Transfer of the Sflt-1 Gene in Morris Hepatoma Results in Decreased Growth and Perfusion and Induction of Genes Associated with Stress Response

2132 Vol. 11, 2132–2140, March 15, 2005 Clinical Cancer Research

Transfer of the sFLT-1 Gene in Morris Hepatoma Results in Decreased Growth and Perfusion and Induction of Genes Associated with Stress Response

Kerstin Schmidt,1,3 Johannes Hoffend,2 tumors. However, the immunohistochemically quantified Annette Altmann,2,3 Ludwig G. Strauss,3 microvascularization and macrovascularization, as indicated by CD31- and a-actin-positive area, revealed no significant Antonia Dimitrakopoulou-Strauss,3 1 6 4 changes, whereas the number of apoptotic cells was Britta Engelhardt, Dirk Koczan, Jo¨rg Peter, increased in sFLT-expressing tumors, although not signifi- 1 3 Silke Vorwald, Helmut Eskerski, cantly. DNA chip analysis of tumors with gene transfer Michael Eisenhut,5 Ju¨rgen Metz,1 Ralf Kinscherf,1 showed an increase of genes related to apoptosis, signal and Uwe Haberkorn2,3 transduction, and oxidative stress. Departments of 1Anatomy and Cell Biology III and 2Nuclear Medicine, Conclusion: Our results suggest that sFLT expression University of Heidelberg; 3Clinical Cooperation Unit Nuclear Medicine inhibits tumor growth and perfusion and enhances expres- and Departments of 4Biophysics and Medical Radiation Physics, and sion of apoptosis-related genes in this model. Enhanced 5Radiopharmaceutical Chemistry, German Cancer Research Center, expression of genes for signal transduction, stress, and 6 Heidelberg, Germany; and Department of Immunology, University of metabolism indicates tumor defense reactions. Rostock, Rostock, Germany

INTRODUCTION ABSTRACT Malignant tumors are dependent on angiogenesis, showing Purpose: Inhibition of tumor angiogenesis is emerging a correlation of the microvessel density with the grade of as a promising target in the treatment of malignancies. invasiveness, frequency of metastasis, and clinical prognosis Therefore, monitoring of antiangiogenic approaches with (1, 2). For tumor angiogenesis, angiogenic factors such as functional imaging and histomorphometrical analyses are vascular endothelial growth factor (VEGF), fibroblast growth desirable to evaluate the biological effects caused by this factor (FGF), and interleukin 8 need to be produced within treatment modality. tumors either by cancer cells or by infiltrating lymphocytes, Experimental Design: Using a bicistronic retroviral macrophages, or fibroblasts (3, 4). Out of these, VEGF may be vector for transfer of the soluble receptor for the vascular one of the most important factors for tumor angiogenesis and is endothelial growth factor (sFLT) hepatoma (MH3924A) cell suggested as an angiogenic factor, which augments tumor lines with sFLT expression were generated. In human vascularity (5, 6). Second, it may act as a permeability factor to umbilical vein endothelial cells cultured with conditioned promote the supply of nutrients to the tumor by diffusion (7, 8). medium of sFLT-expressing hepatoma cells, the inhibitory In this context, tumor suppression has been achieved in animal action of secreted sFLT was determined using a Coulter experiments by inhibiting VEGF or its receptor using neutral- counter and a thymidine incorporation assay. Furthermore, izing VEGF antibodies, blocking VEGF receptor antibodies, in vivo experiments were done to measure the effects on antisense oligonucleotides against VEGF, VEGF antisense tumor growth and perfusion. Finally, the tumors were expression plasmids, VEGF-diphtheria toxin conjugate, and a examined by immunohistochemistry (including computer- truncated soluble form of the VEGF receptor that inhibits the assisted morphometry) and DNA chip analysis. functioning of the wild-type (WT) receptor (9–16). Because the Results: Stable sFLT-expressing hepatoma cells abovementioned methods require either a substantial amount of inhibited endothelial cell proliferation in vitro. In vivo, protein or a direct insertion of the molecules into cancer cells, the 15 growth and perfusion, as measured by H2 O positron transfer of antiangiogenic genes into tumor cells or regional emission tomography, were reduced in genetically modified expression near tumor sites should be more effective in suppressing tumor growth (17, 18). Several groups have used the gene of the soluble VEGF receptor (sFLT) in a variety of Received 10/19/04; revised 12/15/04; accepted 12/23/04. Grant support: Wilhelm Sander Foundation grant 1999.085.1, tumor entities (17, 19–23). sFLT may form a heterodimeric Tumorzentrum Heidelberg/Mannheim, Bequest of Herbert Dauss, and complex with a WT VEGF receptor and function as a dominant- Foundation for Cancer and Scarlatina Research. negative receptor (15, 16). Furthermore, sFLT should also be The costs of publication of this article were defrayed in part by the secreted from infected cells into the blood stream and should payment of page charges. This article must therefore be hereby marked reach most sites of angiogenesis within the tumor and sequester advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. VEGF from receptors on the target cells, thereby achieving an Note: K. Schmidt and J. Hoffend contributed equally to the paper. effective suppression of tumor growth. R. Kinscherf and U. Haberkorn are senior authors. Because intervention with angiogenesis should result in Requests for reprints: Uwe Haberkorn, Department of Nuclear functional consequences with respect to blood supply, monitor- Medicine, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg, 69120, Germany. Phone: 49-6221-567731; Fax: 49-6221- ing of antiangiogenic approaches with functional imaging and 565473; E-mail: [email protected]. histomorphometrical analyses are desirable to deliver informa- D2005 American Association for Cancer Research. tion about the biochemical and physiologic effects caused by this

