Monitoring of tumor burden in vivo by optical imaging in a xenograft SCID mouse model: evaluation of two fluorescent proteins of the GFP-superfamily.

BACKGROUND: Mouse models of human-malignant-melanoma (MM) are important tools to study tumor dynamics. The enhanced green fluorescent protein (EGFP) is widely used in molecular imaging approaches, together with optical scanners, and fluorescence imaging. PURPOSE: Currently, there are no data available as to whether other fluorescent proteins are more suitable. The goal of this preclinical study was to analyze two fluorescent proteins of the GFP superfamily under real-time in vivo conditions using fluorescence reflectance imaging (FRI). MATERIAL AND METHODS: The human melanoma cell line MeWo was stable transfected with one plasmid: pEGFP-C1 or pDsRed1-N1. We investigated two severe combined immunodeficiency (SCID)-mice groups: A (solid xenografts) and B (xenografts as metastases). After three weeks, the animals were weekly imaged by FRI. Afterwards the mice were euthanized and metastases were imaged in situ: to quantify the cutis-dependent reduction of emitted light, we compared signal intensities obtained by metastases in vivo with signal intensities obtained by in situ liver parenchyma preparations. RESULTS: More than 90% of cells were stable transfected. EGFP-/DsRed-xenograft tumors had identical growth kinetics. In vivo the emitted light by DsRed tumors/metastases was much brighter than by EGFP. DsRed metastases were earlier (3 vs. 5 weeks) and much more sensitive detectable than EGFP metastases. Cutis-dependent reduction of emitted light was greater in EGFP than in DsRed mice (tenfold). Autofluorescence of DsRed was lower than of EGFP. CONCLUSION: We established an in vivo xenograft mouse model (DsRed-MeWo) that is reliable, reproducible, and superior to the EGFP model as a preclinical tool to study innovative therapies by FRI under real-time in vivo conditions.

Intravenous Iodinated Contrast Agents Amplify DNA Radiation Damage at CT.

PURPOSE: To determine the effect of the use of iodinated contrast agents on the formation of DNA double-strand breaks during chest computed tomography (CT). MATERIALS AND METHODS: This study was approved by the institutional review board, and written informed consent was obtained from all patients. This single-center study was performed at a university hospital. A total of 179 patients underwent contrast material-enhanced CT, and 66 patients underwent unenhanced CT. Blood samples were taken from these patients prior to and immediately after CT. In these blood samples, the average number of phosphorylated histone H2AX (γH2AX) foci per lymphocyte was determined with fluorescence microscopy. Significant differences between the number of foci that developed in both the presence and the absence of the contrast agent were tested by using an independent sample t test. RESULTS: γH2AX foci levels were increased in both groups after CT. Patients who underwent contrast-enhanced CT had an increased amount of DNA radiation damage (mean increase ± standard error of the mean, 0.056 foci per cell ± 0.009). This increase was 107% ± 19 higher than that in patients who underwent unenhanced CT (mean increase, 0.027 foci per cell ± 0.014). CONCLUSION: The application of iodinated contrast agents during diagnostic x-ray procedures, such as chest CT, leads to a clear increase in the level of radiation-induced DNA damage as assessed with γH2AX foci formation.

Influence of different iodinated contrast media on the induction of DNA double-strand breaks after in vitro X-ray irradiation.

The objective of this work was to examine differences in DNA double-strand break induction in peripheral blood lymphocytes after in vitro X-ray irradiation between iodinated contrast agents. Four different iodinated X-ray contrast agents--three of them with two different iodine concentrations--and mannitol (negative control; concentration of 150 mg mannitol per ml blood) were pipetted into blood samples so that there was a concentration of 0, 7.5 or 15 mg of iodine per ml blood in the samples. Negative controls without contrast medium (0 mg of iodine per ml blood) were also processed for every irradiation dose. The tubes were exposed to 0, 20 or 500 mGy in vitro X-ray irradiation. After that, the lymphocytes were separated by using density-gradient centrifugation. Fluorescence microscopy was applied to determine the average number of γH2AX-foci per lymphocyte in the presence or absence of different contrast media or mannitol. Differences in the number of γH2AX-foci were statistically analysed by one-way ANOVA and post-hoc Tukey's honestly significant difference test. Iodinated contrast agents led to a statistically significant increase in DNA double-strand breaks after in vitro irradiation. This effect increased statistically significant with rising radiation dose and appeared independent of the contrast agent used (iopromid, iodixanol, iomeprol, iopamidol). A statistically significant difference in DNA damage between the different tested contrast agents was not found. Therefore, the increase in DNA double-strand breaks depends solely on the amount of iodine applied. For evaluation of clinical consequences, our findings could be tested in further animal studies.

