The role of the cancer stem cell marker CD271 in DNA damage response and drug resistance of melanoma cells

Several lines of evidence have suggested that stemness and acquired resistance to targeted inhibitors or chemotherapeutics are mechanistically linked. Here we observed high cell surface and total levels of nerve growth factor receptor/CD271, a marker of melanoma-initiating cells, in sub-populations of chemoresistant cell lines. CD271 expression was increased in drug-sensitive cells but not resistant cells in response to DNA-damaging chemotherapeutics etoposide, fotemustine and cisplatin. Comparative analysis of melanoma cells engineered to stably express CD271 or a targeting short hairpin RNA by expression profiling provided numerous genes regulated in a CD271-dependent manner. In-depth analysis of CD271-responsive genes uncovered the association of CD271 with regulation of DNA repair components. In addition, gene set enrichment analysis revealed enrichment of CD271-responsive genes in drug- resistant cells, among them DNA repair components. Moreover, our comparative screen identified the fibroblast growth factor 13 (FGF13) as a target of CD271, highly expressed in chemoresistant cells. Further we show that levels of CD271 determine drug response. Knock-down of CD271 in fotemustine-resistant cells decreased expression of FGF13 and at least partly restored sensitivity to fotemustine. Together, we demonstrate that expression of CD271 is responsible for genes associated with DNA repair and drug response. Further, we identified 110 CD271-responsive genes predominantly expressed in melanoma metastases, among them were NEK2, TOP2A and RAD51AP1 as potential drivers of melanoma metastasis. In addition, we provide mechanistic insight in the regulation of CD271 in response to drugs. We found that CD271 is potentially regulated by p53 and in turn is needed for a proper p53-dependent response to DNA-damaging drugs. In summary, we provide for the first time insight in a CD271-associated signaling network connecting CD271 with DNA repair, drug response and metastasis

Cancer stem cells in melanoma

The identification of cancer stem cells in various malignancies led to the hypothesis that these cells have the exclusive ability of self-renewal, contribute to the plasticity of the tumours and may be the cause for ineffective cancer therapies. Several markers of melanoma stem cells have been described in recent studies including CD133, CD166, Nestin and BMI-1. Further studies are necessary to identify, better define and understand the origin and function of cancer stem cells. If confirmed that cancer stem cells play an important role in malignancy, therapeutic strategies may need to be redirected towards these cells to circumvent the failure of conventional therapies

Action du sulfite de sodium sur la concentration en composés organohalogénés et sur l'activité mutagène de solutions chlorées de substances humiques