treatment modality. Positron emission tomography (PET) with from WT-MH3924A or sFLTMH-3924A cells in 6-well plates. 15O-labeled water has been used in several trials that investigated The medium consisted of 75% medium 199 (Life Technologies; potential antiangiogenic effects of razoxane, endostatin, or supplemented with 20% FCS, 2 mmol/L glutamine, 100 IU/mL combretastatin A4 phosphate (24–26). Tumors responding to penicillin, 100 IU/mL streptomycin, and 2 ng bFGF/mL) and treatment showed a dose-dependent reduction in tumor perfusion 25% medium from WT-MH3924A or sFLTMH-3924A cells. and blood volume (24–26). However, in renal carcinomas no The cells were cultured in the absence of growth factors or in the significant differences of perfusion, blood volume and fractional presence of 20 ng/mL VEGF or 20 ng/mL bFGF. Proliferation of volume of distribution were seen prior and after treatment with the endothelial cells was measured after 5 days by incorpora- razoxane. Furthermore, no change was seen during tumor tion of (methyl-[3H])-thymidine (Amersham-Buchler, Ismaning, progression, too (24). Germany) in DNA as previously described (31). The experiment In the following experiments, stable Morris hepatoma was repeated twice. (MH3924A) cell lines expressing human sFlt (sFLT-MH3924A) Animal Studies. ACI rats and RNU (nude rats) were were established. In vitro the effect of conditioned medium from supplied by Charles Rivers Laboratories (Kingston, NY). All sFLT-MH3924A on the proliferation of human umbilical vein animal studies were done in compliance with the national laws cells (HUVEC) was measured. Furthermore, in rats, bearing WT- relating to the conduct of animal experimentation. The in vitro MH3924A or genetically modified hepatomas, tumor growth, doubling time of the cell lines used was 18.7 and 17.7 hours for and tumor perfusion were determined. These functional data WT-MH3924A and sFLT-MH3924A cells, respectively. After were combined with histomorphometrical investigations of the inoculation of WT or sFLT cell suspensions (2 Â 106 cells per vascularization, apoptosis, proliferation, oxidative stress, and animal) into the thigh of ACI or RNU rats, tumor growth was inflammation as well as gene array analysis of the expression measured using calipers. PET studies of tumor perfusion were pattern in cells and tumors. done in separate animals weighing 200 to 250 g. Perfusion in tumors with diameters between 10 and 13 mm was examined 15 after i.v. bolus injection of 70 to 150 MBq H2O in 0.3 mL by MATERIALS AND METHODS dynamic PET (20 Â 3 seconds, 6 Â 10 seconds, 4 Â 15 seconds, Cell Culture and Generation of Recombinant Cells. 6 Â 30 seconds) acquired in two-dimensional mode using a Cells were cultured in RPMI 1640 (Life Technologies, matrix of 256 Â 256 on an ECAT HR+ (Siemens CTI, Eggenstein, Germany) supplemented with 292 mg/L glutamine, Knoxville, TN) scanner (pixel size, 2.277 Â 2.777 Â 100,000 IE/L penicillin, 100 mg/L streptomycin, and 20% FCS. 2.425 mm; transaxial resolution, 4.3 mm). A transmission scan The cell lines were cultured at 37jC, in an atmosphere of 95% was done for 10 minutes before tracer administration with three air and 5% CO2. rotating germanium pin sources to obtain cross sections for sFLT Gene Transfer and Northern Blots. For transfer attenuation correction. After iterative reconstruction (OSEM, of the sFLT gene, a bicistronic retroviral vector was used 8 subsets, 16 iterations) the dynamic PET data were evaluated as consisting of the sFLT gene (cloned from pBacPAK8sFLT-1, described earlier using the PMOD software package (32). Time- obtained from H. Weich 27) and the hygromycin resistance gene activity curves were created using volumes of interest. A volume cloned downstream of the elongation factor 1a (EF1a) promoter. of interest consists of several regions of interest over the target To ensure simultaneous expression of the genes coding for the area. Irregular regions of interest were drawn manually. sFLT and for the hygromycin resistance and stabilization of the Perfusion studies with 15O-water can be evaluated using a one- mRNA a synthetic intron and an internal ribosomal entry site tissue-compartment model (33). For the input function, we used from encephalomyocarditis virus was inserted between the genes the mean value of the volumes of interest data obtained from the (28, 29). heart. Regions of interest were defined at 3 to 6 seconds after For transient packaging of the retroviral particles, a bolus injection in at least three consecutive slices (2.4-mm lipofection of the transient packaging cell line BOSC23 was thickness) surrounded by two slices showing the heart in each done. After 2 days, the medium was