Standard Western Blot Research Articles

Histone Deacetylase Inhibitors Enhance Cell Death In Burkitt Lymphoma Cells By Modulating Expression Of The WNT/β-Catenin Pathway and Survivin

BackgroundBurkitt Lymphoma (BL) is an aggressive non-Hodgkin B cell neoplasm primarily affecting children. While overall cure rates are high, prognosis for the 20% of patients with relapsed or refractory is dismal with a 16% survival rate at four years, indicating the need for novel therapeutic approaches. While epigenetic modulators have shown potential therapeutic benefit in other hematologic malignancies, their use in BL has been limited. Here, we evaluate whether histone deacetylase (HDAC) inhibitors could enhance cell death in chemotherapy resistant BL cells. MethodsThe BL cell lines Ramos, Raji, or previously generated/characterized immunochemotherapy resistant Raji BL cell lines (Raji 2R and Raji 4RH) (Barth et al., Br J Haematol, 2012) were treated for 48 hours with 4-hydroperoxycyclophosphamide (4-HC, the active metabolite of cyclophosphamide), cytarabine, prednisolone, HDAC inhibitors (suberoyanilide hydroxamic acid (SAHA, vorinostat), and romidepsin), or vehicle control. Cell viability was measured on a Via Cell, by Alamar Blue staining (Invitrogen), or by measuring Caspase-3 activation by flow cytometry. Levels of pro- (survivin, XIAP, MCL-1, livin, and BCL-XL) and anti- (Bax, Bak) apoptotic proteins were evaluated using standard western blot techniques. To evaluate the wiring of signaling networks in the basal state or in response to drug treatment, samples were fixed, permeabilized, and simultaneously stained for Caspase-3, phospho GSK-3 (pGSK3, the inactive form of this protein), active β-catenin, and survivin. Cells were processed on a BD FACSVerse and analyzed by gating on Caspase-3 negative (chemotherapy resistant) cells and monitoring the activity of the pGSK3, active β-catenin, and survivin using FlowJO (V9.6) software. ResultsRelative to the BL cell line Ramos, Raji BL cells were resistant to all doses of conventional chemotherapy (cyclophosphamide, ara-c, and prednisolone) tested. Because hyper-activation of the WNT/b-catenin network due to epigenetic modulation has been implicated in chemotherapy resistance in other B-lineage pediatric malignancies (Hogan et al., Blood 2011), we treated cells with chemotherapy or vehicle and used phosphoflow cytometry to evaluate the expression of pGSK-3, active β-catenin, and its downstream target survivin. Relative to vehicle control, we found that exposure to cytotoxic chemotherapy resulted in rewiring of the cellular networks with increased levels of pGSK3, β-catenin, and survivin. Western blot analyses of the immunochemotherapy resistant Raji 2R and Raji 4RH cells revealed similar upregulation of anti-apoptotic proteins including survivin, downregulation of pro-apoptotic proteins Bak and Bax, and increased expression of pGSK3 compared to Raji cells. To test whether alterations in these signaling axes might enhance the survival of chemorefractory BL cells, we treated Raji cells with the HDAC inhibitor SAHA and found induction of apoptosis at nanomolar doses. Sensitivity to SAHA correlated with down-regulation of active β-catenin and survivin in a dose-dependent manner. To extend these results, we also tested whether immunochemotherapy resistant Raji 2R and Raji 4RH cells were sensitive to HDAC inhibition and found similar robust induction of cell death upon treatment with a panel of HDAC inhibitors. ConclusionsActive β-catenin and survivin are upregulated in chemotherapy resistant BL cells. HDAC inhibition results in their rapid down regulation and enhanced apoptotic cell death. Further evaluation of inhibitors of the WNT/ β-catenin pathway and HDAC inhibitors is warranted in chemotherapy resistant BL and could have a role in treating refractory or relapsed BL patients. Disclosures:No relevant conflicts of interest to declare.

