41 research outputs found
Foetal bovine serum-derived exosomes affect yeld and phenotype of human cardiac progenitor cell culture
No full textIntroduction: Cardiac Progenitor Cells represent a powerful tool in cardiac regenerative medicine. Pre-clinical studies suggest that most of the beneficial effects promoted by the injected cells are due to their paracrine activity exerted on endogenous cells and tissue. Exosomes are candidate mediators of this paracrine effects. According to their potential, many researchers are focusing on characterizing exosomes derived from specific cell types, but, up to date, only few studies have analysed the possible in vitro effects of bovine serum-derived exosomes on cell proliferation or differentiation.
Methods: Aim of this study was to analyse, from a qualitative and quantitative point of view, the in vitro effects of bovine serum exosomes on human cardiac progenitor cells cultured either as cardiospheres or as monolayers of cardiosphere-forming cells.
Results: Effects on proliferation, yield and molecular patterning were detected. We show, for the first time, that exogenous bovine exosomes support human cardiosphere-forming cells proliferation and migration, and that their depletion affects cardiospheres formation, in terms of size, yield and extra-cellular matrix production.
Conclusion: These results stress the importance of considering differential biological effects of exogenous cell culture supplements on the final phenotype of primary human cell cultures
Inhibition of in vitro myogenic differentiation by a polyomavirus early function
No full textIn the present work we report on the role of a polyomavirus (Py) early function in interfering with both morphological and biochemical differentiation of the myogenic C2 cell line. The analysis of cell clones stably transfected with a plasmid carrying an ORI- Py genome showed that in the presence of the whole viral early region myogenesis is blocked and a transformed phenotype is evident. By using a plasmid that only encodes large-T function, the involvement of this individual early viral gene product was determined. Inhibition of myogenic differentiation by Py large T is proportional to the level of its expression. This inhibition does not appear to require alteration of cell growth properties. The analysis of muscle-specific functions expressed at different steps in the myogenic pathway showed that Py large T blocks the expression of terminal differentiation markers without altering the expression of the regulatory gene MyoD
Inhibition of in vitro muscle differentiation by the immortalizing oncogene Py LT-Ag
No full textThe interference of Polyomavirus (Py) early functions with in vitro myogenic differentiation is the object of this study. Single cell analysis of C2 myogenic Py infected cells showed a mutual exclusion between Py early functions and muscle gene expression. The morphological and biochemical analysis of clones obtained from C2 cells stably transfected with a plasmid carrying an ORI- Py genome, showed that myogenesis is blocked and cells display the transformed phenotype. By using plasmids separately encoding Middle T or Large T functions, the involvement of individual early viral gene products was determined. Py Middle T alone does not inhibit myotube formation even though cells are morphologically transformed. Myogenic differentiation, on the other hand, is inhibited by Py Large T. This inhibition, which is proportional to the level of Py Large T expression, does not entail to require alteration of cell growth properties and acts by blocking the expression of myogenin and terminal differentiation markers without altering the expression of the regulatory gene MyoD
Methods to Study the BECN1 Interactome in the Course of Autophagic Responses
No full textAutophagy is an extremely dynamic process that mediates the rapid degradation of intracellular components in response to different stress conditions. The autophagic response is executed by specific protein complexes, whose function is regulated by posttranslational modifications and interactions with positive and negative regulators. A comprehensive analysis of how autophagy complexes are temporally modified upon stress stimuli is therefore particularly relevant to understand how this pathway is regulated. Here, we describe a method to define the protein-protein interaction network of a central complex involved in autophagy induction, the Beclin 1 complex. This method is based on the quantitative comparison of protein complexes immunopurified at different time points using a stable isotope labeling by amino acids in cell culture approach. Understanding how the Beclin 1 complex dynamically changes in response to different stress stimuli may provide useful insights to disclose novel molecular mechanisms responsible for the dysregulation of autophagy in pathological conditions, such as cancer, neurodegeneration, and infections
The impact of mevastatin on HCV replication and autophagy of non-transformed HCV replicon hepatocytes is influenced by the extracellular lipid uptake