centrifuged to remove direction. This approach was based on the findings of Ohtake detached BOSC23 cells and used for the infection of MH3924A et al. demonstrating that the input function can be retrieved from cells in the presence of 8 Ag polybrene/mL over night. The cells the image data with acceptable accuracy (34). The heart weight were treated with 425 Ag/mL hygromycin for 4 weeks until and heart volume of a 250-g rat are 1.0 g and 1.2 mL, resistant cell lines were established. These were further respectively (35). Furthermore, the size of the heart was incubated in hygromycin-containing medium. determined in our rats showing a median value of 1.0 Â 1.3 cm. Total RNA was isolated using Trizol reagent (Roche, This results in an estimated recovery error of 10%, which can be Mannheim, Germany) and mRNA expression was assessed by neglected for the purposes of this study. The transport constant Northern blots using radioactive labeled probes delivered by K1 and the rate constant k2 were calculated using a one-tissue- digestion from the sFLT plasmid. compartment model for 15O-water based on a method imple- Measurement of Cell Proliferation. Human umbilical mented in the PMOD software taking into account the vascular vein endothelial cells (HUVEC) were isolated from umbilical fraction, which is associated with the volume of blood cords as previously described (30) and cultured in supplemented exchanging with tissue in a volume of interest. RPMI 1640 [with 292 mg/L glutamine, 100,000 IE/L penicillin, Tissue Preparation and Immunohistochemistry. Rat 100 mg/L streptomycin, 20% FCS, and 4 ng/mL basic FGF tumors were fixed with immunohistochemistry zinc fixative (BD (bFGF)]. To determine the effects of sFLT on HUVEC Biosciences, Heidelberg, Germany) or 4% paraformaldehyde proliferation, HUVECs were cultured in conditioned medium (and routinely embedded in paraffin) or shock-frozen in liquid

nitrogen-cooled isopentane and kept at À70jC in a freezer until Data Analysis and Statistics. Results are presented as used for immunohistochemical investigations. Immunohisto- mean F SD. Statistical comparisons between groups were done chemistry of the tumors was routinely done according to by the Mann Whitney U-Wilcoxon rank sum W test or by the procedures as described earlier (31). In detail, 6-Am kryostat unpaired Student’s t test using the SIGMASTAT program sections were cut on a Microm Microtome, placed on Superfrost (Jandel Scientific, Erkrath, Germany). P V 0.05 was considered microscope slides (Fisher Scientific, Pittsburgh, PA), exposed to statistically significant. acetone (10 minutes, À20jC), and dried (30 minutes). Nonspecific sites were blocked with 1% normal swine serum (Life Technologies) in PBS. For immunohistochemical inves- RESULTS tigations the monoclonal antibodies mouse anti-rat-CD31 (1:500; Inhibition of Proliferation of Human Umbilical Vascular BD PharMingen, San Diego, CA), mouse anti-a-actin Endothelial Cells Cultured with Conditioned Medium of (1:100; Roche), mouse anti-proliferating cell nuclear antigen sFLT-MH3924A. The successful transfer of the sFLT gene (1:500; DakoCytomation, Glostrup, Denmark), mouse anti-rat into MH3924A (sFLT-MH3924A) and the overexpression of this CD11b (1:500; BD PharMingen), rabbit anti-mouse-cycloox- gene was verified by Northern blotting in different hygromycin- ygenase (COX)-2 (1:300; Cayman Chemical Company, Ann resistant cell lines (data not shown). Of these, the cell line with Arbor, Michigan), and mouse anti-human-MIF (1:50; R&D the highest signal as evaluated by the sFLT/h-actin ratio was Systems GmbH, Wiesbaden-Nordenstadt, Germany) were used. chosen for further experiments. Next, we investigated the effects As a negative control, the primary antibody was omitted or of culture with conditioned medium from WT-MH3924A and control immunoglobulins were used. sFLT-MH3924A on proliferation of HUVECs. The proliferation Apoptosis was measured on 4% paraformaldehyde-fixed was measured by (methyl-[3H])-thymidine incorporation into the paraffin-embedded tumor cross-sections by the terminal deoxy- DNA of HUVECs after perchloric acid extraction. The acid- nucleotidyl transferase–mediated nick end labeling (TUNEL) insoluble fraction in this assay represents nucleic acids and technique using a commercially available kit (Oncor, Heidelberg, proteins, whereas the acid-soluble fraction represents unbound Germany). radioactivity in acid-soluble molecules, which are not in DNA The sections were photographed using an Axioplan2 and proteins. In the presence of conditioned medium from WT- imaging microscope (Carl Zeiss GmbH, Jena, Germany) and MH3924A cells, bFGF and VEGF caused a 2-fold and 1.5-fold the digital high resolution imaging system AxioCam/AxioVision increase in thymidine uptake in the acid-insoluble fraction (Carl Zeiss). Digitalized images were processed, arranged and compared with the value obtained without these growth factors. lettered by standard imaging software (Adobe Photoshop 6.0/ Using conditioned medium from sFLT-MH3924A cells, a 60% Illustrator 10.0). (without growth factor), 89% (with bFGF), and 83% (with Morphometric Analysis. Immunoreactive cells in tumor VEGF) decrease of thymidine (P < 0.001) uptake in the acid- cross sections were recorded by a video camera (Olympus insoluble fraction