Silencing cytokeratin 18 gene inhibits intracellular replication of Trypanosoma cruzi in HeLa cells but not binding and invasion of trypanosomes

<p>Abstract</p> <p>Background</p> <p>As an obligatory intracellular parasite, <it>Trypanosoma cruzi</it>, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells. Cytokeratin 18 (CK18) is among the host molecules that have been suggested as a mediator of important events during <it>T. cruzi</it>-host cell interaction. Based on that possibility, we addressed whether RNA interference (RNAi)-mediated down regulation of the CK18 gene could interfere with the parasite life cycle <it>in vitro</it>. HeLa cells transiently transfected with CK18-RNAi had negligible levels of CK18 transcripts, and significantly reduced levels of CK18 protein expression as determined by immunoblotting or immunofluorescence.</p> <p>Results</p> <p>CK18 negative or positive HeLa cells were invaded equally as well by trypomastigotes of different <it>T. cruzi </it>strains. Also, in CK18 negative or positive cells, parasites recruited host cells lysosomes and escaped from the parasitophorous vacuole equally as well. After that, the growth of amastigotes of the Y or CL-Brener strains, was drastically arrested in CK18 RNAi-treated cells. After 48 hours, the number of amastigotes was several times lower in CK18 RNAi-treated cells when compared to control cells. Simultaneous staining of parasites and CK18 showed that in HeLa cells infected with the Y strain both co-localize. Although the amastigote surface protein-2 contains the domain VTVXNVFLYNR previously described to bind to CK18, in several attempts, we failed to detect binding of a recombinant protein to CK-18.</p> <p>Conclusion</p> <p>The study demonstrates that silencing CK18 by transient RNAi, inhibits intracellular multiplication of the Y and CL strain of <it>T. cruzi </it>in HeLa cells, but not trypanosome binding and invasion.</p

Src kinase is a cytoplasmic protein tyrosine kinase that interacts with a variety of receptors for growth factors and integrins, playing an important role in cellular differentiation, proliferation and survival

BV UNIFESP: Teses e dissertaçõe

Protein tyrosine phosphatase alpha regulates cell detachment and cell death profiles induced by nitric oxide donors in the A(431) human carcinoma cell line

We investigated the role of protein tyrosine phosphatase-alpha (PTP alpha) expression in the cell death profile of the A431 human carcinoma cell line that was induced by cytotoxic concentrations of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18). Both NO donors promoted extensive cell detachment in A431 parental cells as compared to the detachment observed for A431 cells that ectopically expressed PTP alpha (A431 (A27B(PTP alpha)) cells). the NO-induced cell death characteristics for both cell lines were examined. After incubation for 10 hours with 2.0 mM SNP, attached or detached A431 cells underwent apoptosis. Cells were highly positive for Annexin-V, featured increased cleavage of procaspase-8, activation of downstream caspase-3, and activation of poly-ADP-ribose polymerase 1 (PARP-1). in contrast, exposure of A431 (A27B(PTP alpha)) cells to 2.0 mM SNP produced an increase in the release of lactate dehydrogenase and enhanced incorporation of propidium iodide. in addition, A431 (A27B(PTP alpha)) cells showed partial inhibition of the activities of caspase-8, caspase-3, and PARP-1 upon detachment and cell death induced by SNP treatment. Results indicate that necrotic cell damage was induced, characterized by cellular swelling and lysis. We conclude from these results that PTP alpha regulates the A431 tumor cell death profile mediated by NO donors. Expression of PTP alpha or its absence may determine the occurrence of NO-induced cell death with necrotic or apoptotic features, respectively.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Biochem Mol Biol, Ctr Cellular & Mol Therapy CTCMol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Clin Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Expt Oncol Unit UNONEX, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem Mol Biol, Ctr Cellular & Mol Therapy CTCMol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Clin Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Expt Oncol Unit UNONEX, BR-04044020 São Paulo, BrazilFAPESP: 06/56311-0FAPESP: 07/59617-6CNPq: 42011/2005Web of Scienc

The low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine, promotes cell cycle progression in rabbit aortic endothelial cells

S-Nitrosylation reactions are considered to be a major mechanism by which NO-related bioactivities are regulated in vivo. in the present study, we show the effects of the low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), on cell cycle progression of rabbit aortic endothelial cells (RAEC). SNAP at low concentrations (0.1 mM) stimulated the p21Ras-ERK1/2 MAP kinase signaling pathway. Activation of this signaling pathway was strongly inhibited in cells stably transfected with S-nitrosylation insensitive p21Ras (p21(Ra(C118S))). Furthermore, the SNAP-induced effects on cell cycle progression were eliminated in RAEC expressing N17Ras, a negative dominant mutant of p21Ras. Upon stimulation with SNAP, ERK1/2 MAP kinases become phosphorylated and translocate to the nucleus promoting the phosphorylation of the transcription factor E1k1. Synthesis of Cyclin D1 and stimulation of the cyclin-dependent kinases cdk4 and cdk6 resulted in the phosphorylation of the nuclear protein Rb and its dissociation from the E2F family of transcription factors. Cells then pass the restriction point in the late G1 phase. Cyclins E and A were expressed as the cell cycle progressed through the S phase upon stimulation with SNAP. Further transition in the cell cycle from the G2 to M phase was evidenced by the G2/M peak found in a histogram of the cell-phase distribution in SNAP-treated RAEC. These observations suggest that low molecular weight S-nitrosothiols may promote cell cycle progression possibly through the transnitrosation of p21Ras, and activation of the Ras-ERK1/2 MAP kinases signaling pathway. (C) 2008 Elsevier Inc. All rights reserved.Universidade Federal de São Paulo UNIFESP, CINTERGEN, Dept Biochem Mol Biol, São Paulo, BrazilUniversidade Federal de São Paulo, Expt Oncol Unit UNONEX, São Paulo, BrazilNYU, Sch Med, Dept Pharmacol, New York, NY USAUniversidade Federal de São Paulo UNIFESP, CINTERGEN, Dept Biochem Mol Biol, São Paulo, BrazilUniversidade Federal de São Paulo, Expt Oncol Unit UNONEX, São Paulo, BrazilWeb of Scienc

S-Nitrosoglutathione and Endothelial Nitric Oxide Synthase-Derived Nitric Oxide Regulate Compartmentalized Ras S-Nitrosylation and Stimulate Cell Proliferation

Aims: S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane. Results: Ras signaling pathway stimulation by 50 and/or 100 mu M of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 mu M GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. in this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras. Innovation: the present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways. Conclusion: S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. the nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane. Antioxid. Redox Signal. 18, 221-238.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, CTCMOL, Dept Biochem Mol Biol, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Parasitol & Immunol, BR-04044010 São Paulo, BrazilUniv São Paulo, Sch Med, São Paulo Canc Inst, São Paulo, BrazilNYU, Sch Med, Dept Pharmacol, New York, NY USAUniversidade Federal de São Paulo, CTCMOL, Dept Biochem Mol Biol, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Parasitol & Immunol, BR-04044010 São Paulo, BrazilFAPESP: 07/59617-6FAPESP: 09/52730-7FAPESP: 11/14392-2Web of Scienc