Cette étude a eu pour but de déterminer l'effet d'un traitement par le sulfite de sodium sur la concentration en composés organohalogénés totaux (TOX) et sur l'activité mutagène de solutions chlorées de substances humiques d'origine aquatique (SHA), après avoir cherché à préciser l'influence du pH et du temps sur la concentration en TOX.Les résultats obtenus à partir d'échantillons chlorés de SHA en absence de chlore résiduel ont permis de mettre en évidence une diminution de la concentration en composés organohalogénés totaux, soit par stockage en milieu neutre ou basique, soit par addition de sulfite de sodium. L'intensité de cette réduction de la concentration en TOX augmente avec le pH, le temps de réaction et la dose de sulfite de sodium introduite.Les résultats obtenus à partir d'échantillons contenant du chlore libre indiquent que seule une déchloration totale avec un excès de sulfite de sodium peut conduire, en milieu neutre, à une diminution de l'activité mutagène et de la concentration en TOX des solutions diluées de SHA. La comparaison des pourcentages d'abattement obtenus sur le paramètre TOX et sur l'activité mutagène indique que la diminution de la génotoxicité par déchloration totale est due à l'action du sulfite sur des composés mutagènes non chlorés ou sur des composés chlorés fortement mutagènes et ne représentant qu'une très faible fraction du TOX.If is a well known tact that mimerous organohalogenated compounds are formed during the chlorination (preoxidation or final disinfection) of drinking water. Some of these compounds have been shown to be mutagenic. Recent studies have suggested that a treatment with oxygenated derivatives of SIV (SO2, NaHSO3 and Na2SO3) could reduce the genotoxicity of chlorinated drinking water.The general aim of Ibis study was to determine the effect of dechlorination treatments on the mutagenic activity of chlorinated drinking water. The following experiments were carried out in order to point out the effect of a treatment with sodium sulfite on the concentration of total organohalogenated compounds (TOX) and on the mutagenic activity of chlorinated dilute solutions of Aquatic Humic Substances (AHS).At first, the affects of pH, sodium sulfite dose and contact time on TOX concentration were investigated. Then, the importance of the dechlorination rate (partial or complete) on TOX concentration and also on the mutagenic activity could be studied.ExperimentalAquatic Humic Substances (natural mixture of fulvic and humic acids) were dissolved in phosphate-buffered ultra-pure water at 5 and 15 mg l-1 concentrations (pH 6.1 and 6.9 respectively). Stock solutions of chlorine were prepared in the laboratory and titrated by iodometry. Chlorination and dechlorination treatments were carried out in headspace-free baffles, at 20± 1 °C in the dark. Residual chlorine was determined by spectrophotometric measurements at 510 nm, following the calorimetric method using N,N-diethylphenylene-1,4-diamine (DPD). To avoid the slow oxidation of Slv into Svl by dissolved oxygen, the sodium sulfite solutions were prepared freshly before use. TOX concentrations were measured using a DOHRMAN DX-20 TOX analyser equipped with a MC-1 microcoulometric cell and with an AD-2 adsorption module. Before analysis, the residual chlorine was neutralized with sodium thiosulfate and samples were acidified to pH 1.4.The mutagenic activity was determined using acetone-dichloromethane extracts (AMBERLITE XAD-8 and XAD-2 resins) of the aqueous samples of chlorinated and dechlorinated solutions of AHS, acidified to pH 2.0 before extraction. The mutagenicity tests were carried out on TA 98 and TA 100 tester strains, following the method described by MARON and AMES (1983).Results-Effect of pH, addition of sodium sulfite and storage time on the TOX concentrationThe experiments carried out with dilute solutions of AHS ([AHS] = 5 mg 1-1; DOC = 2.5 mg Cl-1; pH = 6.1) showed a linear relationship between TOX production and chlorine consumption in the range 0-2.0 mg Cl2 l-1 (fig. 2).15 % of the chlorine demand was incorporated as organic chlorine in molecules.Experiments performed on solutions containing no residual free chlorine showed that organohatogenated compounds could be partially destroyed upon storage at neutral or basic pH (table 1). Reductions in TOX concentrations of 10 % at pH 6.1-8.5 in 24 hours and of 20 % at pH 11.5 in 2 hours were observed. This was enhanced by increasing the storage time.The addition of sodium sulfite (100 µmol l-1) in solutions containing no residual free chlorine significantly reduced the TOX concentration (10 % in 2 hours at pH 6.1-8.5; table 1). This reduction was enhanced by increasing sulfite dose and storage time and by increasing pH (30 % in 2 hours at pH 11.5). Furthermore, at a given pH value and for a reaction time of 2 hours, the decrease in TOX concentration was larger in presence of sulfite.- Effect of a dechlorination treatment on the TOX concentrationAs shown in figure 3, a dechlorination treatment (reduction of the residual free chlorine concentration) with sodium sulfite could significantly reduce the TOX concentration of the dilute solutions of AHS at pH 6.1 only if an excess of the dechlorinating agent was added. This effect was enhanced by increasing the excess of sulfite but nevertheless seemed to be limited (less than 15 % of reduction for the highest doses used; table 2).The free chlorine residuals measured after a 2 hours partial dechlorination confirmed the stoichiometric factor of 1 mole/mole for the reaction between chlorine and sodium sulfite.- Effect of a dechlorination treatment on the mutagenic activity and on the TOX concentrationThe dechlorination treatments were carried out on chlorinated dilute solutions of AHS ([AHS] = 15 mg l-1; DOC 7.5 mg C l-1; pH = 6.9). The TOX concentrations were measured on aqueous solutions and mutagenicity tests were performed on the corresponding acetone-dichloromethane extracts following a solvent exchange (dimethylsulfoxide). The results obtained showed again that only a total dechlorination treatment could reduce the TOX concentration of the aqueous chlorinated solutions and was able to destroy a significant part of the mutagenic activity of the extracts (table 3 and fig. 4).Although the effect of sulfite on TOX concentration seemed limited (less than 7 % reduction for the highest sulfite dose tested), the reduction in the genotoxicity was more important when the excess of sulfite was increased. No correlation between the TOX concentration and the mutagenic activity could be established. The mutagenic compounds destroyed by sodium sulfite do not appear to be organohalogenated ones. If they are, they are present at trace levels and thus are extremely patent and account for a very little part of the TOX concentration

Molecular characterization of signalling pathways in cancer stem cells

To avoid artefacts introduced by culturing cells for extended periods of time, it is crucial to use low-passage patient-derived tumour cells. The ability to enrich, isolate and assay sub-populations of cells that behave as cancer stem cells (CSCs) from these primary cell lines is essential before performing characterizations such as gene-expression profiling. We have isolated cells from glioblastomas which show characteristics of CSCs. Although glioblastomas contain only a relatively small amount of putative CSCs, these cells express many genes which seem to be worthy targets for future therapies

Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV Core DII protein

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV

Recalibration of the limiting antigen avidity EIA to determine mean duration of recent infection in divergent HIV-1 subtypes.

