Evidence for a mitochondrial localization of the retinoblastoma protein

<p>Abstract</p> <p>Background</p> <p>The retinoblastoma protein (Rb) plays a central role in the regulation of cell cycle, differentiation and apoptosis. In cancer cells, ablation of Rb function or its pathway is a consequence of genetic inactivation, viral oncoprotein binding or deregulated hyperphosphorylation. Some recent data suggest that Rb relocation could also account for the regulation of its tumor suppressor activity, as is the case for other tumor suppressor proteins, such as p53.</p> <p>Results</p> <p>In this reported study, we present evidence that a fraction of the total amount of Rb protein can localize to the mitochondria in proliferative cells taken from both rodent and human cells. This result is also supported by the use of Rb siRNAs, which substantially reduced the amount of mitochondrial Rb, and by acellular assays, in which [³⁵S]-Methionine-labeled Rb proteins bind strongly to mitochondria isolated from rat liver. Moreover, endogenous Rb is found in an internal compartment of the mitochondria, within the inner-membrane. This is consistent with the protection of Rb from alkaline treatment, which destroys any interaction of proteins that are weakly bound to mitochondria.</p> <p>Conclusion</p> <p>Although a few data regarding an unspecific cytosolic localization of Rb protein have been reported for some tumor cells, our results are the first evidence of a mitochondrial localization of Rb. The mitochondrial localization of Rb is observed in parallel with its classic nuclear location and paves the way for the study of potential as-yet-unknown roles of Rb at this site.</p

Cold-Atmospheric Plasma Induces Tumor Cell Death in Preclinical In Vivo and In Vitro Models of Human Cholangiocarcinoma

Through the last decade, cold atmospheric plasma (CAP) has emerged as an innovative therapeutic option for cancer treatment. Recently, we have set up a potentially safe atmospheric pressure plasma jet device that displays antitumoral properties in a preclinical model of cholangiocarcinoma (CCA), a rare and very aggressive cancer emerging from the biliary tree with few efficient treatments. In the present study, we aimed at deciphering the molecular mechanisms underlying the antitumor effects of CAP towards CCA in both an in vivo and in vitro context. In vivo, using subcutaneous xenografts into immunocompromised mice, CAP treatment of CCA induced DNA lesions and tumor cell apoptosis, as evaluated by 8-oxoguanine and cleaved caspase-3 immunohistochemistry, respectively. The analysis of the tumor microenvironment showed changes in markers related to macrophage polarization. In vitro, the incubation of CCA cells with CAP-treated culture media (i.e., plasma-activated media, PAM) led to a dose response decrease in cell survival. At molecular level, CAP treatment induced double-strand DNA breaks, followed by an increased phosphorylation and activation of the cell cycle master regulators CHK1 and p53, leading to cell cycle arrest and cell death by apoptosis. In conclusion, CAP is a novel therapeutic option to consider for CCA in the future

Technical report: liquid overlay technique allows the generation of homogeneous osteosarcoma, glioblastoma, lung and prostate adenocarcinoma spheroids that can be used for drug cytotoxicity measurements

Introduction: The mechanisms involved in cancer initiation, progression, drug resistance, and disease recurrence are traditionally investigated through in vitro adherent monolayer (2D) cell models. However, solid malignant tumor growth is characterized by progression in three dimensions (3D), and an increasing amount of evidence suggests that 3D culture models, such as spheroids, are suitable for mimicking cancer development. The aim of this report was to reaffirm the relevance of simpler 3D culture methods to produce highly reproducible spheroids, especially in the context of drug cytotoxicity measurements.Methods: Human A549 lung adenocarcinoma, LnCaP prostate adenocarcinoma, MNNG/HOS osteosarcoma and U251 glioblastoma cell lines were grown into spheroids for 20 days using either Liquid Overlay Technique (LOT) or Hanging Drop (HD) in various culture plates. Their morphology was examined by microscopy. Sensitivity to doxorubicin was compared between MNNG/HOS cells grown in 2D and 3D.Results: For all cell lines studied, the morphology of spheroids generated in round-bottom multiwell plates was more repeatable than that of those generated in flat-bottom multiwell plates. HD had no significant advantage over LOT when the spheroids were cultured in round-bottom plates. Finally, the IC50 of doxorubicin on MNNG/HOS cultured in 3D was 18.8 times higher than in 2D cultures (3D IC50 = 15.07 ± 0.3 µM; 2D IC50 = 0.8 ± 0.4 µM; *p &lt; 0.05).Discussion: In conclusion, we propose that the LOT method, despite and because of its simplicity, is a relevant 3D model for drug response measurements that could be scaled up for high throughput screening

Implication du métabolisme dans l'apoptose et la différenciation des cellules souches cancéreuses de gliome murin