occurred compared with the uptake in the HCC-3600 P high gain) and quantified using a computer- presence of the corresponding WT-MH3924A medium. The assisted image analysis system developed by our group uptake in the acid-soluble fraction decreased by 71%, 95%, and (VIBAM 0.0-VFG 1 frame grabber; ref. 36). Cells were 92%, respectively (P < 0.001; Table 1). counted positive when the typical cytoplasmic immunoreac- Inhibition of Tumor Growth and Perfusion in sFLT- tivity was associated with a cell nucleus. For quantifying MH3924A Tumors In vivo: Positron Emission Tomography tumor vascularization, we measured microvessel density, mean and Immunohistomorphometry of Vascularization. After number of vessels, and luminal area. Morphometrical analyses inoculation, we examined the growth of WT-MH3924A or sFLT- were done by two independent observers (interobserver MH3924A tumors in ACI and athymic (nude) RNU rats, variability below 5%). including PET measurements and histologic analyses of Gene Arrays. WT-MH3924A and sFLT-MH3924A cells vascularization. RNU rats were used to exclude immunologic as well as aliquots of the respectively explanted tumors were effects as possible causes for growth inhibition. stored in TRIZOL to extract RNA according to the manufacturer’s specifications. RNA was purified using the RNeasy kit (Qiagen, Inc., Hilden, Germany). Afterwards, the quality of isolated RNA Table 1 Proliferation of HUVECs cultured with conditioned medium was photometrically tested with the 280:260 ratio and on agarose from WT-MH3924A or sFLT-MH3924 cells gel. Gene chip expression arrays were done using The Rat Acid-insoluble fraction Acid-soluble fraction Genome U34 Set (Affymetrix, Inc., Santa Clara, CA) including Conditioned medium from WT-MH3924A cells f7,000 known genes was used and the RNAs of sFLT- Without growth factor 2,689.1 F 58.22 333.6 F 46.72 MH3924A (n = 6) and WT-MH3924A (n = 7) tumors were bFGF 5,287.82 F 390.4 270.7 F 3.62 pooled and evaluated on separate gene chips as suggested by the VEGF 3,975.17 F 197.3 264.43 F 13.27 manufacturer. Because a very low signal intensity results in Conditioned medium from sFLT-MH3924A cells Without growth factor 1,120.2 F 81.77 99.96 F 7.11 increased error rate only genes above a cutoff value of 0.5% of F F h bFGF 594.52 85.26 15.61 1.24 the -actin signal were used for further analysis. The ratios of VEGF 698.98 F 77.51 21.44 F 1.03 modified tumors versus WT tumors were calculated and values NOTE. Incubation of HUVECs with conditioned medium from >2 and <0.5 were interpreted as significant. The whole sFLT-MH3924A cells significantly inhibited their proliferation as experiment was repeated revealing a high correlation of the indicated by diminished (methyl-[3H])-thymidine uptake (Bq/105 cells) signals in both experiments (r = 0.97). in the insoluble fraction after bFGF or VEGF treatment with P < 0.001.

sFLT-MH3924A (0.31 F 0.07) in comparison with WT- MH3924A (0.43 F 0.10; Fig. 2A). Similar changes were obtained

for the k2 value with 0.26 F 0.09 and 0.73 F 0.24 and for sFLTand WT tumors, respectively (P = 0.005; Fig. 2B). The vascular fraction showed no difference (Fig. 2C), whereas the fractional volume of distribution increased significantly (0.65 F 0.26 for WT and 1.26 F 0.24 for sFLT-expressing tumors with P = 0.001; Fig. 2D). The most commonly used vascular marker, a-actin, is expressed by mural cells of most arterioles and venules but not in capillaries (37). In tumors of ACI rats, the macrovascularization, as indicated by the a-actin immunoreactive area (measured in the center and in the periphery of the tumors), is similar in sFLT- MH3924A and WT-MH3924A tumors (Fig. 3A). Furthermore, we found that microvascularization, as determined by CD31 Fig. 1 Tumor growth after transplantation of WT-MH3924A, or sFLT- immunoreactivity, is unchanged in sFLT-MH3924A tumors in MH3924 cells in RNU rats. Points, mean (n = 5); bars, SD. sFLT- comparison with WT-MH3924A (Fig. 3B). The vascularization expressing tumors revealed showed a significantly decreased K1 (P = 0.035) and k2 (P < 0.005). identified by CD31 immunoreactivity was twice higher in the periphery in comparison with the center in both tumor types. sFLT Gene Transfer, Induction of Apoptosis, and Changes At 18 days after inoculation in RNU rats, in vivo growth of of Markers Associated with Oxidative Stress or Inflammation: sFLT-MH3924A tumors was reduced by 43% in comparison Terminal Deoxynucleotidyl Transferase–Mediated Nick End with WT-MH3924A (Fig. 1). Similar results were obtained with Labeling, CD11b, and Cyclooxygenase 2. Next, we examined ACI rats (data not shown). in situ apoptosis, inflammation, necrosis, proliferation, and Because sFLT has been described as a strong inhibitor of markers of oxidative stress inducing cells in WT-MH3924A and angiogenesis, we expected a significant difference in tissue sFLT-MH3924A tumors by histomorphometrical analyses. perfusion in tumors expressing this gene. Therefore, dynamic PET Immunohistochemical quantifications revealed that in 15 measurements with H2 O were done. The diameters of WT and sFLT-MH3924A tumors the density of apoptotic cells shows a sFLT-expressing tumors were not significantly different. The 17% (nonsignificant with P = 0.1) increase (Table 2). The pharmakokinetic analysis of the PET data showed that tumor proliferation rate as identified by proliferating cell nuclear perfusion, represented as the transport rate K1 (in mL Â mL antigen staining is also not significantly changed in comparison tissueÀ1 Â minuteÀ1), was significantly decreased (P = 0.035) in with the WT-MH3924A tumors (Table 2). In addition, the

