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  • 1
    Keywords: CANCER ; CELLS ; EXPRESSION ; tumor ; TUMOR-CELLS ; CELL ; Germany ; KINASE ; THERAPY ; GENE ; GENE-EXPRESSION ; PROTEIN ; cell line ; TUMORS ; LINES ; DNA ; INFECTION ; REDUCTION ; CELL-LINES ; PHOSPHORYLATION ; treatment ; PARTICLES ; virus ; ISOFORM ; gene expression ; PROMOTER ; DECREASE ; ELEMENTS ; NUMBER ; CELL-LINE ; LINE ; TRANSFORMATION ; GLUCOSE ; FLUORESCENCE ; REGULATORY ELEMENTS ; adeno-associated virus ; ADENOASSOCIATED VIRUS ; VIRUS THYMIDINE KINASE ; TUMOR-CELL-LINES ; HIGH-LEVEL ; HaCaT ; Ras ; THYMIDINE KINASE ; SRC ONCOGENE ; ADENOASSOCIATED VIRUS VECTORS ; CYSTIC-FIBROSIS ; glucose transporter promoter,HSV thymidine kinase,suicide gene,AAV
    Abstract: In order to achieve tumor-specific targeting of adeno-associated virus (AAV)-mediated gene expression, the promoter of the glucose transporter isoform 1 (GLUT1) gene was cloned upstream of the enhanced green fluorescence protein (EGFP) and the herpes simplex virus thymidine kinase (HSVtk) gene. FACS analysis performed at 48 h after transient infection with rAAV/cytomegalovirus (CMV)egfp viral particles revealed an increase of fluorescence in all the cell lines tested. However, EGFP expression under control of the GLUT1 promoter element (rAAV/GTI-1.3egfp) was limited to the tumor cells and oncogene-transformed cells. Evidence for phosphorylation of the HSVtk substrates ganciclovir (GCV) and I-125-deoxycytidine was found in all transfected tumor cell lines compared to noninfected controls (HCT116: 111%; MH3924A: 130%; HaCaT-RT3: 257% increase), but not in HaCaT and HUVEC cells. Furthermore, tumor cells and the oncogene-transformed (ras) cell line HaCaT-RT3 showed a GCV-induced reduction in cell number (HCT116:-71%; MH3924A:-43% and HaCaT-RT3:-31%). No statistically relevant cytotoxic effect was observed in HaCaT (6% decrease) and HUVEC cells (2% decrease). Furthermore, a reduction of H-3-thymidine incorporation into the DNA was seen after treatment with GCV (HCT116: 38%; MH3924A: 33% and HaCaT-RT3: 37% decrease). In a therapy study of HSVtk-expressing tumors with GCV, we achieved total tumor remission
    Type of Publication: Journal article published
    PubMed ID: 14681725
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  • 2
    Keywords: CELLS ; IN-VITRO ; tumor ; TUMOR-CELLS ; AGENTS ; Germany ; IN-VIVO ; VITRO ; VIVO ; imaging ; MARKER ; TRANSPORT ; UP-REGULATION ; MAMMALIAN-CELLS ; IMAGING AGENTS ; FLUORESCENCE ; RE ; TRANSPORTER ; tumor imaging ; TUMOR-CELL ; uptake ; ANALOGS ; in vivo ; intraoperative imaging ; POLYAMINE TRANSPORT ; SERUM-ALBUMIN ; spermidine
    Abstract: Up-regulation of polyamine transporters on the surface of tumor cells and the internalization of biogenic polyamines by active transport processes may be exploited for the accumulation of spermidine derivatives as reporter molecules. We have synthesized and tested fluorophor-labeled spermidine derivatives for the development of a new class of intraoperative tumor imaging agents. In vitro uptake experiments and initial in vivo imaging studies illustrated that fluorophor tagged spermidine derivatives show tumor accumulation. (c) 2006 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16621552
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  • 3
