ABSTRACT
Herpesvirus saimiri (HVS) is capable of infecting a range of human carcinoma cell types with high efficiency and the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny upon cell division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Moreover, the insertion of a bacterial artificial chromosome cassette into the HVS genome simplifies the incorporation of large amounts of heterologous DNA for gene delivery. Herein we have produced a recombinant HVS-based vector containing full-length human TRAIL under the control of the α-survivin promoter, and subsequently challenged a variety of cancer cell lines with this vector. The TRAIL transgene was expressed in infected colorectal SW480 cells, causing considerable apoptosis induction. Apoptosis was also observed when several other cancer cell lines derived from different tissues were infected. Moreover, co-treatment with Jak inhibitor AG490 led to the disruption of spheroid cultures grown from the melanoma Mel888 line. These data suggest that an HVS gene therapy vector expressing TRAIL could be an effective treatment against cancer.
Subject(s)
Apoptosis , Herpesvirus 2, Saimiriine/genetics , Neoplasms/therapy , TNF-Related Apoptosis-Inducing Ligand/biosynthesis , Antineoplastic Agents/pharmacology , Caspases/metabolism , Cell Line, Tumor , Cloning, Molecular , Genetic Therapy , Genetic Vectors , HEK293 Cells , Humans , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Sequence Deletion , Spheroids, Cellular , TNF-Related Apoptosis-Inducing Ligand/genetics , Transcription, Genetic , Tyrphostins/pharmacologyABSTRACT
This manuscript describes cell-uptake studies with HEK 293T cells on a series of ruthenium complexes shown previously to act as receptors for protein surface recognition and was motivated by a desire to establish if these receptors represent suitable templates for further elaboration as inhibitors of protein-protein interactions. The results illustrate that large (>3000Da) highly functionalized anionic ruthenium complexes are efficiently transfected via endocytosis to lysosomes with negligible toxicity.
Subject(s)
2,2'-Dipyridyl/analogs & derivatives , Molecular Mimicry/drug effects , 2,2'-Dipyridyl/chemical synthesis , 2,2'-Dipyridyl/chemistry , 2,2'-Dipyridyl/pharmacokinetics , Cell Survival/drug effects , Coordination Complexes , Dose-Response Relationship, Drug , Endocytosis/drug effects , HEK293 Cells , Humans , Lysosomes/drug effects , Molecular Structure , Protein Binding/drug effects , Structure-Activity Relationship , Surface PropertiesABSTRACT
Colorectal cancer (CRC) is a major cause of cancer-related mortality. A contributing factor to the progression of this disease is sporadic or hereditary mutation of the adenomatous polyposis coli (APC) gene, a negative regulator of the Wnt signalling pathway. Inherited mutations in APC cause the disorder familial adenomatous polyposis (FAP), which leads to CRC development in early adulthood. However, the gene is also disrupted in some 60% of sporadic cancers. Restoration of functional APC may slow the growth of CRC by negatively regulating proliferation-associated genes such as c-myc. Therefore, we have cloned the cDNA of the APC tumour suppressor gene into a replication competent Herpesvirus saimiri (HVS)-based vector to assess APC gene delivery in SW480 and SW620 CRC cell lines. Our results demonstrate that full length APC protein was efficiently expressed from the HVS vector and that transgene expression inhibited proliferation of both the SW480 and the metastatic SW620 cancer cell lines. Moreover, a sustained effect could be observed for at least 8 weeks after initial infection in SW480 cells. In addition, monolayer wounding assays showed a marked reduction in proliferation and migration in HVS-GFP-APC infected cells. We believe that this is the first instance of infectious delivery and APC cDNA expression from a virus-based vector.
Subject(s)
Colorectal Neoplasms/genetics , Genes, APC , Genetic Vectors/genetics , Herpesvirus 2, Saimiriine/genetics , Animals , Aotidae , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/metabolism , Gene Expression , Gene Expression Regulation, Neoplastic , Gene Order , Gene Transfer Techniques , HCT116 Cells , Humans , Transduction, Genetic , TransgenesABSTRACT
Herpesvirus saimiri (HVS) is a gamma herpesvirus with several properties that make it an amenable gene therapy vector; namely its large packaging capacity, its ability to persist as a nonintegrated episome, and its ability to infect numerous human cell types. We used RecA-mediated recombination to develop an HVS vector with a mutated virion protein. The heparan sulphate-binding region of HVS ORF51 was substituted for a peptide sequence which interacts with somatostatin receptors (SSTRs), overexpressed on hepatocellular carcinoma (HCC) cells. HVS mORF51 showed reduced infectivity in non-HCC human cell lines compared to wild-type virus. Strikingly, HVS mORF51 retained its ability to infect HCC cell lines efficiently. However, neutralisation assays suggest that HVS mORF51 has no enhanced binding to SSTRs. Therefore, mutation of the ORF51 glycoprotein has specifically targeted HVS to HCC cell lines by reducing the infectivity of other cell types; however, the mechanism for this targeting is unknown.