ArticleBackground: Mean duration of recent infection (MDRI) and misclassification of long-term HIV-1 infections, as proportion false recent (PFR), are critical parameters for laboratory-based assays for estimating HIV-1 incidence. Recent review of the data by us and others indicated that MDRI of LAg-Avidity EIA estimated previously required recalibration. We present here results of recalibration efforts using >250 seroconversion panels and multiple statistical methods to ensure accuracy and consensus. Methods: A total of 2737 longitudinal specimens collected from 259 seroconverting individuals infected with diverse HIV-1 subtypes were tested with the LAg-Avidity EIA as previously described. Data were analyzed for determination of MDRI at ODn cutoffs of 1.0 to 2.0 using 7 statistical approaches and sub-analyzed by HIV-1 subtypes. In addition, 3740 specimens from individuals with infection >1 year, including 488 from patients with AIDS, were tested for PFR at varying cutoffs. Results: Using different statistical methods,MDRI values ranged from 88-94 days at cutoff ODn = 1.0 to 177-183 days at ODn = 2.0. The MDRI values were similar by different methods suggesting coherence of different approaches. Testing formisclassification among long-terminfections indicated that overall PFRs were 0.6%to 2.5%at increasing cutoffs of 1.0 to 2.0, respectively. Balancing the need for a longer MDRI and smaller PFR (<2.0%) suggests that a cutoff ODn = 1.5, corresponding to an MDRI of 130 days should be used for cross-sectional application. The MDRI varied among subtypes from 109 days (subtype A&D) to 152 days (subtype C). Conclusions: Based on the new data and revised analysis, we recommend an ODn cutoff = 1.5 to classify recent and long-term infections, corresponding to an MDRI of 130 days (118-142). Determination of revised parameters for estimation of HIV-1 incidence should facilitate application of the LAg-Avidity EIA for worldwide use.This research has been supported by the President’s Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and Prevention (CDC)

HIV Incidence in Rural South Africa: Comparison of Estimates from Longitudinal Surveillance and Cross-Sectional cBED Assay Testing

The original publication is available at http:/www.plosone.orgBackground: The BED IgG-Capture Enzyme Immunoassay (cBED assay), a test of recent HIV infection, has been used to estimate HIV incidence in cross-sectional HIV surveys. However, there has been concern that the assay overestimates HIV incidence to an unknown extent because it falsely classifies some individuals with non-recent HIV infections as recently infected. We used data from a longitudinal HIV surveillance in rural South Africa to measure the fraction of people with nonrecent HIV infection who are falsely classified as recently HIV-infected by the cBED assay (the long-term false-positive ratio (FPR)) and compared cBED assay-based HIV incidence estimates to longitudinally measured HIV incidence. Methodology/Principal Findings: We measured the long-term FPR in individuals with two positive HIV tests (in the HIV surveillance, 2003-2006) more than 306 days apart (sample size n = 1,065). We implemented four different formulae to calculate HIV incidence using cBED assay testing (n = 11,755) and obtained confidence intervals (CIs) by directly calculating the central 95th percentile of incidence values. We observed 4,869 individuals over 7,685 person-years for longitudinal HIV incidence estimation. The long-term FPR was 0.0169 (95% CI 0.0100-0.0266). Using this FPR, the cross-sectional cBED-based HIV incidence estimates (per 100 people per year) varied between 3.03 (95% CI 2.44-3.63) and 3.19 (95% CI 2.57-3.82), depending on the incidence formula. Using a long-term FPR of 0.0560 based on previous studies, HIV incidence estimates varied between 0.65 (95% CI 0.00-1.32) and 0.71 (95% CI 0.00-1.43). The longitudinally measured HIV incidence was 3.09 per 100 people per year (95% CI 2.69-3.52), after adjustment to the sex-age distribution of the sample used in cBED assay-based estimation. Conclusions/Significance: In a rural community in South Africa with high HIV prevalence, the long-term FPR of the cBED assay is substantially lower than previous estimates. The cBED assay performs well in HIV incidence estimation if the locally measured long-term FPR is used, but significantly underestimates incidence when a FPR estimate based on previous studies in other settings is used. © 2008 Bärnighausen et al.Publishers' Versio

Quantitative Analysis of Lipid Droplet Fusion: Inefficient Steady State Fusion but Rapid Stimulation by Chemical Fusogens

Lipid droplets (LDs) are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (<10 s), followed by the slower merging (100 s) of the neutral lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types

Adenovirus vector delivery stimulates natural killer cell recognition

We report that delivery of first-generation replication-deficient adenovirus (RDAd) vectors into primary human fibroblasts is associated with the induction of natural killer (NK) cell-mediated cytolysis in vitro. RDAd vector delivery induced cytolysis by a range of NK cell populations including the NK cell clone NKL, primary polyclonal NK lines and a proportion of NK clones (36 %) in autologous HLA-matched assays. Adenovirus-induced cytolysis was inhibited by antibody blocking of the NK-activating receptor NKG2D, implicating this receptor in this function. NKG2D is ubiquitously expressed on NK cells and CD8+ T cells. Significantly, γ-irradiation of the vector eliminated the effect, suggesting that breakthrough expression from the vector induces at least some of the pro-inflammatory responses of unknown aetiology following the application of RDAd vectors during in vivo gene delivery