ABSTRACT
For cancer gene therapy, cancer-specific over-expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.
Subject(s)
Female , Humans , Breast Neoplasms/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cloning, Molecular , Cytomegalovirus , Dependovirus , Gene Targeting , Genetic Therapy , Genetic Vectors , Green Fluorescent Proteins , Promoter Regions, Genetic/genetics , Ribonucleoside Diphosphate Reductase/genetics , Transcriptional ActivationABSTRACT
Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif 10PQENDE15 in STP-A11 reveals that Glu (E)12 residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.
Subject(s)
Humans , Transcription, Genetic , Transcription Factor AP-1/agonists , TNF Receptor-Associated Factor 6/metabolism , Solubility , STAT3 Transcription Factor/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Protein Binding , Oncogene Proteins, Viral/metabolism , NF-kappa B/agonists , Ions , Herpesvirus 2, Saimiriine/metabolism , Detergents , Cell LineABSTRACT
To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound-healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.
Subject(s)
Animals , Humans , Male , Mice , Cell Movement , Cells, Cultured , Dependovirus/genetics , Endothelial Cells/metabolism , Gene Transfer Techniques , Genetic Vectors , Mice, Inbred C57BL , Neovascularization, Physiologic , Ribonuclease, Pancreatic/biosynthesis , Umbilical Veins/cytology , Vascular Endothelial Growth Factor A/biosynthesisABSTRACT
No abstract available.
Subject(s)
Animals , Isomerism , Saliva/metabolism , Sodium-Hydrogen Exchangers/metabolism , Sodium-Hydrogen Exchangers/physiology , Sodium-Hydrogen Exchangers/chemistryABSTRACT
No abstract available.