Expression of toll-like receptor 4 in maternal monocytes of patients with gestational diabetes mellitus

Toll-like receptors (TLRs) are pattern recognition receptors and play an important role in innate immune responses and the occurrence of inflammatory disease. TLR4 is a member of the TLR family and its activation is capable of inducing inflammatory responses, reflecting a relationship between the innate and adaptive immune systems. However, whether TLR4 is expressed in patients with gestational diabetes mellitus (GDM) has not been elucidated. The aim of the present study was to investigate whether TLR4 is expressed in maternal peripheral blood monocytes of patients with GDM. A case-control study, using standard quantitative polymerase chain reaction and western blotting, was performed to assess the TLR4 expression in 30 females with GDM and 32 healthy pregnant females at similar gestational ages. Serum tumor necrosis factor (TNF)-α levels were assessed using ELISA in all the females. The TLR4 expression levels in the maternal peripheral blood monocytes and the serum TNF-α levels were increased in females with GDM compared with healthy pregnant females (P<0.05). Additionally, there was a positive correlation between the TLR4 expression level in peripheral blood monocytes and serum TNF-α levels in all the females. These results indicate that TLR4-mediated release of inflammatory cytokines may represent one factor leading to increased glucose levels in patients with GDM. In addition, TLR4 may be involved in the pathogenesis of GDM.

Evaluation of the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test as an alternative to Western blot for confirmation of HIV infection

BackgroundIn the United States, a new HIV diagnostic algorithm has been proposed that uses an HIV-1/HIV-2 antibody differentiation immunoassay instead of Western blot or immunofluoresence for confirmatory testing. ObjectivesTo evaluate the Multispot HIV-1/HIV-2 Rapid Test (Multispot) as an alternative to Western blot analysis for confirmation of HIV infection. Study designA series of 205 serum and plasma specimens positive for HIV-1 or HIV-2 were used to compare the performance of Multispot to a standard HIV-1 Western blot. Positive samples included 63 specimens from patients>18 months of age, 33 proficiency survey specimens, and 109 specimens from nine commercial seroconversion and performance panels. In addition, 63 specimens from 51 HIV-exposed, uninfected children≤18 months of age in various stages of seroreversion and 192 HIV-negative samples were tested. Specimens were initially screened using a 4th generation HIV Ag/Ab Combo assay. ResultsMultispot readily discriminated between individuals with HIV-1 or HIV-2 infection and those who were uninfected. Of the 205 samples repeatedly reactive by the 4th generation screening assay, infection status was correctly confirmed by Multispot in 83.9% (172/205) compared to 68.8% (141/205) for Western blot. Multispot detected HIV-1 earlier in 27.6% of low-titer antibody specimens called indeterminate by Western blot, and effectively reduced the number of indeterminate results in seroreverting HIV-1 exposed, uninfected infants and for HIV-2 infections misinterpreted as indeterminate or positive by HIV-1 Western blot. ConclusionsMultispot offers speed and simplicity over Western blot and has an excellent performance for differentiation and confirmation of antibodies to HIV-1 and HIV-2.

Neurotrophin signaling and melanoma brain metastases.

e13024 Background: Adult metastatic brain tumors occur more frequently than primary intracranial neoplasms. Melanoma is the third most common tumor type that metastasizes to the brain. Therefore, elucidating the underlying biological mechanisms of melanoma metastases is critical. Melanocytes and neurons share a neural ectodermal origin, and thus, melanoma may preferentially travel to the brain due to the expression of common neurotrophin receptors, namely p75 neurotrophin receptor (p75NTR) and Trks. Further, high concentrations of neurotrophic factors present in the brain may recruit metastatic melanoma. We hypothesize that neurotrophin signaling through p75NTR is required for the malignant phenotype of melanoma and melanoma brain metastases (MBM). Methods: We investigated the expression of several neutrophin receptors, signaling molecules, and neurotrophins that we hypothesize to be important in the process of MBM. Using 14 malignant melanoma cell lines and two primary MBMs grown under neurosphere conditions, we investigated the expression of these molecules using standard western blotting, flow cytometry, and RT-PCR. We also performed an in depth microarray using of 14 primary MBM patients from Moffitt’s Total Cancer Care project. Results: All melanoma lines examined have robust expression of p75NTR, Trks, and neurotrophins. We find melanoma cell lines resistant to the BRAF inhibitor, vemurafenib, have elevated expression of neurotrophins and neurotrophin receptors when compared to lines sensitive to vemurafenib. We also detect high levels of p75NTR in patient MBM, at both mRNA and protein levels. Finally, we find a significant level of expression of all 11 genes tested by microarray. Conclusions: Previous studies have demonstrated that p75NTR has a role in melanoma cell survival in vitro. Here we demonstrate p75NTR and Trk receptors have high expression in melanoma cell lines and importantly, primary MBM. These data suggest that signaling through neurotophin receptors may be important for melanoma metastases and survival in the brain. Moreover, increased expression by vemurafenib resistant lines may suggest that these cells upregulate the expression of neurotrophin signaling as a means of treatment resistant.

