CANCER TARGETED GENE THERAPY II inadequate for testing gene therapies targeting STI571-resistant CML. Alternate tumor models, such as cells with bcr-abl kinase domain mutations, will be required in order to test the effectiveness of inhibiting bcr-abl expression for gene therapy of STI571-resistant leukemia.
766. Prostate Targeting Peptides Via the Prostate Specific Membrane Antigen Shawn E. Lupold,1 Ronald Rodriguez.1 1 Urology, Johns Hopkins University School of Medicine, Baltimore, MD, United States. There is a great need for agents that target diagnostic and therapeutic agents to prostate cancer cells. One method to identify such agents is to evolve random peptide libraries, displayed on filamentous phage, to bind to cancer cell lines or tumor markers. Here we have used phage display to identified short disulfide-constrained heptapeptides that bind specifically to the extracellular-portion of the Prostate Specific Membrane Antigen (PSMA), a type II integral membrane protein over-expressed on the surface of prostate cancer cells. A library of approximately 1.2 x 109 random sequence heptapetides, displayed on the pIII protein of M13 filamentous bacteriophage, was selected to bind to a fusion protein containing the extracellular portion of PSMA. A series of PSMA targets and phage selection techniques were applied to identify several unique PSMA binding peptides. The resulting peptides specifically bind purified PSMA protein and PSMA expressing prostate cancer cell lines. One of the most promising peptides, termed R5-XC1, represented all sequences found in the fifth selection round. Preliminary Surface Plasmon Resonance (SPR) studies indicate that R5-XC1 phage bind PSMA with affinities in the 100-500 nM range. The included flanking cysteines should provide a stable structure for targeting both synthetic and viral gene therapy vectors with these peptides.
Anti-Angiogenic Activity of Thymosin beta
Seung-Hoon Lee,1 Myung-Jin Son,1 Je-Ho Lee.1 1 Molecular Therapy Research Center, Samsung Medical Center, Sungkyunkwan University, Seoul, Korea. We recently reported that thymosin beta 10 (Tb10) expression was decreased in human ovarian cancer tissues and that overexpression of Tb10 gene led to apoptosis of human ovarian cancer cells. In the present study, overexpression of Tb10 decrease tumor growth accompanied with marked reduction of tumor vessels in mouse xenograft model. We investigate the antiangiogenic activity of Tb10 in vitro and in vivo. Adenovirus-mediated overexpression of Tb10 in cultured endothelial cells potently inhibited basic fibroblast growth factor (bFGF)-induced endothelial cell proliferation, migration, invasion and tube formation. In contrast, inhibition of endogenous Tb10 by short interfering RNAs (si RNAs) maintained proliferative and chemotactic effects of bFGF. In addition, the expression of prime angiogenic factor, vascular endothelial growth factor (VEGF), Tb10 and of platelet endothelial cell adhesion molecule (PECAM) was observed by immunohitochemistry in control tumor and Tb10-treated tumors in vivo. Microvessels stained with PECAM were markedly reduced in Tb10-treated tumors. Taken together, these findings demonstrate that Tb10 potently inhibits bFGF-induced angiogenesis and Tb10 is a viable target in therapeutic approaches to promote or inhibit angiogenesis.
768. The Potential Role of BimS as a Therapeutic Gene in Human Nasopharyngeal Carcinoma (NPC) Kenneth W. Yip,1 Jian-Hua Li,1 Shahnaz Al Rashid,1 Dolly Huang,2 Fei-Fei Liu.1 1 Medical Biophysics and Radiation Oncology, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, ON, Canada; 2 Chinese University of Hong Kong, Hong Kong. Introduction: Our lab has successfully achieved nasopharyngeal carcinoma (NPC) specific gene expression by transcriptional targeting through EBNA-1 (Cancer Res 62:171, 2002). In the context of an adenoviral vector (adv.oriP), we demonstrated a 1000-fold induction of gene expression in Epstein Barr Virus (EBV)-positive NPC cells. This strategy now provides us with the platform from which we can explore the use of potent pro-apoptotic genes, such as the novel BimS. Methods and Materials:BimS was sub-cloned downstream of the oriP-FR promoter sequence (poriP.BimS), and the plasmid was recombined with pJM17 to generate the novel adv.oriP.BimS. The cellular effects of adv.oriP.BimS on the EBVpositive C666-1 human NPC cell line were determined using the MTS assay. Apoptosis was evaluated utilizing AO-EB staining, and subcellular localization of BimS was assessed using confocal microscopy. Results: We observed a time and dose-dependent reduction in C666-1 cell viability, with 9% survival four days after infection with adv.oriP.BimS (25 ifu/cell). The addition of ionizing radiation (6 Gy) caused a further reduction in cell viability. There was a clear induction of apoptosis in a time and dose-dependent manner. Three days after infection, the proportion of apoptotic cells under control, 5, or 25 ifu/cell adv.oriP.BimS conditions were 17%, 46%, and 61%, respectively. Confocal microscopy clearly demonstrated cytoplasmic distribution of BimS, which co-localized to the mitochondria within 24 h of adv.oriP.Bim S infection. Conclusion: We have successfully constructed a novel adenovirus expressing BimS by utilizing a tumour-specific promoter. This vector appears to be effectively cytotoxic to an EBV-positive NPC cell line, mediated primarily through induction of apoptosis.
769. Transcriptional Control of Adenoviral Vector Expression in Tumor Cells through the CXCR4 Gene Promoter Zeng B. Zhu,1 Bao G. Lu,1 Ming H. Wang,1 Lioudmila Kaliberova,1 Angel A. Rivera,1 Dirk M. Nettelbeck,1 Parameshwar J. Mahasreshti,1 Charles A. Leath III,2 Sharmila K. Makhija,2 David T. Curiel.1 1 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 2Department of Obatetrics & Gynecology, University of Alabama at Birmingham, Birmingham, AL, United States. Regional gene therapy appears to be an attractive cancer treatment modality. However, most gene therapy approaches utilize nonspecific gene promoters, which can lead to potential limitations such as toxicity to normal tissues. Recently, several references have documented that a novel gene, CXCR4 gene, was over-expressed in most tumors including breast cancer. Our objective was to test the promoter activity of the CXCR4 gene promoter in different tumor cell lines, including pancreatic cancer, breast cancer, ovarian cancer, and melanoma. We also tested this promoter for “liver off” status in vivo using a mouse model. We anticipate that the CXCR4 gene promoter might be of potential use as a tumor specific promoter for transgene delivery in cancer therapy with low systemic toxicity. A recombinant adenovirus (reAdGL3BCXCR4) was constructed by inserting a luciferase reporter gene, and a CXCR4 promoter, which drives the reporter expression, into the deleted E1 region of adenovirus type 5. The reAdGL3BCXCR4 was used to infect Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
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