Fig. 2 PET. Tumor perfusion [K 1, k 2, vascular fraction (VB), and fractional volume of distribution (DV) values] measured of WT-MH3924A, or sFLT-MH3924A in ACI rats; two-dimensional acquisi- tion (one-tissue compartment). sFLT-expressing tumors showed a significantly decreased K 1 (P = 0.035; A )andk 2 (P < 0.005; B). The vascular fraction showed no difference (C), whereas the fractional volume of distribution increased significantly (P = 0.001; D).

Because the transfer of selection markers such as the hygromycin gene and the selection procedure may cause changes in the genetic program, we analyzed the expression pattern in the tumor cell lines as well as in the tumors obtained after transplantation of these cell lines. In general, we found changes in the expression of genes related to signal transduction, cellular matrix, and apoptosis (Table 3).

DISCUSSION Transfer of antiangiogenic genes leading to local produc- tion of the therapeutic agent at the tumor site has been suggested as a new strategy of tumor therapy. In this study, we examined the effects of sFLT gene transfer into rat hepatoma on tumor growth, perfusion/vascularization, and apoptosis/inflammation/ proliferation. In vitro a significant inhibition of thymidine incorporation into the DNA of HUVECs was seen, when conditioned medium of sFLT producing cells was added, thus indicating an inhibition of endothelial cell proliferation. These data are consistent with recently published results showing that adenoviral transfer of the sFlt-1 gene in human embryo kidney 293 cells as well as a sFLT- expressing 293 embryonic kidney cell line (293-Flt1-3d) inhibited the in vitro growth of VEGF-stimulated HUVECs (38, 39). Furthermore, in vivo an inhibition of tumor growth in animals with sFLT-MH3924A tumors was observed. These data are in line with results by Ye et al. showing that inoculation of 293-Flt1-3d cells at a site remote to a follicular thyroid carcinoma tumor transplant significantly inhibited the growth of these tumors (39). As expected, we found a reduction of tumor 15 perfusion (decreased K1 and k2 values), measured by H2 O dynamic PET, in sFLT-MH3924A tumors. The fractional Fig. 3 Macrovascularization and microvascularization identified by a- volume of distribution increased significantly, indicating actin or CD31 staining. Immunohistochemical staining of Morris changes in the membrane permeability. Because the diameters hepatomas using anti-a-actin (A) or anti-CD31 (B) antibodies and of WT and sFLT-expressing tumors were not different, this computer-assisted morphometry. WT-MH3924A and sFLT-MH3924 difference is not due to partial volume effects. However, the tumors were stained with a-actin or CD31. Control immunoglobulins (data not shown) were used as negative controls. Immunoreactivities histologic findings revealed that the CD31-immunoreactive were quantified by analyzing five hot spots per tumor. Columns, mean area, as a marker for endothelial cells and microvascularization, (each group n = 6); bars, SD. Center, center of the tumor; per, tumor is not significantly changed in sFLT-expressing tumors in periphery. Note that there is no significant difference in the macro- comparison with WT hepatoma. The area of a-actin immuno- vascularization and microvascularization in the tumors of sFLT- reactive smooth muscle cells, as a marker for larger vessels MH3924A and WT-MH3924A. (e.g., arterioles), was also comparable between WT-MH3924A and sFLT-MH3924A. These data are consistent with the fact necrotic area was unchanged in sFLT-MH3924A in comparison that the vascular fraction as measured by PET remained with WT-MH3924A with 21.9 F 4.1% in WT and 21.7 F 5.4% unchanged. This may be due to the fact that the measurement in sFLT-expressing tumors. of perfusion is more sensitive for the detection of changes The percentage of CD11b immunoreactive macrophages, as major source of oxidative stress, decreased from 14.1 F F Table 2 Histochemical analyses of Morris hepatomas transplanted 1.0% in WT-MH3924A to 7.1 1.0% in sFLT-MH3924A into ACI rats: apoptosis, proliferation, oxidative stress, with P = 0.001. As a marker for inflammation, we used COX- and inflammatory cells 2 and found that in sFLT-MH3924A tumors the percentage of Variable WT-MH3924A sFLT-MH3924A P COX-2 immunoreactive cells were significantly reduced to 2 F F 18% (P = 0.036). MIF staining showed a 26% increase with TUNEL-positive cells/mm 414.31 27.92 482.74 31.81 NS % PCNA-positive cells 26.86 F 1.52 21.68 F 4.61 NS P < 0.001 (Table 2). % CD11b-positive cells 14.09 F 2.25 7.08 F 2.22 0.001 Effects of sFLT Gene Transfer on Expression of Other % COX-2-positive cells 10.68 F 3.44 1.93 F 0.56 0.03 Genes In vitro and In vivo: Gene Arrays. To assess general % MIF-positive cells 69.30 F 5.01 87.44 F 3.12 0.001 effects of sFLT gene transfer on the tumor cells in vitro and NOTE. Mean F SD (n = 6). in vivo the gene expression pattern was studied using arrays. Abbreviation: NS, not significant.

