ABSTRACT
Our recent studies have revealed that among the 10 different commonly used adeno-associated virus (AAV) serotypes, AAV3 vectors transduce human liver cancer cells extremely efficiently because these cells express high levels of human hepatocyte growth factor receptor (hHGFR), and AAV3 utilizes hHGFR as a cellular co-receptor for viral entry. In this report, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression. We also document that AAV3 vectors are targeted for degradation by the host cell proteasome machinery, and that site-directed mutagenesis of surface-exposed tyrosine (Y) to phenylalanine (F) residues on AAV3 capsids significantly improves the transduction efficiency of Y701F, Y705F and Y731F mutant AAV3 vectors. The transduction efficiency of the Y705+731F double-mutant vector is significantly higher than each of the single mutants in liver cancer cells in vitro. In immunodeficient mouse xenograft models, direct intratumoral injection of AAV3 vectors also led to high-efficiency transduction of human liver tumor cells in vivo. We also document here that the optimized tyrosine-mutant AAV3 vectors lead to increased transduction efficiency following both intratumoral and tail-vein injections in vivo. The optimized tyrosine-mutant AAV3 serotype vectors containing proapoptotic genes should prove useful for the potential gene therapy of human liver cancers.
Subject(s)
Carcinoma, Hepatocellular/genetics , Dependovirus/genetics , Genetic Vectors , Liver Neoplasms/genetics , Proto-Oncogene Proteins c-met/genetics , Transduction, Genetic , Animals , Breast Neoplasms/genetics , Cell Line, Tumor , Female , Humans , Mice , Mutagenesis, Site-Directed , Proteasome Inhibitors , Protein Structure, Tertiary , Transplantation, Heterologous , Tyrosine/genetics , Virus IntegrationABSTRACT
The c-erbB-2 oncogene encodes a tyrosine kinase that constitutes the internal and transmembrane part of the epidermal growth factor receptor (EGFR). ErbB-2 overexpression has been reported in 20% to 30% of human adenocarcinomas of the breast and ovary, and has been linked to an unfavorable prognosis in patients. Hypericin is a protein tyrosine kinase inhibitor that has been exploited in models for anti-tumor and anti-viral activity. In this study, we investigated the effects of hypericin on the activity of the c-erbB-2 oncoprotein and its downstream kinases. We also investigated the effect of hypericin on metastasis. We used ovarian SK-OV-3 cells as a model to determine whether hypericin-induced cell death was associated with inhibition of c-erbB-2 expression and activation. The IC50 of hypericin after 72 hrs exposure was 7.5 microM as determined by the MTT assay. Apoptosis, which was assessed by morphological changes and a flow cytometric assay, was observed at 24 h after continuous exposure to 5 microM hypericin. Inhibition of expression of the c-erbB-2 protein was detected, using a monoclonal anti-erbB-2 antibody after 12-48 hrs of exposure to hypericin. Hypericin was found to inhibit autophosphorylation of the erbB-2 protein and downstream kinases such as MEK and ERK1/2. We also found up-regulation of p21WAF1 expression and down-regulation of Bcl-2 in hypericin treated cells. An invasion assay showed that hypericin inhibited the movement of SK-OV-3 cells into the Matrigel. However, gelatin zymography showed that hypericin had no effect on the secretion of matrix metalloproteinases (MMPs) in SK-OV-3 cells. From these results, we conclude that hypericin inhibits the growth of SK-OV-3 ovarian cancer cells, inhibits the autophosphorylation of c-erbB-2, induces apoptosis, and may inhibit invasion.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/enzymology , Perylene/analogs & derivatives , Perylene/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Anthracenes , Apoptosis/drug effects , Cell Division/drug effects , Cell Movement/drug effects , Cell Movement/physiology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Female , Humans , Neoplasm Invasiveness , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Receptor, ErbB-2/metabolism , Signal Transduction/drug effects , Tumor Cells, CulturedABSTRACT
The feasibility of using salivary ABH substance, covalently coupled to an affinity gel as an immunoadsorbent for anti-A and -B in sera containing antibodies to high-frequency antigens was investigated. Several examples of anti-Hy (n = 4), -Jra (n = 3), -U (n = 3), -Lub (n = 3), -Tja (n = 8), and -Vel (n = 6), plus one anti-Coa, were treated by batch adsorption with the affinity gel. Titration of the adsorbed sera with ABO-incompatible cells lacking the relevant high-frequency antigen, in parallel with ABO-compatible cells expressing the high-frequency antigen, revealed that ABO antibodies could be adsorbed to exhaustion without loss of the sera's activity against cells bearing high-frequency antigens.