Abstract 309: Osteogenic Induction of p-ERK 1/2 Signaling Associated with NOTCH1 Inactivation in Human Aortic Valvar Interstitial Cells

Introduction Heart valve disease affects over 2% of the population, leading to at least 20,000 deaths and $1 billion in direct costs in the US each year. Calcific aortic valve disease (CAVD) is linked to loss of function mutations in NOTCH1 that result in osteogenic gene induction. In vitro animal studies have established that multiple osteogenic targets are mediated through induction of p-ERK 1/2 signaling; however, the relationship between NOTCH1 and p-ERK has not been examined in primary human aortic valvar interstitial cells (AVICs). Hypothesis We tested the hypothesis that chemically-induced NOTCH1 suppression results in mineralization due to p-ERK 1/2 activation in human AVICs. Methods Human AVICs were isolated from CAVD specimens at the time of valve replacement. AVICs were cultured for two weeks in complete media containing DMEM and 10% FBS, and treated with calcific media (10mM b-glycerol phosphate; 60μM ascorbic acid-2-phosphate; 1μM dexamethasone), which has been shown to suppress NOTCH1 signaling. Standard real-time RT-PCR, western blot, and immunocytochemistry were used to compare groups. Results Treatment with calcific media resulted in a 2.3 fold decrease in the level of NOTCH1 expression and a 3 fold increase in p-ERK 1/2 expression, assessed with both western blots and immunocytochemistry. Interestingly, calcific media also induced a 3.5 fold decrease in VEGF, consistent with early osteogenic differentiation. Additionally, the AVICs treated with calcific media demonstrated overt calcification by Alizarin Red staining. Conclusions Human AVICs demonstrated calcification associated with p-ERK 1/2 activation and reduced NOTCH1 levels, suggesting p-ERK inhibition may mitigate the effects of NOTCH loss of function, thereby representing a potential pharmacologic therapy for CAVD. Further investigation of the molecular mechanisms linking NOTCH1 and p-ERK are warranted, including studies that specifically inhibit NOTCH1 signaling and define the impact of mechanical forces on molecular pathways, thereby identifying mechanisms underlying CAVD progression.

Role of MEK and RSK in Blood Pressure Regulation

A current hypothesis underlying Angiotensin II (Ang II) and norepinephrine (NE) mediated hypertension is that the extracellular regulated kinase cascade (MEK and ERK) is involved in phosphorylation of the regulatory myosin light chain (rMLC). However, the signal transduction is not fully elucidated. We hypothesized that in renal microvascular smooth muscle cells (RμVSMCs) MEK/ERK‐mediated phosphorylation of rMLC is through p90RSK, and this pathway contributes to hypertension. We utilized standard and PhosTag Western blots to measure Ang II and NE‐mediated phosphorylation of ERK, RSK, and rMLC in RμVSMCs. Additionally, CI‐1040 and SL 0101–1, which inhibit MEK and RSK, respectively, were utilized to interrogate this pathway. Simultaneously, an in vivo study was conducted to analyze the anti‐hypertensive effects of the MEK inhibitor CI‐1040 via regular tail cuff measurements. Osmotic minipumps were implanted in mice, delivering 1.0 μg/kg/min Ang II and 5 μg/kg/min NE. Two weeks after insertion of the pumps the mice were gavaged daily with 100 mg/kg of CI‐1040 for an additional 2 weeks. The molecular data suggest that the MEK/ERK/RSK pathway is involved in phosphorylation of rMLC, but contrary to previous studies the preliminary in vivo component failed to show a significant effect of CI‐1040 on blood pressure. TJW and this study were supported by the APS STEP‐UP program.