Récemment, la présence de cellules souches cancéreuses (CSC) a été mise en évidence dans les gliomes. Ces cellules, de par leur résistance à l'apoptose, seraient responsables de la résurgence des tumeurs après radio et chimiothérapie. Le but de cette étude a été de rechercher des marqueurs/voies de signalisation ciblant ces cellules souches issues de gliomes chimio-induits chez le rat, via une comparaison avec des cellules souches neurales (NSC) murines. Une analyse protéomique comparative a montré que le métabolisme des CSC était différent de celui des NSC : les CSC ont un métabolisme essentiellement glycolytique. Le dichloroacétate (DCA) est une molécule ciblant ce métabolisme particulier. Nous avons mis en évidence qu'elle était capable de potentialiser l'apoptose induite par l'Etoposide ou les irradiations dans les CSC, mais pas dans les NSC. Cette potentialisation est corrélée avec une surexpression de p53, Foxo3 et de certains de leurs gènes-cibles : Bad, Puma et Noxa, qui sont des protéines pro-apoptotiques à domaine BH3-seul. De plus, le DCA induit la différenciation des CSC mais pas celle des NSC, via la modulation de l'interaction entre PKM2 (enzyme de la glycolyse) et Oct4 (facteur de transcription impliqué dans le maintien de l'état indifférencié). Le DCA pourrait donc être un bon adjuvant aux traitements classiques contre les gliomes.The so-called cancer stem cells are thought to be involved in some of the basic features of tumors, especially brain tumors .These cells are known to be extremely resistant to apoptosis and may be responsible for the resurgence of tumors after chemo and radio-therapy. We aimed to compare brain cancer stem cells (CSC) versus neural stem cells (NSC) to find some markers/pathways specific of these CSC. We used rat neural stem cells (NSCs) and cancer stem cells (CSCs) in a model of ENU-induced rat brain tumors. A proteomic comparison of these cells demonstrated that a major difference between CSCs and NSCs is due to metabolism: contrary to NSC, CSCs have a glycolytic metabolism. DCA is a drug involved in this particular metabolism. DCA treatment of CSCs led to a decrease in their resistance to apoptosis induced by etoposide and radiation. This decrease is correlated with an increase of p53, Foxo3 and some of their targets: the pro-apoptotic agents Puma, Noxa, and Bad. Moreover, DCA forces CSCs to differentiate, probably by modulating M2PK (glycolysis enzyme) and Oct4 (transcription factor involved in stemness) interaction. To conclude, DCA may improve the efficiency of usual treatments on patients with glioma.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

CONTROLE DES MECANISMES D'ACTIVATION DE LA CASPASE-3 (DOCTORAT BIOLOGIE CELLULAIRE ET BIOCHIMIE)

NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

A conformation-dependent monoclonal antibody against active chicken acetylcholinesterase

International audienceWe show that the C-131 monoclonal antibody, directed against chicken AChE, recognizes active chicken AChE, but not the SDS-denatured or heat-inactivated protein. Previous results indicated that C-131 only binds to the active enzyme, and not to inactive molecules which also occur in the embryonic chicken brain. In contrast with C-131, other monoclonal antibodies obtained in the same series, such as C-6 and C-54, also recognize denatured or inactive AChE. It is noteworthy that these antibodies all seem to react with a trypsin-sensitive peptide which is present in chicken but not in mammalian or Torpedo AChE, whereas the C-131 antibody binds trypsin-modified as well as intact molecules. These results show that C-131 is highly conformation-dependent, specific for active AChE. They confirm our previous conclusion that active and inactive molecules arise from different folding processes

Congenital Isolated Adrenocorticotropin Deficiency: An Underestimated Cause Of Neonatal Death, Explained By Tpit Gene Mutations

Tpit is a T box transcription factor important for terminal differentiation of pituitary proopiomelanocortin-expressing cells. We demonstrated that human and mouse mutations of the TPIT gene cause a neonatal-onset form of congenital isolated ACTH deficiency (IAD). In the absence of glucocorticoid replacement, IAD can lead to neonatal death by acute adrenal insufficiency. This clinical entity was not previously well characterized because of the small number of published cases. Since identification of the first TPIT mutations, we have enlarged our series of neonatal IAD patients to 27 patients from 21 unrelated families. We found TPIT mutations in 17 of 27 patients. We identified 10 different TPIT mutations, with one mutation found in five unrelated families. All patients appeared to be homozygous or compound heterozygous for TPIT mutations, and their unaffected parents are heterozygous carriers, confirming a recessive mode of transmission. We compared the clinical and biological phenotype of the 17 IAD patients carrying a TPIT mutation with the 10 IAD patients with normal TPIT-coding sequences. This series of neonatal IAD patients revealed a highly homogeneous clinical presentation, suggesting that this disease may be an underestimated cause of neonatal death. Identification of TPIT gene mutations as the principal molecular cause of neonatal IAD permits prenatal diagnosis for families at risk for the purpose of early glucocorticoid replacement therapy.Wo

DNA Methylation and Apoptosis Resistance in Cancer Cells

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