    Abstract: Autonomous parvoviruses possess an intrinsic oncotropism based on viral genetic elements controlling gene expression and genome replication. We constructed a hybrid vector consisting of the H1 parvovirus-derived expression cassette comprising the p4 promoter, the ns1 gene and the p38 promoter flanked by the adeno-associated viruses 2 ( AAV2) inverted terminal repeats and packaged into AAV2 capsids. Gene transduction using this vector could be stimulated by coinfection with adenovirus, by irradiation or treatment with genotoxic agents, similar to standard AAV2 vectors. However, the latter were in most cases less efficient in gene transduction than the hybrid vector. With the new vector, tumor cell-selective increase in transgene expression was observed in pairs of transformed and non-transformed cells, leading to selective killing of the transformed cells after expression of a prodrug-converting enzyme. Preferential gene expression in tumor versus normal liver tissue was also observed in vivo in a syngeneic rat model. Comparative transduction of a panel of different tumor cell lines with the H1 and the H1/AAV hybrid vector showed a preference of each vector for distinct cell types, probably reflecting the dependence of the viral tropism on capsid determinants
    Type of Publication: Journal article published
    PubMed ID: 18202715
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  • 4
    Keywords: CANCER ; CELLS ; EXPRESSION ; IN-VITRO ; IN-VIVO ; THERAPY ; GENE ; gene therapy ; LINES ; NUCLEAR-MEDICINE ; TIME ; TRANSDUCTION ; ACTIVATION ; RAT ; PROSTATE-CANCER ; TRANSFORMATION ; GLUCOSE ; ESTABLISHMENT ; sodium iodide symporter ; glucose transporter promoter ; SODIUM-IODIDE SYMPORTER ; SRC ONCOGENE ; THYROID-CELLS
    Abstract: Targeted transfer of a functionally active sodium iodide symporter (NIS) into tumour cells may be used for radioiodine therapy of cancer. Therefore, we investigated radioiodine uptake in a hepatoma cell line in vitro and in vivo after transfer of the sodium iodide symporter (hNIS) gene under the control of a tumour-specific regulatory element, the promoter of the glucose transporter 1 gene (GTI-1.3). Employing a self- inactivating bicistronic retroviral vector for the transfer of the hNIS and the hygromycin resistance genes, rat Morris hepatoma (MH3924A) cells were infected with retroviral particles and hNIS-expressing cell lines were generated by hygromycin selection. I-125(-) uptake and efflux were determined in genetically modified and wild type hepatoma cells. In addition, the iodide distribution in rats bearing wild type and genetically modified hepatomas was monitored. hNIS-expressing MH3924A cell lines accumulated up to 30 times more iodide than wild type hepatoma cells, with a maximal iodide uptake after 30 min incubation time. Competition experiments in the presence of sodium perchlorate revealed a decrease in the iodide uptake (80-84% decrease). Moreover, ouabain led to a loss of accumulated I- (81% decrease) whereas 4,4'-diisothiocyano-2,2'-disulphonic acid stilbene (DIDS) increased the I- uptake into cells (87% increase). However a rapid efflux of the radioactivity (70%) was observed 20 min after I-125-containing medium had been replaced by non- radioactive medium. Lithium had no significant effect on iodide efflux. In rats, the hNIS-expressing tumours accumulated 22 times more iodide than the contralateral wild type tumour. In accordance with the in vitro data, we also observed a rapid efflux of the radioactivity out of the tumour in vivo. Dosimetric calculations resulted in an absorbed dose of 85 mGy in the wild type tumour and 830 mGy in the hNIS-expressing tumour after administration of 18.5 MBq I-131. In conclusion, transduction of the hNIS gene under the control of the GLUT1 promoter element induces iodide transport in Morris hepatoma cells in vitro and in vivo. However, for therapeutic application additional conditions need to be defined which inhibit the iodide efflux out of the tumour cells
    Type of Publication: Journal article published
    PubMed ID: 12541134
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  • 5
