and cilia moving to 47 and 52{\%},respectively,when compared to control air. CS and EC (1.2{\%}) increased cell velocity compared to air control by 2.5 and 2.6 times,respectively. The expression of E-cadherin reduced to 39{\%} of control air levels by CS exposure shows an insight into a plausible molecular mechanism. Altogether,EC (0{\%}) and TW exposures resulted in more moderate decreases in epithelial integrity,while EC (1.2{\%}) substantially decreased airway epithelial barrier function comparable with CS exposure. CONCLUSIONS The results support a toxic effect of sub-chronic exposure to EC (1.2{\%}) as evident by disruption of the bronchial epithelial cell barrier integrity,whereas further research is needed to address the molecular mechanism of this observation as well as TW and EC (0{\%}) toxicity in chronic exposures. View Publication

Deregulation of apoptosis,the physiological form of cell death,is closely associated with immunological diseases and cancer. Apoptosis is activated either by death receptor-driven or mitochondrial pathways,both of which may provide potential targets for novel anticancer drugs. Although several ligands stimulating death receptors have been described,the actual molecular events triggering the mitochondrial pathway are largely unknown. Here,we show initiation of apoptosis by gradual depletion of the intracellular coenzyme NAD+. We identified the first low molecular weight compound,designated FK866,which induces apoptosis by highly specific,noncompetitive inhibition of nicotinamide phosphoribosyltransferase (NAPRT),a key enzyme in the regulation of NAD+ biosynthesis from the natural precursor nicotinamide. Interference with this enzyme does not primarily intoxicate cells because the mitochondrial respiratory activity and the NAD+ -dependent redox reactions involved remain unaffected as long as NAD+ is not effectively depleted by catabolic reactions. Certain tissues,however,have a high turnover of NAD+ through its cleavage by enzymes like poly(ADP-ribose) polymerase. Such cells often rely on the more readily available nicotinamide pathway for NAD+ synthesis and undergo apoptosis after inhibition of NAPRT,whereas cells effectively using the nicotinic acid pathway for NAD+ synthesis remain unaffected. In support of this concept,FK866 effectively induced delayed cell death by apoptosis in HepG2 human liver carcinoma cells with an IC(50) of approximately 1 nM,did not directly inhibit mitochondrial respiratory activity,but caused gradual NAD+ depletion through specific inhibition of NAPRT. This enzyme,when partially purified from K562 human leukemia cells,was noncompetitively inhibited by FK866,and the inhibitor constants were calculated to be 0.4 nM for the enzyme/substrate complex (K(i)) and 0.3 nM for the free enzyme (K(i)'),respectively. Nicotinic acid and nicotinamide were both found to have antidote potential for the cellular effects of FK866. FK866 may be used for treatment of diseases implicating deregulated apoptosis such as cancer for immunosuppression or as a sensitizer for genotoxic agents. Furthermore,it may provide an important tool for investigation of the molecular triggers of the mitochondrial pathway leading to apoptosis through enabling temporal separation of NAD+ decrease from ATP breakdown and apoptosis by several days. View Publication

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence,this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes,Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution,the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay,which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2{\%} vs. 9.9{\%},p {\textgreater} 0.05). However,when 100 mM Z-VAD-FMK was added to the post-thaw culture media,there was a significant enhancement in the survival rate from 9.9{\%} to 14.4{\%} (p {\textless} 0.05),which was further increased to 18.7{\%} when Z-VAD-FMK was also added to the freezing solution as well (p {\textless} 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy,and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture. View Publication

The recent emergence of the novel,pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here,we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally,we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention. View Publication

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density,but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF). In an LQT type 1 (LQT1) cardiomyocyte model,enDUB treatment restored delayed rectifier potassium currents and normalized action potential duration. CF-targeted enDUBs synergistically rescued common ($\Delta$F508) and pharmacotherapy-resistant (N1303K) CF mutations when combined with the US Food and Drug Administation (FDA)-approved drugs Orkambi (lumacaftor/ivacaftor) and Trikafta (elexacaftor/tezacaftor/ivacaftor and ivacaftor). Altogether,targeted deubiquitination via enDUBs provides a powerful protein stabilization method that not only corrects diverse diseases caused by impaired ion channel trafficking,but also introduces a new tool for deconstructing the ubiquitin code in situ. View Publication

Perhexiline is a potent prophylactic anti-anginal agent that has been shown to inhibit myocardial utilization of long-chain fatty acids and to inhibit the mitochondrial enzyme carnitine palmitoyltransferase (CPT)-1. We compared the hemodynamic and biochemical effects of perhexiline (0.5 and 2.0 microM) and of another CPT-1 inhibitor,oxfenicine (0.5 mM),in Langendorff-perfused rat hearts subjected to 60 min of low-flow ischemia (95{\%} flow reduction) followed by 30 min of reperfusion. Both perhexiline (2 microM only) and oxfenicine attenuated (p {\textless} 0.003,p {\textless} 0.0002,respectively) increases in diastolic tension during ischemia,without significant effects on developed tension,or on cardiac function during reperfusion. Myocardial concentrations of long-chain acylcarnitines (LCAC),products of CPT-1 action,were decreased (p {\textless} 0.05) by oxfenicine,unaffected by 2 microM perhexiline,and increased slightly by 0.5 microM perhexiline. Perhexiline,but not the active metabolite of oxfenicine,also inhibited cardiac CPT-2 with similar IC50 and Emax,although lower Hill slope,compared with CPT-1. Oxfenicine,but not perhexiline,reduced concentrations of the endogenous CPT-1 inhibitor,malonyl-CoA. Perhexiline,but not oxfenicine,inhibited myocardial release of lactate during normal flow. We conclude that (a) perhexiline protects against diastolic dysfunction during ischemia in this model,independent of major changes in LCAC accumulation and (b) this may result from simultaneous effects of perhexiline on myocardial CPT-1 and CPT-2. View Publication