Anticancer chemotherapy and radiotherapy trigger both non-cell-autonomous and cell-autonomous death.

Even though cell death modalities elicited by anticancer chemotherapy and radiotherapy have been extensively studied, the ability of anticancer treatments to induce non-cell-autonomous death has never been investigated. By means of multispectral imaging flow-cytometry-based technology, we analyzed the lethal fate of cancer cells that were treated with conventional anticancer agents and co-cultured with untreated cells, observing that anticancer agents can simultaneously trigger cell-autonomous and non-cell-autonomous death in treated and untreated cells. After ionizing radiation, oxaliplatin, or cisplatin treatment, fractions of treated cancer cell populations were eliminated through cell-autonomous death mechanisms, while other fractions of the treated cancer cells engulfed and killed neighboring cells through non-cell-autonomous processes, including cellular cannibalism. Under conditions of treatment with paclitaxel, non-cell-autonomous and cell-autonomous death were both detected in the treated cell population, while untreated neighboring cells exhibited features of apoptotic demise. The transcriptional activity of p53 tumor-suppressor protein contributed to the execution of cell-autonomous death, yet failed to affect the non-cell-autonomous death by cannibalism for the majority of tested anticancer agents, indicating that the induction of non-cell-autonomous death can occur under conditions in which cell-autonomous death was impaired. Altogether, these results reveal that chemotherapy and radiotherapy can induce both non-cell-autonomous and cell-autonomous death of cancer cells, highlighting the heterogeneity of cell death responses to anticancer treatments and the unsuspected potential contribution of non-cell-autonomous death to the global effects of anticancer treatment

Résultats anatomiques et fonctionnels de la colpoplastie sigmoïde dans le traitement chirurgical du syndrome de Mayer Rokitansky Kuster Hauser

AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Involvement of the Cdc42 Pathway in CFTR Post-Translational Turnover and in Its Plasma Membrane Stability in Airway Epithelial Cells.

publicationCystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation

Pharmacological inhibitors of Cdc42 pathway impair CFTR channel activation.

<p>(A) Iodide efflux curves obtained in CFBE-wtCFTR cells treated with 10 μM wiskostatin for 120 min, 10 μM ML141 for 30 min or corresponding vehicle, prior to stimulation of CFTR activity by forskolin (Fsk, 10 μM) + genistein (Gst, 30 μM), n = 4 in each condition. (B) Histograms show the mean relative rates of CFTR activity. The result obtained with wiskostatin (resp. ML141) was compared with DMSO 1/1000 (resp. DMSO 1/100) (v/v) treatment. Means ± SEM are indicated. ***: p<0.001, *: p<0.05, ns: non significant.</p

Analysis of CFTR maturation and turnover by metabolic labelling.

<p>CFBE-wtCFTR cells were transfected with negative control or Cdc42 siRNA and cultured 48 h prior to pulse-chase experiments. Cells were pulse-labelled for 15 min with 100 μCi/mL of [³⁵S]methionine and [³⁵S]cysteine mix and then chased for 0, 0.5, 1, 2, and 4 h. CFTR was then immunoprecipited and subjected to SDS-PAGE. Bands corresponding to core-glycosylated (band B) and fully-glycosylated (band C) CFTR were quantified by densitometry for analysis. (A) Representative gels are shown. (B) Maturation of CFTR is evaluated as the ratio of band C detected at a given time relative to total CFTR (bands B+C). (C) CFTR turnover is displayed as the relative total CFTR amount (bands B+C) along the chase. Total CFTR amount is assigned a value of 100 in arbitrary units at the beginning of the chase (0 h), when the cells are transfected with negative control siRNA. (D) The rate of CFTR disappearance is estimated as the natural logarithm of the amount of CFTR (bands B+C) at a given time of chase relative to its amount at the beginning of the experiment (B₀+C₀). Displayed lines are the linear regressions to the data. Symbol and error bars are means ± SEM of the values at each point. The numbers of independent experiments used to build the graphs are indicated on the figures.</p

Cdc42 depletion does not alter fibrillar actin content.

