Epstein, A.L. (Alberto L.)
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- Identification of replication-competent HSV-1 Cgal+ strain signaling targets in human hepatoma cells by functional organelle proteomics(American Society for Biochemistry and Molecular Biology, 2009) Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Epstein, A.L. (Alberto L.); Potel, C. (Corinne); Fernandez-Irigoyen, J. (Joaquín); Carro-Roldan, E. (Elvira); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Santamaria, E. (Enrique)In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.
- Characterization of herpes simplex virus 1 strains as platforms for the development of oncolytic viruses against liver cancer(John Wiley and Sons, 2011) Pourchet, A. (Aldo); Corrales, F.J. (Fernando José); Epstein, A.L. (Alberto L.); Manservigi, R. (Roberto); Bolaños, E. (Elixabet); Goicoechea, I. (Ibai); Brocker, T. (Thomas); Carro-Roldan, E. (Elvira); Volpi, I. (Ilaria); Foschini, M.G. (María Giovanna); Marconi, P. (Peggy); Hernandez-Alcoceba, R. (Rubén); Argnani, R. (Rafaela); Ried, C. (Christine); Santamaria, E. (Enrique)BACKGROUND: Diverse oncolytic viruses (OV) are being designed for the treatment of cancer. The characteristics of the parental virus strains may influence the properties of these agents. AIMS: To characterize two herpes simplex virus 1 strains (HSV-1 17syn(+) and HFEM) as platforms for virotherapy against liver cancer. METHODS: The luciferase reporter gene was introduced in the intergenic region 20 locus of both HSV-1 strains, giving rise to the Cgal-Luc and H6-Luc viruses. Their properties were studied in hepatocellular carcinoma (HCC) cells in vitro. Biodistribution was monitored by bioluminescence imaging (BLI) in athymic mice and immune-competent Balb/c mice. Immunogenicity was studied by MHC-tetramer staining, in vivo killing assays and determination of specific antibody production. Intratumoural transgene expression and oncolytic effect were studied in HuH-7 xenografts. RESULTS: The H6-Luc virus displayed a syncytial phenotype and showed higher cytolytic effect on some HCC cells. Upon intravenous or intrahepatic injection in mice, both viruses showed a transient transduction of the liver with rapid relocalization of bioluminescence in adrenal glands, spinal cord, uterus and ovaries. No significant differences were observed in the immunogenicity of these viruses. Local intratumoural administration caused progressive increase in transgene expression during the first 5 days and persisted for at least 2 weeks. H6-Luc achieved faster amplification of transgene expression and stronger inhibition of tumour growth than Cgal-Luc, although toxicity of these non-attenuated viruses should be reduced to obtain a therapeutic effect. CONCLUSIONS: The syncytial H6-Luc virus has a strong oncolytic potential on human HCC xenografts and could be the basis for potent OV.
- HSV-1 Cgal+ infection promotes quaking RNA binding protein production and induces nuclear-cytoplasmic shuttling of quaking I-5 isoform in human hepatoma cells(American Society for Biochemistry and Molecular Biology, 2011) Greco, A. (Anna); Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Epstein, A.L. (Alberto L.); Dayon, L. (Loïc); Sanchez-Quiles, V. (Virginia); Foschini, M.G. (María Giovanna); Prieto, J. (Jesús); Sánchez, J.C. (Jean-Charles); Segura, V. (Víctor); Santamaria, E. (Enrique)Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.
- Identification of replication-competent HSV-1 Cgal+ strain targets in a mouse model of human hepatocarcinoma xenograft(Elsevier, 2009) Greco, A. (Anna); Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Epstein, A.L. (Alberto L.); Manservigi, R. (Roberto); Fernandez-Irigoyen, J. (Joaquín); Carro-Roldan, E. (Elvira); Prieto, J. (Jesús); Molina, M. (Manuela); Marconi, P. (Peggy); Hernandez-Alcoceba, R. (Rubén); Santamaria, E. (Enrique)Recent studies based on animal models have shown the advantages and potential of oncolytic viral therapy using HSV-1 -based replication-competent vectors in the treatment of liver tumors, but little is known about the cellular targets that are modulated during viral infection. In the present work, we have studied the effects of intratumoral injections of HSV-1 Cgal(+) strain in a murine model of human hepatoma xenografts. Viral replication was assessed for more than 1month, leading to a significant reduction of tumor growth rate mediated, in part, by a cyclin B dependent cell proliferation arrest. Early events resulting in this effect were analyzed using a proteomic approach. Protein extracts from xenografted human hepatomas treated with saline or HSV-1 Cgal(+) strain during 24h were compared by 2-D DIGE and differential spots were identified by nanoLC-ESI-MS/MS. Alterations on glutathione S transferase 1 Omega, and ERp29 suggest novel HSV-1 Cgal(+) targets in solid liver tumors. Additionally, ERp29 showed a complex differential isoform pattern upon HSV-1 Cgal(+) infection, suggesting regulatory mechanisms based on post-translational modification events.