Table 3 Changes in gene expression in sFLT-expressing hepatomas Table 3 Continued Gene Tumor ratio Cell ratio Gene Tumor ratio Cell ratio Matrix associated dynein light intermediate chain 53/55 2.45 0.79 clone p12.3 tenascin 10.45 0.34 hepatic nuclear factor one (HNF1) 2.28 1.03 laminin 8.87 1.70 NonO/p54nrb homologue 2.28 2.43 tenascin-C 6.38 1.31 skeletal muscle h-tropomyosin and 2.24 1.00 fibronectin, exons 2b and 3a 4.18 1.58 fibroblast tropomyosin 1 tenascin-X 3.62 2.39 dithiolethione-inducible gene-1 (DIG-1) 2.22 2.72 fibronectin (fn-1) 2.70 0.63 silencer factor B 2.20 0.70 Apoptosis associated receptor for advanced glycosylation 2.15 0.79 ICE-like cysteine protease (Lice) 4.39 1.04 end products (RAGE) cyclin-dependent kinase 2-h 4.08 2.12 protein tyrosine phosphatase 2.11 0.68 Ca2+-independent phospholipase A2 3.28 0.29 nuclear serine/threonine protein kinase 2.09 2.33 Tumor necrosis factor receptor 2.74 2.00 NOTE. Ratios of modified versus WT tumors and cells are shown. a-fodrin (A2A) 2.42 1.31 LIM kinase 1 2.39 1.78 caspase 6 (Mch2) 2.29 2.23 induced by sFLT expression than the morphologic assessment TNF-a converting enzyme 2.20 1.08 of vascularization. In addition, sFLT may induce changes in the Signal transduction and stress vascular permeability (i.e., tight junctions consisting of SOD gene 5.14 0.46 occludins, claudins, and zonula occludens, which contribute PSD-95/SAP90-associated protein-4 3.90 1.77 G-protein beta-subunit 6 3.88 0.80 to the blood-tissue barrier) or the tumor microenvironment. protein kinase PASK 3.57 0.58 This interpretation is in line with the observed increase of the c-fos 3.35 1.25 fractional volume of distribution. adenylyl cyclase type VI 3.19 0.31 In this context, we investigated proliferation, apoptosis/ MEK5alpha-1 (MEK5) 3.12 1.72 heat shock protein 27 3.07 1.73 necrosis, and the presence of indicators of oxidative stress in the complement component C3 3.02 0.18 tumors. Immunohistochemical analyses of WT-MH3924 and scaffold attachment factor B 2.85 1.29 sFLT-MH3924A tumors showed no difference in the percentage phosphatidylinositol 4-kinase 2.73 1.58 of proliferating cells, as measured by proliferating cell nuclear c-jun for transcription factor AP-1 2.53 1.15 antigen staining. The percentage of apoptotic cells was increased S-adenosylmethionine synthetase 2.51 1.87 75 kDa glucose-regulated protein 2.46 0.85 in sFLT expressing tumors, although not significantly. These data choline kinase 2.41 0.79 are also in line with a most recent publication showing that sFLT- adenylyl cyclase type (IV) 2.38 0.74 expressing lung metastases from renal cell carcinoma revealed MEKK1 2.28 0.40 more apoptotic (TUNEL positive) cells per metastatic tumor chemokine CX3C 2.27 0.09 nodule than the control group (38). putative protein kinase C regulatory protein 2.26 1.25 + ischemia-responsive 94-kDa protein 2.26 1.85 CD11b macrophages are suggested to be the major source tyrosine hydroxylase 2.22 0.85 of oxidative stress (40) and their cellular infiltration into the insulin-like growth factor I receptor 2.18 0.73 tissue was found to correspond to an enhanced tumor growth pre-pro-complement C3 2.17 0.31 (41). Vice versa, because the percentage of CD11b+ macro- nuclear serine/threonine protein kinase 2.09 2.33 inositol polyphosphate 4-phosphatase 2.09 0.83 phages is diminished in sFLT-MH3924A tumors, their stimula- insulin-like growth 2.08 0.59 tory effect on tumor growth may be reduced. Further evidence is factor-binding protein (IGF-BP3) obtained by the decrease of COX-2 expression, as an Smad1 protein (Smad1) 2.05 0.50 inflammation marker, in sFLT-MH3924A tumors in comparison nucleolin 2.03 0.80 Metabolism/synthesis with WT-MH3924A. The involvement of COX-2 in tumor Dihydroorotase 5.15 2.95 growth and angiogenesis has been recently shown, because lecithin-cholesterol acyltransferase 4.45 2.54 treatment with celecoxib, a COX-2-specific inhibitor, reduced branched chain a-ketoacid 3.83 0.22 breast cancer progression and microvessel density (42). COX-2 dehydrogenase E1-a subunit derived prostaglandin E stimulated the expression of angiogenic putative peroxisomal 2,4-dienoyl-CoA 3.51 1.47 2 reductase (DCR-AKL) regulatory genes such as VEGF, Flt, Ang2, and Tie-2 in stearyl-CoA desaturase 2 2.80 0.42 mammary tumor cells isolated from COX-2 transgenic mice. fatty acid translocase/CD36 2.69 0.63 Consequently, COX-2 selective nonsteroidal anti-inflammatory hexokinase II 2.66 0.49 drugs reduced the expression of VEGF and FLT-1 (42, 43). The mevalonate kinase 2.54 0.69 fatty acid translocase/CD36 2.12 0.79 potential roles of the proinflammatory and carcinogenic cytokine steroid 5 a-reductase 2.03 1.20 MIF in regulating hepatoma cell migration and the expression of