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Volume 4, Issue 4, Pages 365-371 (October 2001)
High-Level, β-Catenin/TCF-Dependent Transgene Expression in Secondary Colorectal Cancer Tissue Kai S. Lipinski, A.Hakim Djeha, Tariq Ismail, Andrew Mountain, Lawrence S. Young, Christopher J. Wrighton Molecular Therapy Volume 4, Issue 4, Pages (October 2001) DOI: /mthe Copyright © 2001 American Society for Gene Therapy Terms and Conditions
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FIG. 1 Activity and specificity of an artificial β-catenin-dependent promoter (CTP1). (A) Composition of the CTP1 promoter. The basal SV40 promoter (nt 49–244 from the plasmid pGL3promoter) was combined with five upstream TCF-4 consensus binding sites (filled boxes). Numbers indicate the distances between the TCF sites and between the most proximal TCF site and start of the TATA box. (B) Comparison of CTP1 and CMV promoter directed β-gal expression levels in cell lines with deregulated (SW480) and regulated (HeLa) β-catenin. SW480 and HeLa cells were infected with Ad.CMV-LacZ or Ad.CTP1-LacZ at the indicated MOIs (pfu/cell). CTP1 activity is expressed as a percentage of CMV activity. Three independent experiments are shown. Molecular Therapy 2001 4, DOI: ( /mthe ) Copyright © 2001 American Society for Gene Therapy Terms and Conditions
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FIG. 2 Comparison of CTP1 and CMV promoter activity in cultured normal human primary cells. The indicated cell types were infected with Ad.CMV-LacZ or Ad.CTP1-LacZ at the MOIs (pfu/cell) indicated on the vertical axis. β-Gal activity is expressed as a multiple of the respective mock value. A representative experiment is shown for each cell type. Molecular Therapy 2001 4, DOI: ( /mthe ) Copyright © 2001 American Society for Gene Therapy Terms and Conditions
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FIG. 3 CTP1 activity in freshly resected biopsies of human colon cancer. (A) Segments of freshly excised secondary colorectal cancer biopsies obtained from five independent donors were incubated with Ad.CMV-LacZ, Ad.CTP1-LacZ (7 × 108 pfu), or vehicle only (L, liver; O, ovarian; M, mesenteric). After allowing gene expression to proceed, all samples were fixed and stained for β-gal activity by X-gal staining. A representative segment of each biopsy is shown. (B) Tumor-specific expression of CTP1 in tissue samples containing both tumor and normal liver cells. Tumor segments containing associated normal liver tissue were prepared from a fresh biopsy of secondary colon cancer and infected and stained for β-gal activity as described above (T, tumor tissue; L, normal liver). Representative tumor segments are shown. Molecular Therapy 2001 4, DOI: ( /mthe ) Copyright © 2001 American Society for Gene Therapy Terms and Conditions
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FIG. 4 Analysis of CTP1 activity and β-catenin expression in biopsies of primary colorectal cancer. CTP1 was active in the majority of these tumors; representative CTP1-permissive and non-permissive tumors are shown (A). The level and sub-cellular distribution of β-catenin in each biopsy was determined by immunocytochemistry. (B) Distinct β-catenin staining patterns observed with CTP1-permissive and non-permissive tumors (bottom and top, respectively). Molecular Therapy 2001 4, DOI: ( /mthe ) Copyright © 2001 American Society for Gene Therapy Terms and Conditions
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FIG. 5 The CTP1 promoter drives therapeutic levels of tumor-specific E. coli nitroreductase gene expression in vivo. Subcutaneous SW480 tumors were injected with a single bolus of CTL102 or CTL501 at the indicated doses of virus particles (note: the preparations used contained equivalent proportions of infectious particles) or with vehicle only, followed by systemic CB1954 treatment. Individual tumor sizes are shown for each treatment, before and 80 days after virus injection. Horizontal bars indicate mean tumor sizes before injection and at the end of the study. Molecular Therapy 2001 4, DOI: ( /mthe ) Copyright © 2001 American Society for Gene Therapy Terms and Conditions
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FIG. 6 Systemic exposure of mice to CTL501/CB1954 is significantly less toxic than exposure to CTL102/CB1954. (A) The indicated doses of CTL102, CTL501, or virus vehicle were administered as a single bolus by tail vein injection, followed by systemic CB1954 treatment. The graph shows the maximum average mouse body weight loss recorded for each treatment group that occurred during the 15 days of monitoring and the percentage of mouse survival on day 15. (B) Lack of toxicity after systemic CTL501/CB1954 treatment correlates with low NTR expression in mouse liver. The indicated doses of CTL102 or CTL501 or vehicle were administered by tail vein injection, with or without CB1954 treatment. (Top) A representative hematoxylin and eosin stained liver section from vehicle- or virus-injected mice that subsequently received the standard CB1954 treatment. A separate group of mice were injected with vehicle, CTL102, or CTL501 and sacrificed 2 days later without CB1954 treatment. (Bottom) Representative liver sections analyzed for NTR expression by immunocytochemistry. Molecular Therapy 2001 4, DOI: ( /mthe ) Copyright © 2001 American Society for Gene Therapy Terms and Conditions
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