Lucia Binó

New Role for L-Arginine in Regulation of Inducible Nitric-Oxide-Synthase-Derived Superoxide Anion Production in Raw 264.7 Macrophages

Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O2∙− formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O2∙− and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O2∙− soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O2∙− formation in inflammatory macrophages.

Simple non-invasive analysis of embryonic stem cell-derived cardiomyocytes beating in vitro.

The analysis of digital video output enables the non-invasive screening of various active biological processes. For the monitoring and computing of the beating parameters of cardiomyocytes in vitro, CB Analyser (cardiomyocyte beating analyser) software was developed. This software is based on image analysis of the video recording of beating cardiomyocytes. CB Analyser was tested using cardiomyocytes derived from mouse embryonic stem cells at different stages of cardiomyogenesis. We observed that during differentiation (from day 18), the beat peak width decreased, which corresponded to the increased speed of an individual pulse. However, the beating frequency did not change. Further, the effects of epinephrine modulating mature cardiomyocyte functions were tested to validate the CB Analyser analysis. In conclusion, data show that CB Analyser is a useful tool for evaluating the functions of both developing and mature cardiomyocytes under various conditions in vitro.

Hypoxia inducible prolyl hydroxylase PHD3 maintains carcinoma cell growth by decreasing the stability of p27

BackgroundHypoxia can halt cell cycle progression of several cell types at the G1/S interface. The arrest needs to be overcome by cancer cells. We have previously shown that the hypoxia-inducible cellular oxygen sensor PHD3/EGLN3 enhances hypoxic cell cycle entry at the G1/S boundary.MethodsWe used PHD3 knockdown by siRNA and shRNA in HeLa and 786–0 renal cancer cells. Flow cytometry with cell synchronization was used to study cell growth at different cell cycle phases. Total and phosphospecific antibodies together with cycloheximide chase were used to study p27/CDKN1B expression and fractionations for subcellular protein localization.ResultsHere we show that PHD3 enhances cell cycle by decreasing the expression of the CDK inhibitor p27/CDKN1B. PHD3 reduction led to increased p27 expression under hypoxia or VHL mutation. p27 was both required and sufficient for the PHD3 knockdown induced cell cycle block. PHD3 knockdown did not affect p27 transcription and the effect was HIF-independent. In contrast, PHD3 depletion increased the p27 half-life from G0 to S-phase. PHD3 depletion led to an increase in p27 phosphorylation at serine 10 without affecting threonine phosphorylation. Intact serine 10 was required for normal hypoxic and PHD3-mediated degradation of p27.ConclusionsThe data demonstrates that PHD3 can drive cell cycle entry at the G1/S transition through decreasing the half-life of p27 that occurs by attenuating p27S10 phosphorylation.

Apocynin and Diphenyleneiodonium Induce Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk Activity in Mouse Embryonic Stem Cells.

Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.

Plasticity and intratumoural heterogeneity of cell surface antigen expression in breast cancer

This corrects the article DOI: 10.1038/bjc.2017.85

The acceleration of cardiomyogenesis in embryonic stem cells in vitro by serum depletion does not increase the number of developed cardiomyocytes

The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.

Hypoxia favors myosin heavy chain beta gene expression in an Hif-1alpha-dependent manner

The potentiation of the naturally limited regenerative capacity of the heart is dependent on an understanding of the mechanisms that are activated in response to pathological conditions such as hypoxia. Under these conditions, the expression of genes suggested to support cardiomyocyte survival and heart adaptation is triggered. Particularly important are changes in the expression of myosin heavy chain (MHC) isoforms. We propose here that alterations in the expression profiles of MHC genes are induced in response to hypoxia and are primarily mediated by hypoxia inducible factor (HIF). In in vitro models of mouse embryonic stem cell-derived cardiomyocytes, we showed that hypoxia (1% O2) or the pharmacological stabilization of HIFs significantly increased MHCbeta (Myh7) gene expression. The key role of HIF-1alpha is supported by the absence of these effects in HIF-1alpha-deficient cells, even in the presence of HIF-2alpha. Interestingly, ChIP analysis did not confirm the direct interaction of HIF-1alpha with putative HIF response elements predicted in the MHCalpha and beta encoding DNA region. Further analyses showed the significant effect of the mTOR signaling inhibitor rapamycin in inducing Myh7 expression and a hypoxia-triggered reduction in the levels of antisense RNA transcripts associated with the Myh7 gene locus. Overall, the recognized and important role of HIF in the regulation of heart regenerative processes could be highly significant for the development of novel therapeutic interventions in heart failure.The potentiation of the naturally limited regenerative capacity of the heart is dependent on an understanding of the mechanisms that are activated in response to pathological conditions such as hypoxia. Under these conditions, the expression of genes suggested to support cardiomyocyte survival and heart adaptation is triggered. Particularly important are changes in the expression of myosin heavy chain (MHC) isoforms. We propose here that alterations in the expression profiles of MHC genes are induced in response to hypoxia and are primarily mediated by hypoxia inducible factor (HIF). In in vitro models of mouse embryonic stem cell-derived cardiomyocytes, we showed that hypoxia (1% O2) or the pharmacological stabilization of HIFs significantly increased MHCbeta (Myh7) gene expression. The key role of HIF-1alpha is supported by the absence of these effects in HIF-1alpha-deficient cells, even in the presence of HIF-2alpha. Interestingly, ChIP analysis did not confirm the direct interaction of HIF-1alpha with putative HIF response elements predicted in the MHCalpha and beta encoding DNA region. Further analyses showed the significant effect of the mTOR signaling inhibitor rapamycin in inducing Myh7 expression and a hypoxia-triggered reduction in the levels of antisense RNA transcripts associated with the Myh7 gene locus. Overall, the recognized and important role of HIF in the regulation of heart regenerative processes could be highly significant for the development of novel therapeutic interventions in heart failure.