Miscellaneous angiogenic factors by hepatoma cells have not been studied yet plasminogen activator inhibitor 2 type A 3.63 2.00 (44). In colorectal tumors, the immunohistologically determined DNA binding protein C/EBP 3.02 1.69 MIF level is related to their levels of biological aggressiveness GABA-A receptor delta-subunit 2.76 0.74 transcription factor GATA-1 2.70 1.06 (45) and MIF is suggested to act as an autocrine factor that stimu gap junction protein, connexin (CXN-37) 2.56 2.87 lates angiogenesis and metastasis in hepatoma by promoting transcription factor UBF2 2.53 2.85 expression of angiogenic factors and migration of tumor cells receptor-linked protein tyrosine phosphatase 2.48 0.53 (44). Thus, a possible association between overexpression of myosin regulatory light chain isoform C 2.47 0.61 MIF and hepatocarcinogenesis is suggested (46). However, our

data show that although the percentage of MIF-positive cells is which have been shown to be increased (e.g., after experimental increased in sFLT-MH3924A in comparison with WT- ischemia; refs. 54, 55). We also found that hsp27 and scaffold MH3924A tumors, the microvascularization/macrovasculariza- attachment factor B (SAF-B) gene expression was increased in tion and proliferation rate are unchanged, but the growth of these sFLT-MH3924A tumors. These data are in accordance with tumors is inhibited. Because MIF is a modulator of prooxidative others suggesting an involvement of HAP (hnRNP A1– stress-induced apoptosis (47), it may also function in this way in associated protein), which was shown to be identical to SAF- sFLT-MH3924A. B, in the cellular response to heat shock, possibly at the RNA Additionally, changes in gene expression as a result of metabolism level (56). tumor defense reactions are possible. To assess general escape In line with an increase of histomorphometrically analyzed phenomena of the tumors by changes in the expression pattern, apoptotic (TUNEL positive) cells in sFLT-MH3924A tumors, gene array analyses were done. We analyzed the expression gene array analyses revealed an enhanced expression of fac- pattern in the tumor cell lines as well as in the tumors obtained tors, which are involved in apoptosis induction like Lice, after transplantation of these cell lines and found changes in a caspase-6, tumor necrosis factor receptor, and tumor necrosis variety of genes related to matrix proteins, apoptosis, signal factor a converting enzyme. Because apoptosis is a fast event, transduction, stress, or metabolism. A comparison with the the apoptotic cells are removed quickly which results in results of the gene expression pattern in RNA obtained in vitro difficulties of detection. This may explain that the changes (from the tumor cells) showed that most of these genes were observed in histology were not significant. A link between elevated only after transplantation of the cells into animals and, apoptosis induction in hepatoma cells and activation of Smads, therefore, represent changes induced in vivo. A contribution of mitogen-activated protein kinases, caspases has been shown nontumor cells such as macrophages or endothelial cells to the recently (57); however, until now there were no data available signal obtained is unlikely for two reasons: immunohistochem- about the influence of sFLT gene expression and induction of istry revealed that CD11b+ macrophages were reduced signifi- apoptosis by activation of these pathways. Because apoptosis is cantly in sFLT-expressing tumors which excludes a contribution an energy dependent process and hexokinase II mRNA of macrophages. Furthermore, the CD31 immunoreactivity was expression was significantly higher in metastatic liver cancers, unchanged which is evidence against a significant contribution glycolysis is suggested to be the predominant energy source of endothelial cells. under the hypoxic environment (58). In addition, we found an In sFLT-MH3924A tumors, several matrix proteins showed induction of the expression of several genes coding for an elevated expression, which may be a sign of increased enzymes involved in metabolism/synthesis of cholesterol/fatty oxidative stress: tenascin-C, tenascin-X, and laminin. Tenascins acids/triglycerides possibly indicating a high energy expendi- contribute to extracellular matrix structure and influence the ture or cell transformation. Furthermore, cyclin-dependent physiology of the cells in contact with the tenascin-containing kinases represent potentially promising molecular targets for environment. Tenascin-C expression is regulated by mechanical cancer therapeutic strategies, because cyclin-dependent kinase 2 stress and shows highest expression in connective tissue in seems to be involved in massive tumor cell apoptosis