Synergistic combination of valproic acid and oncolytic parvovirus H‐1PV as a potential therapy against cervical and pancreatic carcinomas VPA treatment.

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Synergistic combination of valproic acid and oncolytic parvovirus H‐1PV as a potential therapy against cervical and pancreatic carcinomas VPA treatment of HeLa cells increases NS1 acetylation and NS1‐dependent transcriptional activity Source data is available for this figure in the Supporting Information. The H‐1PV nonstructural protein NS1 is acetylated in vivo. Mock or H‐1PV‐infected HeLa cells (MOI of 0.5 pfu/cell) were treated with VPA (1 mM) or NaB (1 mM) for 16 or 32 h before to be lysed. Immunoprecipitated NS1 was analysed by Western blotting with antibody against acetylated lysines. For detection of total NS1 protein, the membrane was stripped and re‐probed with anti‐NS1 antiserum. VPA treatment enhances NS1 transcriptional activities. HeLa cells were transfected with pCDNA4/TO‐Flag‐HA‐NS1‐H1 (NS1) or pCDNA4/TO empty vector (−), together with the pGL3‐H‐1PV‐P38 and the pRL‐TK plasmid. Transfected cells were grown in a medium containing or not containing VPA (1 mM) for 48 h. Cells were then lysed and subjected to Dual Luciferase Assay. Values represent the mean ratios of firefly to Renilla luciferase activity with standard deviation bars, for three replicates. VPA treatment increases NS1‐driven gene expression. HeLa cells seeded into 6‐cm dishes in a medium supplemented or not with VPA (1 mM) were mock‐treated (−) or infected with an H‐1 recombinant parvovirus (recH1‐EGFP) at a MOI 1 transduction unit per cell (+). The recH‐1‐EGFP vector carries the EGFP‐encoding transgene under the control of the NS1‐inducible viral promoter P38. After 48 h, cells were collected, total RNA was isolated and reverse transcribed. Quantitative PCR was carried out using specific primers for EGFP, NS1 and GAPDH (used as a housekeeping gene). The EGFP and NS1 mRNA expression levels were normalized to those of GAPDH mRNA. Normalized expression values presented are means with standard deviation bars from three measurements. VPA treatment increases viral protein synthesis. HeLa cells were infected with H‐1PV (MOI of 1 pfu/cell) and grown for 48 h in the presence or absence of VPA (1 mM). Cell lysates were analysed by Western blotting for the presence of viral NS1 and capsid (VP1 and VP2) proteins. Actin was used as a loading control. VPA enhances binding of NS1 to the P38 promoter. Chromatin immunoprecipitation and PCR amplification of the precipitated viral P38 promoter region was performed as described in Materials and Methods Section. Results are presented as average Ct values with standard deviation bars, computed from duplicate measurements per experimental conditions. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO Mol Med, Volume: 5, Issue: 10, Pages: 1537-1555, First published: 17 September 2013, DOI: (10.1002/emmm.201302796)