    Keywords: CELLS ; EXPRESSION ; IN-VITRO ; Germany ; IN-VIVO ; GENE ; GENE-EXPRESSION ; transcription ; DIFFERENTIATION ; MICE ; ACTIVATION ; TRANSCRIPTION FACTOR ; INDUCTION ; RAT ; CELL-LINES ; ELEMENT-BINDING PROTEIN ; TRANSCRIPTION FACTORS ; LINE ; protein-protein interaction ; FACTOR-I ; cell lines ; interaction ; FACTOR GENE ; bioluminescence imaging ; HISTONE DEACETYLASE INHIBITORS ; LIVING SUBJECTS ; PAIRED BOX GENE ; protein-protein ; radioiodine ; SODIUM/IODIDE-SYMPORTER GENE ; SYNERGISTICALLY ACTIVATE ; THYROGLOBULIN GENE ; thyroid transcription ; thyroid transcription factors human Pax8 and dog TTF-1 ; TRANSCRIPTIONAL ACTIVATION
    Abstract: The thyroid transcription factors TTF-1 and Pax8 cooperate in the transcriptional activation of thyroid-specific genes such as thyroglobulin (Tg), thyroperoxidase (TPO), and sodium/iodide symporter (NIS). Methods: Dog TTF-1 (dTTF-1) and the human Pax8 (hPax8) gene were transfected in Morris hepatoma (MH3924A) cells to investigate (a) the possible visualization of functional protein-protein interaction and (b) the induction of thyroid-specific gene expression. In MH3924A call lines expressing dTTF-1, hPax8, or both, the activation of human Tg (hTg), human TPO (hTPO), or rat NIS (rNIS) promoter/enhancer was measured using firefly luciferase reporter constructs. Furthermore, the possible induction of thyroid-specific genes was investigated in iodide uptake and reverse transcription polymerase chain reaction (RT-PCR) experiments. Results: Low transcriptional activation of these constructs was observed in cells expressing either hPax8 or dTTF-1 alone. In contrast, the hTg and hTPO and, to a lesser extent, the rNIS regulatory region were significantly activated in cell lines expressing both transcription factors. Imaging the transcriptional activation of the thyroid-specific regulatory regions by Pax8 and TTF-1 was possible in nude mice implanted with MHhPax8dTTF-1 cells using a cooled charge-coupled device camera. (NaI)-I-125 uptake experiments and RT-PCR showed no effect of hPax8 and dTTF-1 on endogenous thyroid-specific gene expression in genetically modified cells. Conclusion: The activation of thyroid-specific promoter/enhancer elements in Morris hepatoma cells depends on the functional interaction of hPax8 and dTTF-1. The cooperation of these 2 transcription factors can be visualized in vitro as well as in vivo. With regard to a possible application for radioiodine therapy, further modifications are required
    Type of Publication: Journal article published
    PubMed ID: 15872358
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  • 6
    Keywords: RECEPTOR ; ANGIOGENESIS ; APOPTOSIS ; CELLS ; ENDOTHELIAL-CELLS ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; IN-VITRO ; proliferation ; tumor ; CELL-PROLIFERATION ; FACTOR RECEPTOR ; Germany ; IN-VIVO ; INHIBITION ; MODEL ; PERFUSION ; VITRO ; imaging ; HEPATOCELLULAR-CARCINOMA ; GENE ; GENES ; METABOLISM ; TUMORS ; LINES ; TRANSDUCTION ; gene transfer ; GENE-TRANSFER ; DNA ; INDUCTION ; CELL-LINES ; signal transduction ; immunohistochemistry ; MALIGNANCIES ; ASSAY ; DESIGN ; VECTOR ; NUMBER ; STRESS ; SIGNAL-TRANSDUCTION ; LINE ; positron emission tomography ; POSITRON-EMISSION-TOMOGRAPHY ; tomography ; OXIDATIVE STRESS ; cell lines ; MALIGNANCY ; OXIDATIVE-STRESS ; TUMOR-GROWTH ; monitoring ; endothelial cells ; cell proliferation ; MIGRATION INHIBITORY FACTOR ; CHIP ; computer-assisted ; functional imaging ; ANGIOGENESIS IN-VIVO ; GLIOBLASTOMA GROWTH ; IRRADIATED SKIN ; SOLUBLE FORM ; SYMPORTER GENE
