Studies on bladder cancer cell lines have shown that low adenoviral (Ad) infectivity is associated with low-level coxsackie adenovirus receptor (CAR) expression. Recently, we and others demonstrated ...a tumor stage- and grade-dependent downregulation of CAR expression in a large series of clinical bladder cancer specimens. Here, we demonstrate adenoviral gene transfer can be markedly enhanced in bladder cancer cells by upregulation of CAR through the use of certain differentiating agents, including the histone deacetylase inhibitors (HDACI) trichostatin A and sodium phenylbutyrate. CAR upregulation to supraphysiologic levels was demonstrated by quantitative rt-PCR, Western blotting, flow cytometry and adenoviral gene transfer. Normal urothelial cells and CAR-positive papilloma cells (RT4) failed to demonstrate upregulation under the same conditions. Upregulation was cell cycle dependent, associated with increased adenoviral gene transfer and persisted for at least 7 days after a single treatment. Such upregulation, however, appears to be tumor cell specific, as other CAR-negative cell lines failed to demonstrate enhanced adenoviral gene transfer with the same treatments. These results provide a rational basis for combining HDACI therapy with gene therapy as a method of augmenting activity in bladder cancer, but this strategy may not be universally applicable to other cell types.
Adenoviral vectors for gene transfer Kovesdi, Imre; Brough, Douglas E; Bruder, Joseph T ...
Current opinion in biotechnology,
10/1997, Letnik:
8, Številka:
5
Journal Article
Recenzirano
Adenoviruses began to be developed into highly effective gene expression vectors in the early 1980s. Recently, the increased interest in utilizing this transfer system
in vivo has posed new problems ...for heterologous gene-transfer, spurring a renewed effort in the field of vector development toward solving the structural, immunological and targeting problems posed by gene therapy applications.
Ovarian cancer is the fourth most common cancer among women and existing treatment is not routinely curative. One new strategy for cancer therapy is the selective delivery of TNFalpha to tumors via ...adenovirus vectors. We have tested the combination of two modifications to adenovirus vectors designed to limit delivery to tumors, capsid modification and expression control. To target alpha(v)beta(3/5) integrin receptors that are highly expressed in tumor and sparsely expressed in the epithelial layer of peritoneum, we modified the capsid fiber and penton base to remove native receptor binding and incorporated an RGD-4C motif in the fiber knob (Ad.PB*F*RGD). This vector exhibits effective gene transfer in all of the alpha(v)beta(3/5)-positive ovarian cancer cells tested in vitro and in vivo. Importantly, the Ad.PB*F*RGD vector is able to transduce ovarian tumor nodules and avoid infecting the normal mesothelial cells that line the intraperitoneal space following intraperitoneal administration. To further increase selectivity, different promoters were incorporated into the capsid-modified vector to confer the expression of the hTNFalpha therapeutic gene. We analyzed both constitutive (CMV or RSV) and potentially tumor selective promoters (MUC-1, E2F or hTERT) in terms of efficacy, selectivity and safety. TNF-expressing Ad.PB*F*RGD vectors containing the MUC-1 promoter showed anti-tumor activity in two ovarian cancer xenograft models (Caov3 and Igr-ov1) with little evidence of toxicity or systemic TNF. The data indicate that combination of capsid modification and transcriptional regulation of expression is a promising strategy for development of a new ovarian cancer treatment.
To create tumor-targeted Ad vectors, ablation of native CAR and integrin receptor binding is crucial to enhance the specificity of tumor transduction. Toward this aim, we have previously created base ...vectors in which binding to CAR (single-ablated) or to both CAR and integrins (double-ablated) has been ablated. In this study, the biodistribution of the conventional (CAR and integrin binding intact), single-ablated, and double-ablated vectors was evaluated following intraperitoneal administration. The mesothelial lining of the peritoneal organs was the principle site of CAR-dependent gene transfer by the conventional vector. Surprisingly, the single-ablated vector strongly transduced the liver parenchyma rather than the mesothelium, while the double-ablated vector did not significantly transduce the parenchyma or mesothelium. The high level of parenchymal transduction by the single-ablated vector suggested that it efficiently entered the bloodstream from the peritoneal cavity. Consistent with this hypothesis, a large proportion of active particles distributed and persisted in the bloodstream following intraperitoneal administration of either the single- or the double-ablated vector. The above results suggest that the double-ablated vector backbone may not only significantly improve targeting to cancers located in the peritoneal cavity, but may also significantly improve targeting to metastatic tumors located throughout the body by virtue of its enhanced bloodstream persistence.
Surfactant proteins play important roles in lung surfactant function and innate immunity. The DNA methylation state of 11 CpG sites of surfactant protein (SP)-A1, -B, -C, and -D was determined using ...universal bead arrays. A total of 90 cancerous and non-cancerous tissues from 23 patients with adenocarcinoma and 22 with squamous cell carcinoma were studied. These were divided into a training set and a testing set. The results indicate that DNA methylation profiling of these CpGs is associated with lung cancer. Four CpG sites, SP-A1_370, SP-A1_1080, SP-D_1170, and SP-D_1370, were hypomethylated in cancer and were significantly associated with both adenocarcinoma and squamous cell carcinoma, indicating that they have the potential to be used as biomarkers for lung cancer diagnosis and treatment. Normal lung tissues with a higher level of unmethylated SP-A1_1468 and SP-D_1170 CpG exhibited a higher level of SP-A1 and SP-D gene transcripts indicating that CpG methylation may play a role in gene expression. When the non-cancerous tissues were compared to cancerous tissues in patients with adenocarcinoma, the methylation profile results of these 46 samples (23 cancerous and 23 non-cancerous) could be clustered into 4 groups by agglomerative nesting. The percentage of tumor samples in each group was 0, 58, 91, and 100, respectively. A similar pattern was observed in squamous cell carcinoma patients. We speculate that SP-A1 and SP-D are subject to methylation/demethylation regulatory mechanisms and are involved in lung cancer pathogenesis by virtue of their function in innate host defense and/or regulation of inflammation.
Recombinant adenovirus (Ad) vectors provide a means of local, therapeutic gene delivery to a wide range of neoplasms. Ad-mediated gene therapy trials in malignant glioma models have been limited by ...the need for high viral titers and multiple dosages. In an attempt to improve Ad vector gene transfer, we studied human (U87, D54) and rodent (GL261, C6) malignant glioma cell lines transfected with various doses of unmodified Ad vectors (AdZ), Ad vectors that contain an alteration of the fiber-coat protein and that direct virus binding to heparan sulfate receptors (AdZ.F(pK7)), and Ad vectors with modifications of the fiber-coat protein that direct virus binding to alpha1, integrin cellular receptors (AdZ.F(RGD)). AdZ.F(pK7) increased the frequency of cells expressing the reporter gene, beta-galactosidase, and improved transduction by 2- to 20-fold compared with AdZ in U87, D54, and GL261 cells. In U87, D54, GL261, and C6 tumors, AdZ.F(pK7) increased gene transfer by 10- to 100-fold compared with AdZ. AdZ.F(RGD) increased gene expression in C6 xenografts compared with AdZ, but had reduced transduction compared with the C6 xenografts of AdZ in all other glioma tumors. These findings suggest that the increased tropisms resulting from alterations of the Ad vector fiber-coat protein as in AdZ.F(pK7) and AdZ.F(RGD) offer a feasible approach to improving in vitro and in vivo transduction efficiencies in certain malignant glioma cell lines.
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