The presence of iodinated contrast agents amplifies DNA radiation damage in computed tomography.

The purpose of this study was to determine the influence of iodinated contrast agents on the formation of DNA double-strand breaks in vitro in lymphocytes and to verify these results in patients undergoing diagnostic computed tomography examinations. Blood samples were irradiated in vitro in the presence of iodinated X-ray contrast agent. Controls were irradiated without contrast agent. Fourteen patients were investigated using contrast-enhanced computed tomography (CT), and 14 other patients with unenhanced CT. Blood samples were taken prior to and 5 min and 1, 2 and 24 h after the CT examination. In these blood samples the average number of γH2Ax-foci per lymphocyte was enumerated by fluorescence microscopy. Statistical differences between foci numbers developed in the presence and absence of contrast agent were tested using an independent sample t-test. In vitro foci numbers after irradiation were significantly higher when contrast agent was present during irradiation. In vivo, γH2Ax-foci levels were 58% higher in patients undergoing contrast-enhanced CT compared with those undergoing unenhanced CT. In the presence of iodinated contrast agents DNA, damage is increased and the radiation dose is not the only factor affecting the amount of DNA damage. Individual patient characteristics and biological dosimetry applications, e.g. the analysis of γH2Ax-foci, have to be considered.

Expression of the Wnt antagonist DKK3 is frequently suppressed in sporadic epithelial ovarian cancer.

PURPOSE: The Wnt pathway plays an important role in embryonic development, and defects in this pathway have been implicated in the tumorigenesis. The Dickkopf 3 (DKK3) is a putative Wnt signaling inhibitor that is frequently inactivated in human cancers. However, the expression of DKK3 in ovarian cancer remains unknown. METHODS: We investigated the expression of DKK3 in silico using the Digital Differential Display. DKK3 mRNA expression was also analyzed by real-time RT-PCR in ovarian carcinomas and normal ovarian tissues. DKK3 protein expression was determined by immunohistochemistry in the same ovarian carcinomas and normal ovarian tissues. RESULTS: A significantly reduced expression of DKK3 (P < 0.05) was found after comparison of normal ovary- and tumor-derived libraries in the Cancer Genome Anatomy Project (CGAP). DKK3 mRNA expression was reduced in 63% (35 of 56) of tumors compared with normal ovarian samples (P < 0.02). Analysis of 13 matched pairs of ovarian carcinomas and adjacent normal tissues showed significant transcriptional downregulation of DKK3 (>twofold) in 9 paired carcinomas (69%). Loss or weak membranous expression of DKK3 protein was observed in 66% of ovarian cancers (37 of 56) including all tumors with low transcriptional level of DKK3 gene analyzed by real-time PCR. CONCLUSIONS: To our best knowledge, this is the first time to demonstrate altered expression of DKK3 in ovarian cancer. The latter could be a relevant mechanism for the activation of the Wnt pathway in the carcinogenesis of ovarian cancer but additional studies are required to elucidate the function of DKK3 silencing in ovarian carcinogenesis.

Local administration of NF-kappa B decoy oligonucleotides to prevent restenosis after balloon angioplasty: an experimental study in New Zealand white rabbits.