Increased arrhythmogenesis and expression of left ventricular pS368‐Connexin43 following acute myocardial ischemia (AMI) in female ovariectomized rats

Estrogen has been previously shown to be protective against ventricular arrhythmias following myocardial infarction possibly through changes in connexin 43 (Cx43) expression and phosphorylation. Phosphorylation of Cx43 at S368 is induced with AMI, suggesting that phosphorylation at this site plays a role in arrhythmogenesis. The goal of the current study was to determine if there is a direct correspondence between phosphorylation of left ventricular (LV)‐Cx43 at S368 and the arrhythmogenic response to AMI, and to further examine if this effect is augmented following loss of estrogen. Anesthetized, ventilated female ovariectomized (n=3) and intact (n=3) Sprague‐Dawley rats were subjected to 20‐minutes of AMI produced through ligation of the left coronary artery. The arrhythmic burden in response to AMI was measured using a standard lead II ECG, and quantified through an established scoring method. Total LV‐Cx43 expression and pS368‐Cx43 expression were measured through standard Western blotting techniques. Ovariectomized animals had a greater arrhythmic burden versus intact animals following AMI. Correspondingly, ovariectomized animals also had an increase in the pS368‐Cx43/Total Cx43 ratio. When data were further examined using a linear regression analysis, results show a direct positive correlation between the level of pS368‐Cx43 expression and arrhythmic burden (R2 = 0.94). IOER‐3726.

The involvement of heme oxygenase 1 but not nitric oxide synthase 2 in a hepatoprotective action of quercetin in lipopolysaccharide-induced hepatotoxicity of d-galactosamine sensitized rats

The objective of this study was to evaluate potential hepatoprotective capabilities of quercetin in relation to its modulation of the HO-1 and NOS-2 activities in an experimental model of fulminant liver failure. Liver insult was induced by in vivo administration of d-galactosamine (d-GalN, 400mg/kg, i.p.) and lipopolysaccharide (LPS, 10μg/kg, i.p.). The effects of quercetin (50mg/kg, i.p) on d-GalN toxicity was evaluated by standard biochemical, RT-PCR and Western blot methods. Administration of d-GalN/LPS combination resulted in significantly higher plasma levels of aminotransferases, as well as increased mRNA and protein expressions of both HO-1 and NOS-2 enzymes. Quercetin exhibited cytoprotective effects on the liver, as evidenced by decreased aminotransferase plasma levels. Additionally, quercetin treatment in d-GalN/LPS treated rats significantly increased HO-1 mRNA and its protein expressions. On the contrary, quercetin did not exhibit any significant effects on the levels of nitrites, and NOS-2 mRNA and protein expressions in d-GalN/LPS treated rats. Quercetin when given alone did not have any significant changes on liver enzymes nor HO-1 and NOS-2 mRNA and protein expressions. It can be concluded that the quercetin's induction of HO-1 and its byproducts, without concomitant NOS-2 activity reduction, is among mechanisms contributing to the hepatoprotective effect in d-GalN/LPS hepatotoxicity.

Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons. Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment. Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Neuroprotective effects of hesperidin, a plant flavanone, on rotenone-induced oxidative stress and apoptosis in a cellular model for Parkinson's disease.

Rotenone a widely used pesticide that inhibits mitochondrial complex I has been used to investigate the pathobiology of PD both in vitro and in vivo. Studies have shown that the neurotoxicity of rotenone may be related to its ability to generate reactive oxygen species (ROS), leading to neuronal apoptosis. The current study was carried out to investigate the neuroprotective effects of hesperidin, a citrus fruit flavanol, against rotenone-induced apoptosis in human neuroblastoma SK-N-SH cells. We assessed cell death, mitochondrial membrane potential, ROS generation, ATP levels, thiobarbituric acid reactive substances, reduced glutathione (GSH) levels, and the activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) using well established assays. Apoptosis was determined in normal, rotenone, and hesperidin treated cells, by measuring the protein expression of cytochrome c (cyt c), caspases 3 and 9, Bax, and Bcl-2 using the standard western blotting technique. The apoptosis in rotenone-induced SK-N-SH cells was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, the depletion of GSH, enhanced activities of enzymatic antioxidants, upregulation of Bax, cyt c, and caspases 3 and 9, and downregulation of Bcl-2, which were attenuated in the presence of hesperidin. Our data suggests that hesperidin exerts its neuroprotective effect against rotenone due to its antioxidant, maintenance of mitochondrial function, and antiapoptotic properties in a neuroblastoma cell line.