No full textStatins efficiently inhibit cholesterol synthesis by blocking 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase in the mevalonate pathway. However, the effect of statins on intracellular cholesterol is partially counterbalanced by a consequent increased uptake of extracellular lipid sources. Hepatitis C virus (HCV) infection induces intracellular accumulation of cholesterol by promoting both new synthesis and uptake of circulating lipoproteins, which is required for HCV replication and release. Hepatocytes respond to the increase in intracellular cholesterol levels by inducing lipophagy, a selective type of autophagy mediating the degradation of lipid deposits within lysosomes. In a cellular system of HCV replication based on HuH7 hepatoma cells, statin treatment was shown to be sufficient to decrease intracellular cholesterol, which is accompanied by reduced HCV replication and decreased lipophagy, and has no apparent impact on endocytosis-mediated cholesterol uptake. To understand whether these results were influenced by an altered response of cholesterol influx in hepatoma cells, we analyzed the effect of statins in non-transformed murine hepatocytes (MMHD3) harboring subgenomic HCV replicons. Notably, we found that total amount of cholesterol is increased in MMHD3 cells upon mevastatin treatment, which is associated with increased HCV replication and lipophagy. Conversely, mevastatin is able to reduce cholesterol amounts only when cells are grown in the presence of delipidated serum to prevent extracellular lipid uptake. Under this condition, HCV replication is reduced and autophagy flux is severely impaired. Altogether, these results indicate that both de novo synthesis and extracellular uptake have to be targeted in non-transformed hepatocytes in order to decrease intracellular cholesterol levels and consequently limit HCV replication. Copyright © 2019 Vescovo, Refolo, Manuelli, Tisone, Piacentini and Fimia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms
Molecular mechanisms of hepatitis C virus–induced hepatocellular carcinoma
No full textAbstractHepatitis C virus (HCV) is a major leading cause of hepatocellular carcinoma (HCC). HCV-induced hepatocarcinogenesis is a multistep process resulting from a combination of pathway alterations that are either caused directly by viral factors or immune mediated as a consequence of a chronic state of inflammation. Host genetic variation is now emerging as an additional element that contribute to increase the risk of developing HCC. The advent of direct-acting antiviral agents foresees a rapid decline of HCC rate in HCV patients. However, a full understanding of the HCV-mediated tumourigenic process is required to elucidate if pro-oncogenic signatures may persist after virus clearance, and to identify novel tools for HCC prevention and therapy. In this review, we summarize the current knowledge of the molecular mechanisms responsible for HCV-induced hepatocarcinogenesis
An immunosurveillance mechanism controls cancer cell ploidy
No full textSenovilla, L., Vitale, I., Martins, I., Tailler, M., Pailleret, C., Michaud, M., Galluzzi, L., Adjemian, S., Kepp, O., Niso-Santano, M., Shen, S., Mariño, G., Criollo, A., Boilève, A., Job, B., Ladoire, S., Ghiringhelli, F., Sistigu, A., Yamazaki, T., Rello-Varona, S., Locher, C., Poirier-Colame, V., Talbot, M., Valent, A., Berardinelli, F., Antoccia, A., Ciccosanti, F., Fimia, G.M., Piacentini, M., Fueyo, A., Messina, N.L., Li, M., Chan, C.J., Sigl, V., Pourcher, G., Ruckenstuhl, C., Carmona-Gutierrez, D., Lazar, V., Penninger, J.M., Madeo, F., López-Otín, C., Smyth, M.J., Zitvogel, L., Castedo, M., Kroemer, G
Molecular definitions of autophagy and related processes
No full textGalluzzi, L., Baehrecke, E.H., Ballabio, A., Boya, P., Bravo-San Pedro, J.M., Cecconi, F., Choi, A.M., Chu, C.T., Codogno, P., Colombo, M.I., Cuervo, A.M., Debnath, J., Deretic, V., Dikic, I., Eskelinen, E.-L., Fimia, G.M., Fulda, S., Gewirtz, D.A., Green, D.R., Hansen, M., Harper, J.W., Jäättelä, M., Johansen, T., Juhasz, G., Kimmelman, A.C., Kraft, C., Ktistakis, N.T., Kumar, S., Levine, B., Lopez-Otin, C., Madeo, F., Martens, S., Martinez, J., Melendez, A., Mizushima, N., Münz, C., Murphy, L.O., Penninger, J.M., Piacentini, M., Reggiori, F., Rubinsztein, D.C., Ryan, K.M., Santambrogio, L., Scorrano, L., Simon, A.K., Simon, H.-U., Simonsen, A., Tavernarakis, N., Tooze, S.A., Yoshimori, T., Yuan, J., Yue, Z., Zhong, Q., Kroemer, G
Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry
No full textThe limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescence intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay’s protocol, obtaining more reproducible and sensitive results when a post-LC3-staining fixation was performed, in either THP-1 cells or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14+ cells from active TB patients’ PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described improve the stability and accura- cy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses. © the Author(s), 2019
Tissue transglutaminase contributes to the formation of disulphide bridges in proteins of mitochondrial respiratory complexes
No full textIn this study we provide the first in vivo evidences showing that, under physiological conditions, "tissue" transglutaminase (TG2) might acts as a protein disulphide isomerase (PDI) and through this activity contributes to the correct assembly of the respiratory chain complexes. Mice lacking TG2 exhibit mitochondrial energy production impairment, evidenced by decreased ATP levels after physical challenge. This defect is phenotypically reflected in a dramatic decrease of motor behaviour of the animals. We propose that the molecular mechanism, underlying such a phenotype.. resides in a defective disulphide bonds formation in ATP synthase (complex V), NADH-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II) and cytochrome c oxidase (complex IV). In addition, TG2-PDI might control the respiratory chain by modulating the formation of the prohibitin complexes. These data elucidate a new pathway that directly links the TG2-PDI enzymatic activity with the regulation of mitochondrial respiratory chain function. (c) 2006 Elsevier B.V. All rights reserved