PURPOSE: To evaluate the efficacy of NF-kappa B oligonucleotides (ODN) administered by local administration with the channeled balloon catheter to prevent restenosis after balloon angioplasty in restenotic iliac arteries of New Zealand white rabbits. MATERIALS AND METHODS: In vitro, 8000 rabbit vascular smooth muscle cells (rVSMC) where transfected with a liposomal carrier (TfX50) with 100 ng of decoy and scrambled ODN. Inhibition of proliferation was measured using a MTT assay after 24 hours in comparison to control. In vivo, 22 male New Zealand White rabbits were fed a 1% cholesterol diet and received denudation of both common iliac arteries with a 3 mm balloon catheter to induce an arterial stenosis. Four weeks after stenosis induction, local application of NF-kappa B in two different concentrations (1 mug: n = 14; 10 mug: n = 8) was performed randomly on one common iliac artery. Scrambled oligonucleotides without specific binding capacities were injected into the contralateral side. The channeled balloon catheter allows simultaneous balloon dilation (8 atm) of the stenosis and local application of a drug solution (2 atm). Four weeks after local drug delivery the animals were killed and the vessels were excised and computerized morphometric measurements were performed. RESULTS: NF-kappa B decoy ODN but not scrambled ODN inhibited proliferation of rVSMC in vitro. Following local ODN application in the animals, no acute vascular complications were seen. NF-kappa B ODN resulted in a statistically non significant reduction of neointimal area compared to the control group. The neointimal area was 0.97 mm(2) using 1 mug NF-kappa B ODN compared to 0.98 mm(2) in the control group. The higher dose resulted in a neointimal area of 0.97 mm(2) compared to 1.07 mm(2) at the control side. CONCLUSIONS: Local drug delivery of NF-kappa B ODN using the "channeled balloon" catheter could not reduce neointimal hyperplasia in stenostic rabbit iliac arteries. Application modalities have to be improved to enhance the effect of the local application to prevent restenosis after balloon angioplasty.

In vitro MR imaging of regulated gene expression.

PURPOSE: To design and evaluate a construct that allows regulated expression of the magnetic resonance (MR) imaging reporter gene human tyrosinase under control of the tetracycline response element. MATERIALS AND METHODS: A breast cancer cell line (MCF-7) was transfected with a plasmid that codes for the tetracycline-controlled transactivator and a new construct. In this construct, the reporter gene human tyrosinase is under control of the tetracycline response element, thus allowing suppression of gene expression by adding doxycycline (tetracycline switched off). A reverse transcription polymerase chain reaction was conducted to evaluate gene expression. Additionally, immunohistochemical investigation of tyrosinase and melanin staining was undertaken to analyze the presence of these molecules. After culture in an iron- and holotransferrin-enriched medium, cells were imaged in a 1.0-T clinical MR imager by using a surface coil and T1-weighted spin-echo and gradient-echo sequences. RESULTS: Two stable transfected cell clones were established. Cells cultured with doxycycline showed no background expression of the human tyrosinase gene, whereas withdrawal of doxycycline resulted in detectable tyrosinase messenger RNA expression. Gene expression results in a detectable tyrosinase protein level and melanin content. Increased signal intensity on T1-weighted MR images in cells that expressed the reporter gene was observed in comparison to genetically identical cells with the reporter gene switched off. CONCLUSION: Our construct enables MR imaging of regulated tyrosinase gene expression in vitro.

Paclitaxel inhibits proliferation of cell lines responsible for metal stent obstruction: possible topical application in malignant bile duct obstructions.

RATIONALE AND OBJECTIVES: To evaluate a dose dependent inhibitory effect of paclitaxel to assess a possible local application for biliary tract malignancies in conjunction with stent placement. METHODS: Cell cultures of the three different cell types (human epithelial gallbladder cells [HEGC], human fibroblasts [HF; PA 314 wt] and pancreatic carcinoma cells [PC; P181]) were incubated for 20 minutes at 37 degrees C with increasing doses of paclitaxel (1.0 x 10(-4) - 1.0 x 10(2) micromol). Half of the cultures were then incubated without paclitaxel, the other half with paclitaxel for 20 minutes, 24 hours, or 72 hours. Cell proliferation was detected by photometric measurements of mitochondrial dehydrogenase activity (MTT assay). RESULTS: Incubation of cell cultures with paclitaxel resulted in a dose dependent and cell specific inhibition of cell proliferation. Concentrations of 1.0 x 10(-4) (and higher) paclitaxel for 20 minutes resulted in a inhibition of cell proliferation of HEGC (28%), PA (26%), and HF (17%). A prolonged paclitaxel incubation (up to 72 hours) resulted in an inhibitory effect on cell proliferation of HEGC (40%), PA (45%), and HF (39%). Cytotoxic effects, manifested by development of vacuoles and damage of cell integrity were seen at concentrations above 1.0 x 10(1) for both the short and long term incubation. CONCLUSIONS: Paclitaxel incubation resulted in a dose dependent inhibition of cell proliferation of human epithelial gallbladder cells, human fibroblasts and pancreatic carcinoma cells. This inhibitory effect of paclitaxel on the cell lines could serve as the basis to develop drug coated or drug eluting stents for malignant biliary strictures.