ITRAQ-based quantitative proteomic analysis of submandibular glands from rats with STZ-induced hyperglycemia

The impairment of salivary glands activity is often connected to the complaints of dry-mouth and subsequent degradation of the periodontium of diabetic patients. In this context, submandibular glands (SMGs) play a central role in saliva production and so the understanding of the molecular pathways affected is of paramount importance. Using a streptozotocin-induced hyperglycemia rat model and two different time points (2 and 4 months), we applied mass spectrometry-based proteomic techniques, validated with standard western blot analysis, to identify and quantify the effect of chronic hyperglycemia on the proteome of SMGs. We observed significant variations of proteins such as kallikreins, protein S100A6 or annexins. After 2 months of hyperglycemia, we observed an early phase response characterized by a significant increase of protein S100A6, linked to the inflammatory response, together with the impairment of metabolic and energy production processes. On the other hand, vesicular transport appeared to be favoured in such conditions. Interestingly, in a long-term response to hyperglycemia after 4 months of exposure, we observed a general attenuation of the variations. In conclusion, we present data that support the existence of an adaptation of the gland to long-term stress.

Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer

BackgroundG-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer.MethodsThe expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed.ResultsAnalysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue.ConclusionThe results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression.

Identification of the Black Tea Polyphenol Theaflavin-3, 3'-Digallate From Screen of Natural Product Inducers of Endoplasmic Reticulum Stress

Abstract 5021 Background:A distinguishing characteristic of myeloma plasma cells is the large quantity of paraprotein that they synthesize and secrete, rendering them especially sensitive to the effects of endoplasmic reticulum (ER) stress. Consistent with this notion, the proteasome inhibitor bortezomib disrupts protein equilibrium in the ER by preventing misfolded proteins from being properly degraded. Given the clinically validated importance of targeting ER stress mediated pathways in the treatment of multiple myeloma (MM), we sought to identify natural products that modulate pathways known to be an effective therapeutic target for MM for potential use to inhibit progression of asymptomatic MM to symptomatic MM without the limiting side effects of current targeted therapies. Methods:Using decreased protein processing in the secretory pathway as a measurable hallmark of ER stress, our screen employed the naturally secreted Gaussia luciferase (Gluc) as a reporter that can be easily monitored through extracellular release of luciferase activity in real time. KMS11 and ARP-1 MM cells expressing Gluc were exposed to compounds in our natural products library in order to identify those which potentially induce ER stress as measured by inhibition of Gluc secretion. The growth inhibitory activity of theaflavin-3, 3’–digallate (TF3) was further characterized by MTS assay. Mechanistic studies of ER stress related pathways including the unfolded protein response (UPR) and apoptotic cascades were analyzed by standard Western blotting techniques. Results:Our screen identified the black tea polyphenol TF3 as a significant inhibitor of GLUC secretion in ARP-1 and KMS-11 cells. TF3 at 0. 5 μM inhibits GLUC secretion by 73 and 68% in ARP-1 and KMS-11 cells, respectively. This inhibition observed is on par with that observed for bortezomib and tunacamycin (a well known inducer of ER stress). TF-3 effectively inhibits cellular proliferation and induces apoptosis in a panel of MM cell lines at physiologically achievable concentrations. Apoptotic induction is at least partially mediated by ER stress mediated pathways as upregulation of the protein chaperone HSP90 and phosphorylation of eIF2-a, a key mediator of the UPR pathway, occurs prior to caspase and PARP cleavage. Conclusions:Our results suggest that TF-3 inhibits protein secretion and MM cell growth through promotion of ER stress generating mechanisms. Based upon these promising results, further mechanistic evaluation and characterization of this safe, natural product as a prophylactic agent in the treatment of asymptomatic conditions like monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) is warranted. Disclosures:No relevant conflicts of interest to declare.

Nanomaterial-based electrochemical biosensors for cytochrome c using cytochrome c reductase

Emerging evidences have pointed out that the release of cytochrome c (cyt c) from mitochondria into cytosol is a critical step in the activation of apoptosis. This article presents a novel approach for the detection of mitochondrial cyt c release for the first time using cytochrome c reductase (CcR) immobilized on nanoparticles decorated electrodes. Two kinds of nanomaterial-based biosensor platforms were used: (a) carbon nanotubes (CNT) incorporated polypyrrole (PPy) matrix on Pt electrode and (b) self-assembled monolayer (SAM) functionalized gold nanoparticles (GNP) in PPy-Pt. Scanning electron microscope was used to characterize the surface morphologies of the nanomaterial modified electrodes. Cyclic voltammograms of both the biosensors showed reversible redox peaks at −0.45 and −0.34V vs Ag/AgCl, characteristic of CcR. In comparison, the CcR-CNT biosensor gave a detection limit of 0.5±0.03μM cyt c, which was 4-fold better than the CcR-GNP biosensor (2±0.03μM). Moreover, the CcR-CNT biosensor achieved a much larger linear range (1–1000μM) over the CcR-GNP biosensor (5–600μM) with 2-fold better sensitivity. The CcR-CNT-PPy-Pt biosensor was further applied to quantify the mitochondrial cyt c released in cytosol of A549 cells upon induction of apoptosis with doxorubicin, the results agreed well with standard western blot analysis.