<p>RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h and F-actin content was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118943#pone.0118943.g005" target="_blank">Fig. 5</a> legend. As displayed in histograms, N-WASP depletion alone elicited a decrease of polymerized actin content, compared with negative control RNAi condition. Data represent means ± SEM of 3–8 independent experiments, each performed in triplicate. **: p<0.01, ns: non-significant.</p

Entosis: The emerging face of non-cell-autonomous type IV programmed death

The present review summarizes recent experimental evidences about the existence of the non-cell-autonomous death entosis in physiological and pathophysiological contexts, discusses some aspects of this form of cell death, including morphological, biochemical and signaling pathways that distinguish non-cell-autonomous demises from other death modalities and propose to define this new modality of death as type IV programmed cell death

ML141 or wiskostatin treatments stimulate CFTR endocytosis.

<p>(A) Representative Western blots and (B and C) histograms summarizing the data are presented. First, cells were submitted to 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min. Treatment with 1% DMSO (v/v) was used as a negative control. Surface proteins were then biotinylated. Total CFTR protein amount was assessed by immunoblot in denatured samples obtained from 20 μg of clarified lysates (α1 Na⁺/K⁺ ATPase was used as a normalization control). Biotinylated proteins were purified from 100 μg of clarified lysates and the amount of labelled CFTR was assessed in the resulting samples. (B) The amount of plasma membrane CFTR, expressed as the percentage of DMSO treatment condition, decreased following both pharmacological treatments. Alternatively, the biotinylated PM proteins were allowed to enter the inner cell compartment through 5 min incubation of cell cultures at 37°C. Surface-exposed biotin moieties were then stripped by MESNA reduction. Biotinylated (internalized) proteins were purified from 600 μg clarified lysates and analyzed by Western blot. The ratio of the densitometric quantification of bands to the relative initial PM-CFTR amounts was then calculated. (C) Relative CFTR internalization, expressed as the percentage of DMSO control condition, appeared to have increased following ML141 or wiskostatin treatments. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05.</p

Pharmacological treatments decrease actin polymerization.

<p>(A) Cell shape, polarity and peripheral actin cytoskeleton pattern appear preserved upon 10 μM wiskostatin or 10 μM L141 treatments compared to vehicle treatment, whereas actin is scattered throughout the cytoplasm with 100 μM wiskostatin. Cells were grown on Transwell permeable support until a monolayer was established. After ML141 or wiskostatin treatments at the indicated concentrations, ZO-1 proteins were immunostained (green) and actin was tracked using phalloidin-TRITC (red). TO-PRO-3 was used as a cell nucleus marker (blue). Transversal section images were acquired with a confocal microscope. Scale bars represent 20 μm. (B) Pharmacological inhibitions of Cdc42 pathway reduce fibrillar (F-) actin content. Cells were incubated with 10 μM ML141 or 10 μM wiskostatin, and 10 μM cytochalasin B was used as an F-actin polymerization inhibition positive control treatment, whereas 1% DMSO (v/v) was used as negative control treatment. Actin-bound phalloidin-FITC was methanol-extracted and fluorescence measurements were performed. Results were normalized to protein amount and the relative F-actin contents are expressed as the percentage of DMSO control condition in histograms. Data represent means ± SEM of 3 independent experiments, each performed in triplicate. **: p<0.01, *: p<0.05.</p

ML141 selectively reduces the GTP-bound form of Cdc42.

<p>Before lysis, cells were submitted to 10 μM ML141 for 30 min and 1% DMSO (v/v) was used as the control condition. After enrichment of the activated forms of GTPases in the clarified lysates, GST-pull-down was performed. Cdc42, Rac1 or RhoA protein amounts were then assessed in the resulting samples. (A) Representative Western blot images are shown. Densitometric quantification of bands was normalized to DMSO condition. (B) Histogram displays relative activated GTPase amounts, expressed as the percentage of control. Data represent means ± SEM of 3 independent experiments each performed in duplicate. *: p<0.05, ns: non significant.</p