    Abstract: Purpose: Inhibition of tumor angiogenesis is emerging as a promising target in the treatment of malignancies. Therefore, monitoring of antiangiogenic approaches with functional imaging and histomorphometrical analyses are desirable to evaluate the biological effects caused by this treatment modality. Experimental Design: Using a bicistronic retroviral vector for transfer of the soluble receptor for the vascular endothelial growth factor (sFLT) hepatoma (MH3924A) cell lines with sFLT expression were generated. In human umbilical vein endothelial cells cultured with conditioned medium of sFLT-expressing hepatoma cells, the inhibitory action of secreted sFLT was determined using a Coulter counter and a thymidine incorporation assay. Furthermore, in vivo experiments were done to measure the effects on tumor growth and perfusion. Finally, the tumors were examined by immunohistochemistry (including computer-assisted morphometry) and DNA chip analysis. Results: Stable sFLT-expressing hepatoma cells inhibited endothelial cell proliferation in vitro. In vivo, growth and perfusion, as measured by (H2O)-O-15 positron emission tomography, were reduced in genetically modified tumors. However, the immunohistochemically quantified microvascularization and macrovascularization, as indicated by CD31- and alpha-actin-positive area, revealed no significant changes, whereas the number of apoptotic cells was increased in sFLT-expressing tumors, although not significantly. DNA chip analysis of tumors with gene transfer showed an increase of genes related to apoptosis, signal transduction, and oxidative stress. Conclusion: Our results suggest that sFLT expression inhibits tumor growth and perfusion and enhances expression of apoptosis-related genes in this model. Enhanced expression of genes for signal transduction, stress, and metabolism indicates tumor defense reactions
    Type of Publication: Journal article published
    PubMed ID: 15788658
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  • 7
    Keywords: CELLS ; EXPRESSION ; GROWTH ; IN-VITRO ; tumor ; TUMOR-CELLS ; Germany ; human ; IN-VIVO ; INHIBITION ; THERAPY ; VITRO ; VIVO ; imaging ; EXPOSURE ; SITE ; GENE ; GENE-EXPRESSION ; TUMORS ; LINES ; DNA ; RAT ; RATS ; CELL-LINES ; ENTRY ; gene expression ; TRANSCRIPTIONAL ACTIVITY ; ASSAY ; resistance ; VECTOR ; PROMOTER ; NUMBER ; PROSTATE-CANCER ; CELL-LINE ; LINE ; HEPATOMA ; BIODISTRIBUTION ; ESTABLISHMENT ; SODIUM/IODIDE SYMPORTER ; FEASIBILITY ; TISSUE-SPECIFIC EXPRESSION ; SODIUM-IODIDE SYMPORTER ; albumin ; ENHANCER ; TUMOR-GROWTH ; INCREASE ; TRANSFECTION ; TRANSPORTER ; HUMAN SODIUM/IODIDE SYMPORTER ; SIZE ; in vivo ; HUMAN HEPATOCELLULAR-CARCINOMA ; RADIONUCLIDE ; radionuclide gene therapy
    Abstract: We investigated the feasibility of radioiodine therapy targeting hepatoma cells (MH3924A) by tissue-specific expression of the human sodium/iodide symporter (hNIS) gene directed by the murine albumin enhancer and promoter (mAlb). Methods: The cell-specific transcriptional activity of mAlb was examined by a luciferase assay in several transiently transfected cell lines. MH3924A cells were stably transfected with the recombinant retroviral vector, in which hNIS complementary DNA expression was driven by mAlb and coupled to hygromycin resistance gene using an internal ribosomal entry site (IRES). Functional hNIS expression in hepatoma cells was confirmed by an iodide uptake assay. In imaging studies, the tumor-bearing ACl rats were intravenously injected with I-131 and imaged with a gamma-camera. Biodistribution was studied at 30 min and at 1, 3, 6, and 25 h after injection of I-131. Toxic effects of I-131 on hepatoma cells were studied in vitro and in vivo. Results: Stably transfected MH3924A cells concentrated I-125 up to 240-fold higher than the wild-type cells. The iodide uptake in stably transfected cells was inhibited by ouabain and sodium perchlorate but increased by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. An in vitro clonogenic assay revealed an 86% decrease in colony number in stably transfected cells after exposure to 3.7 MBq/mL of I-131 and only about 8% in hNIS-negative control cells. Furthermore, the in vivo study showed