Fusion of the BCL9 HD2 domain to E1A increases the cytopathic effect of an oncolytic adenovirus that targets colon cancer cellsReportar como inadecuado




Fusion of the BCL9 HD2 domain to E1A increases the cytopathic effect of an oncolytic adenovirus that targets colon cancer cells - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Cancer

, 6:236

First Online: 04 October 2006Received: 06 June 2006Accepted: 04 October 2006DOI: 10.1186-1471-2407-6-236

Cite this article as: Fuerer, C., Homicsko, K., Lukashev, A.N. et al. BMC Cancer 2006 6: 236. doi:10.1186-1471-2407-6-236

Abstract

BackgroundThe Wnt signaling pathway is activated by mutations in the APC and β-catenin genes in many types of human cancer. β-catenin is stabilized by these mutations and activates transcription in part by acting as a bridge between Tcf-LEF proteins and the HD2 domain of the BCL9 coactivator. We have previously described oncolytic adenoviruses with binding sites for Tcf-LEF transcription factors inserted into the early viral promoters. These viruses replicate selectively in cells with activation of the Wnt pathway. To increase the activity of these viruses we have fused the viral transactivator E1A to the BCL9 HD2 domain.

MethodsLuciferase assays, co-immunoprecipitation and Western blotting, immunofluorescent cell staining and cytopathic effect assays were used to characterize the E1A-HD2 fusion protein and virus in vitro. Growth curves of subcutaneous SW620 colon cancer xenografts were used to characterize the virus in vivo.

ResultsThe E1A-HD2 fusion protein binds to β-catenin in vivo and activates a Tcf-regulated luciferase reporter better than wild-type E1A in cells with activated Wnt signaling. Expression of the E1A-HD2 protein promotes nuclear import of β-catenin, mediated by the strong nuclear localization signal in E1A. Tcf-regulated viruses expressing the fusion protein show increased expression of viral proteins and a five-fold increase in cytopathic effect CPE in colorectal cancer cell lines. There was no change in viral protein expression or CPE in HeLa cells, indicating that E1A-HD2 viruses retain selectivity for cells with activation of the Wnt signaling pathway. Despite increasing the cytopathic effect of the virus in vitro, fusion of the HD2 domain to E1A did not increase the burst size of the virus in vitro or the anti-tumor effect of the virus in an SW620 xenograft model in vivo.

ConclusionDespite an increase in the nuclear pool of β-catenin, the effects on viral activity in colon cancer cells were small, suggesting that factors acting downstream of β-catenin are limiting for viral replication and toxicity in these cells. The approach of fusing E1A to a protein domain implicated in oncogenic signaling could be used to selectively increase the activity of oncolytic viruses targeting several other pathways defective in cancer.

Abbreviations12S-13SAlternatively spliced transcripts of E1A

APCAdenomatous Polyposis Coli

ATFActivating Transcription Factor

BCL9B Cell Lymphoma 9

BSABovine Serum Albumin

CPECytopathic Effect

CR3E1A Conserved Region 3

DBPAdenoviral DNA Binding Protein

E1A-E1B-E2-E4Adenoviral early transcription units

HD2Homology Domain 2

HNPCCHereditary Non-Polyposis Colon Cancer

ICATInhibitor of β-catenin

MTT3-4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide

NESNuclear Export Signal

NHDN-terminal Homology Domain

NLSNuclear Localization Signal

PBSPhosphate Buffered Saline

Tcf-LEFT cell factor-Lymphoid Enhancer Factor

WntWingless Integration site

YAC-BACYeast Artificial Chromosome-Bacterial Artificial Chromosome

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2407-6-236 contains supplementary material, which is available to authorized users.

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Autor: Christophe Fuerer - Krisztian Homicsko - Alexander N Lukashev - Anne-Laure Pittet - Richard D Iggo

Fuente: https://link.springer.com/







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