Trop-2 plasticity is controlled by epithelial-to-mesenchymal transition

The cell surface glycoprotein Trop-2 is commonly overexpressed in carcinomas and represents an exceptional antigen for targeted therapy. Here, we provide evidence that surface Trop-2 expression is functionally connected with an epithelial phenotype in breast and prostate cell lines and in patient tumor samples. We further show that Trop-2 expression is suppressed epigenetically or through the action of epithelial-to-mesenchymal transition transcription factors and that deregulation of Trop-2 expression is linked with cancer progression and poor patient prognosis. Moreover, our data suggest that the cancer plasticity-driven intratumoral heterogeneity in Trop-2 expression may significantly contribute to response and resistance to therapies targeting Trop-2-expressing cells.

Abstract B084: Trop-2 plasticity is driven by epithelial-to-mesenchymal transition in prostate cancer cells

Dissemination of cancer cells to distant organs has fatal consequences to most of the patients with malignant tumors. Patients with prostate and breast cancer show apparent overlap of the most common sites of cancer metastasis, suggesting that breast and prostate tumor cells share common mechanisms of dissemination and colonization. During each step of prostate and breast cancer metastasis, malignant cells display phenotypic plasticity that is associated with the manifestation of epithelial and mesenchymal properties or an epithelial-to-mesenchymal transition (EMT). One of the molecules that most likely interlink processes of pathologic plasticity of cancer cells, their dissemination capability, and response to microenvironmental factors is Tumor-Associated Calcium Signal TransDucer 2 (Trop-2, TACSTD2). Trop-2 is a type-I transmembrane glycoprotein encoded by TACSTD2 gene often associated with tumorigenesis and cancer progression, but the data remain controversial. Trop-2 deregulation has been repeatedly proposed as an event associated with cancer progression and poor patient prognosis. Instead of this simplistic view, our results showed Trop-2 level change as a context-dependent, dynamic event associated with cancer plasticity and dissemination. Using antibody-based surface profiling of selected cancer stem-like cell markers in human and mouse prostate and breast cancer cell lines, we identified subpopulation of Trop-2+ cells within culture of metastatic prostate cell line DU-145 and similarly Trop-2+ subpopulation within mouse mammary cancer cell line 4T1. Gene expression analysis of sorted subpopulations showed significant correlation of Trop-2 with epithelial phenotype, and this finding was further validated in wide panel of human and murine cell lines and independent patient datasets. We further proved that expression of Trop-2 is regulated by EMT transcription factors and DNA methylation. Moreover, immunohistochemical analysis of Trop-2 in pairs of primary prostate tumors and lymph node metastasis showed strong association with E-cadherin and epithelial-mesenchymal plasticity in patient samples. In conclusion, we showed that Trop-2 expression associates with epithelial phenotype and can be suppressed either epigenetically or through the action of EMT master regulators. Acknowledgments: This work was supported by Ministry of Health of the Czech Republic, grant no. 15-33999A and 15-28628A, and by GACR 15-11707S, HistoPARK (CZ.1.07/2.3.00/20.0185), and by project FNUSA-ICRC (no. CZ.1.05/1.1.00/02.0123) and ICRC-ERA-HumanBridge a.k.a. REGPOT (Grant agreement no. 316 345) from the European Regional Development Fund. Citation Format: Jan Remsik, Lucia Bino, Zuzana Kahounova, Gvantsa Kharaishvili, Sarka Simeckova, Radek Fedr, Tereza Nehybova, Eva Slabakova, Lucia Knopfova, Jan Bouchal, Milan Kral, Petr Benes, Karel Soucek. Trop-2 plasticity is driven by epithelial-to-mesenchymal transition in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B084.

Synthesis and profiling of a novel potent selective inhibitor of CHK1 kinase possessing unusual N-trifluoromethylpyrazole pharmacophore resistant to metabolic N-dealkylation

Checkpoint-mediated dependency of tumor cells can be deployed to selectively kill them without substantial toxicity to normal cells. Specifically, loss of CHK1, a serine threonine kinase involved in the surveillance of the G2–M checkpoint in the presence of replication stress inflicted by DNA-damaging drugs, has been reported to dramatically influence the viability of tumor cells. CHK1′s pivotal role in maintaining genomic stability offers attractive opportunity for increasing the selectivity, effectivity, and reduced toxicity of chemotherapy. Some recently identified CHK1 inhibitors entered clinical trials in combination with DNA antimetabolites. Herein, we report synthesis and profiling of MU380, a nontrivial analogue of clinically profiled compound SCH900776 possessing the highly unusual N-trifluoromethylpyrazole motif, which was envisioned not to undergo metabolic oxidative dealkylation and thereby provide greater robustness to the compound. MU380 is a selective and potent inhibitor of CHK1 which sensitizes a variety of tumor cell lines to hydroxyurea or gemcitabine up to 10 times. MU380 shows extended inhibitory effects in cells, and unlike SCH900776, does not undergo in vivo N-dealkylation to the significantly less selective metabolite. Compared with SCH900776, MU380 in combination with GEM causes higher accumulation of DNA damage in tumor cells and subsequent enhanced cell death, and is more efficacious in the A2780 xenograft mouse model. Overall, MU380 represents a novel state-of-the-art CHK1 inhibitor with high potency, selectivity, and improved metabolic robustness to oxidative N-dealkylation. Mol Cancer Ther; 16(9); 1831–42. ©2017 AACR.