intense tracer accumulation in hNIS-expressing tumors after administration of I-131. At 3 h after intraperitoneal injection, the transfected tumors accumulated I-131 19.2-fold higher than the parental tumors in a biodistribution study. Moreover, administration of a therapeutic dose of I-131 resulted in an inhibition of hNIS-expressing tumor growth, whereas control tumors continued to increase in size. Conclusion: A therapeutic effect of I-131 on hepatoma cells in vitro and in vivo has been demonstrated after tumor-specific iodide uptake induced by mAlb-directed hNIS gene expression. Because a stable transformed cell line has been used in these experiments, the clinical potential of this strategy must be evaluated after in vivo transfection of hepatoma cells
    Type of Publication: Journal article published
    PubMed ID: 16644756
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  • 8
    Keywords: TARGET ; MELANOMA ; MALIGNANT-MELANOMA ; malignant melanoma
    Type of Publication: Meeting abstract published
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  • 9
  • 10
    Keywords: CANCER ; CELLS ; EXPRESSION ; tumor ; TUMOR-CELLS ; carcinoma ; CELL ; Germany ; human ; IN-VIVO ; THERAPY ; VIVO ; GENE ; cell line ; TUMORS ; gene therapy ; LINES ; MICE ; NUCLEAR-MEDICINE ; TIME ; TRANSDUCTION ; RAT ; animals ; CELL-LINES ; treatment ; TRANSPORT ; resistance ; PROSTATE-CANCER ; NUDE-MICE ; CELL-LINE ; LINE ; WILD-TYPE ; CARCINOMA-CELLS ; CARCINOMAS ; GENE-THERAPY ; EXPERIMENTAL I-131 THERAPY ; LITHIUM ; RADIOIODINE THERAPY ; nuclear medicine ; nude mice ; THYROID-CARCINOMA-CELLS ; BICISTRONIC RETROVIRAL VECTOR ; HUMAN SODIUM/IODIDE SYMPORTER ; NA+/I-SYMPORTER ; sodium iodide symporter,gene therapy,lithium,thyroid carcinoma
    Abstract: Transfer of the human sodium iodide symporter (hNIS) has been proposed as a new principle of cancer gene therapy. This study evaluates the iodide kinetics and dosimetry of iodide in hNIS-expressing thyroid carcinoma cells under optimized conditions. Methods: Using a bicistronic retroviral vector for the transfer of the hNIS and the hygromycin resistance gene, hNIS-expressing rat thyroid carcinoma cell lines were generated. Afterward, Na(125)1 uptake and efflux were determined in genetically modified and wild-type cells in the presence or absence of modulators of iodide transport. In addition, the (131)1 distribution in thyroidablated nude mice bearing wild-type and genetically modified thyroid carcinomas was monitored after intraperitoneal administration of (131)1 with and without coadministration of lithium carbonate. Results: hNIS-expressing cell lines accumulated up to 49 times more iodide than did noninfected cells, with a maximal iodide uptake after 30 min of incubation. However, a 90% efflux of the radioactivity occurred 20 min after replacement of the medium. In mice, the hNIS-expressing tumors accumulated up to 23 and 19.5 times more iodide than did the wild-type tumors in lithium-treated and control animals, respectively. However, efflux of the radioactivity was also observed in vivo: After 24 h, hNIS-expressing tumors lost 82.5% and 80.4% of the initial activity. Dosimetric calculations showed that 1,650 MBq of (131)1 per square meter resulted in 5.4 and 5.2 Gy in hNIS-expressing tumors and 0.24 and 0.26 in wild-type tumors. Conclusion: Transduction of the hNIS gene in rat thyroid carcinoma cells induces iodide transport, which is associated with rapid efflux. Application of (131)1 in clinically relevant amounts did not result in therapeutically useful absorbed doses in hNIS-expressing tumors in vivo, even under optimized conditions of thyroid ablation and treatment with lithium carbonate
    Type of Publication: Journal article published
    PubMed ID: 15136633
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