A Comparative Study of CdTe Quantum Dots and CdTe@SiO2 Nanoparticles: Fabrication and Cytotoxicity in HEK293 Cells

Quantum Dots have shown remarkable potentials in biomedical research. Herein, we reported the effects of CdTe quantum dots (QDs) and CdTe@SiO2 nanoparticles (NPs) on human embryonic kidney 293 (HEK 293A) cells with the aim of investigating their in vitro cytotoxicity. The CdTe@SiO2 particles were prepared by reverse microemulsion method. The structural morphology of the CdTe and hydrophilic silica-coated CdTe particles were characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV-vis) spectrometry and photoluminescence (PL) spectrometry. The in vitro cytotoxicity of CdTe QDs and CdTe@SiO2 nanoparticles was assessed in 293A cells using standard MTT assay, western blot and fluorescent microscopy. The results showed that the CdTe and CdTe@SiO2 particles were relatively uniform with the diameter of about 3.8 nm, 75 nm respectively. The cell viability and the adhesion ability were similar to the control 293A cells. The level of the fibronectin protein expression was decreased with the increasing concentration of CdTe while the no effects were observed on expression of beta-actin in CdTe as well as CdTe@SiO2 treated cells even at highest concentration of 45 microg/mL which demonstrated their good biocompatibility to 293A cells. The results indicate that the CdTe@SiO2 nanoparticles are attractive candidates for biological imaging studies as expected.

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.

Ca2+ Induces Spontaneous Dephosphorylation of a Novel P5A-type ATPase

P5 ATPases constitute the least studied group of P-type ATPases, an essential family of ion pumps in all kingdoms of life. Although P5 ATPases are present in every eukaryotic genome analyzed so far, they have remained orphan pumps, and their biochemical function is obscure. We show that a P5A ATPase from barley, HvP5A1, locates to the endoplasmic reticulum and is able to rescue knock-out mutants of P5A genes in both Arabidopsis thaliana and Saccharomyces cerevisiae. HvP5A1 spontaneously forms a phosphorylated reaction cycle intermediate at the catalytic residue Asp-488, whereas, among all plant nutrients tested, only Ca(2+) triggers dephosphorylation. Remarkably, Ca(2+)-induced dephosphorylation occurs at high apparent [Ca(2+)] (K(i) = 0.25 mM) and is independent of the phosphatase motif of the pump and the putative binding site for transported ligands located in M4. Taken together, our results rule out that Ca(2+) is a transported substrate but indicate the presence of a cytosolic low affinity Ca(2+)-binding site, which is conserved among P-type pumps and could be involved in pump regulation. Our work constitutes the first characterization of a P5 ATPase phosphoenzyme and points to Ca(2+) as a modifier of its function.

Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53

Herpesviral capsids are assembled in the host cell nucleus and are subsequently translocated to the cytoplasm. During this process it has been demonstrated that the human cytomegalovirus proteins pUL50 and pUL53 interact and form, together with other viral and cellular proteins, the nuclear egress complex at the nuclear envelope. In this study we provide evidence that specific residues of a conserved N-terminal region of pUL50 determine its intranuclear interaction with pUL53. In silico evaluation and biophysical analyses suggested that the conserved region forms a regular secondary structure adopting a globular fold. Importantly, site-directed replacement of individual amino acids by alanine indicated a strong functional influence of specific residues inside this globular domain. In particular, mutation of the widely conserved residues Glu-56 or Tyr-57 led to a loss of interaction with pUL53. Consistent with the loss of binding properties, mutants E56A and Y57A showed a defective function in the recruitment of pUL53 to the nuclear envelope in expression plasmid-transfected and human cytomegalovirus-infected cells. In addition, in silico analysis suggested that residues 3-20 form an amphipathic α-helix that appears to be conserved among Herpesviridae. Point mutants revealed a structural role of this N-terminal α-helix for pUL50 stability rather than a direct role in the binding of pUL53. In contrast, the central part of the globular domain including Glu-56 and Tyr-57 is directly responsible for the functional interaction with pUL53 and thus determines formation of the basic nuclear egress complex.