in vitro inflamed tissues, tumors, and wounds, where it may regulate cell (59). Thus, our data of enhanced expression of cyclin- morphology, growth, and migration by activating diverse dependent kinase 2-h may underline the involvement of this intracellular signaling pathways (48). Furthermore, an associa- enzyme in apoptosis. tion with stress-induced signaling has been described: in dermal For the strategy described in our experiments to be fibroblasts inflammatory cytokines or activation of the c-jun- effective, the target cancer cells need to depend on VEGF for NH2-kinase/stress-activated protein kinase-1 pathway by the tumor angiogenesis. Most authors, using this anti-VEGF addition of sphingomyelinase increased tenascin-C expression approach with sFLT, describe smaller lesions with fewer (49, 50). Laminin is a basement-membrane protein that increases microvessels, slower tumor growth and a longer survival time in liver fibrosis. Recent investigations have shown that of the animals after the transfer of the sFLT gene (17, 19, 20, diffusable CYP2E1-derived oxidative-stress mediators induce 23, 39). However, in all studies the tumors did not disappear synthesis of laminins by a transcriptional mechanism in hepatic completely. We here show that transfer of the sFLT gene in stellate cells (51) and activate the lectin complement pathway MH3924A cells reduces tumor growth and perfusion with no (52). A further hint for a modulation of stress-relevant variables significant change in vascularization. Because tumor angiogen- in sFLT-MH3924A tumors is the increased expression of stress- esis is known to be promoted by several cytokines or growth related genes such as genes of the c-jun-NH2-kinase pathway factors (FGF, VEGF, platelet derived endothelial cell growth (jun, fos, G-protein h-subunit 6), mitogen-activated protein factor, and interleukin 8) and many cancer cells produce kinase kinase kinase-1 (MEKK-1), an upstream activator of the multiple angiogenic factors, the simultaneous targeting of stress-activated protein kinase/c-Jun NH2-terminal kinase path- multiple factors may be more effective. Ciafre et al. used a way, or stress-activated protein kinase-3/p38 pathway (SAP90/ tricistronic retroviral vector to transfer two inhibitors of PSD-95), which are the most prominent changes in these angiogenesis, NH2-terminal fragment of rat prolactin and a tumors. c-jun-NH2-kinase has been described to mediate secreted form of human platelet factor 4 (60). Although in vitro responses to cardiac hypertrophy, ischemia/reperfusion injury endothelial cell locomotion and formation of an endothelial cell to the heart and kidney, and endothelial cell apoptosis caused by tube network were inhibited, in vivo effects on glioblastoma diabetic hyperglycemia (53). Additionally, we found other stress progression were not observed. They suggested to combine genes to be elevated in sFLT-MH3924A tumors, such as Hsp27, even more factors or to combine antiangiogenic factors with SOD, choline kinase, and insulin-like growth factor I receptors, radiation or chemotherapy (61–63).

CONCLUSION soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem Biophys Res Commun Our results suggest that transfer of sFLT in the present tumor 1996;226:324–8. model inhibits tumor growth and perfusion and induces changes 17. Goldman CK, Kendall RL, Cabrera G, et al. Paracrine expression of in the expression of multiple genes related to the matrix, signal a native soluble vascular endothelial growth factor receptor inhibits transduction, apoptosis and metabolism. Because these changes tumor growth, metastasis, and mortality rate. Proc Natl Acad Sci U S A in the gene expression pattern are observed mainly in tumor tissue 1998;95:8795–800. and not in vitro they represent reactions of the tumor to its 18. Kong HL, Hecht D, Song W, et al. Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the microenvironment. Furthermore, inhibition of endothelial cell extracellular domain of the flt-1 vascular endothelial growth factor proliferation in vitro was much stronger than the inhibition of receptor. Hum Gene Ther 1998;9:823–33. tumor growth and perfusion in vivo. This suggests that at least 19. Takayama K, Ueno H, Nakanishi Y, et al. Suppression of tumor some of the changes are part of tumor defense mechanisms for angiogenesis and growth by gene transfer of a soluble form of vascular survival in a less permissive microenvironment. endothelial growth factor receptor into a remote organ. Cancer Res 2000; 60:2169–77. 20. Mori A, Arii S, Furutani M, et al. Soluble Flt-1 gene therapy for ACKNOWLEDGMENTS peritoneal metastases using HVJ-cationic liposomes. Gene Ther 2000; We thank U. Schierbaum and K. Leotta for their help in performing 7:1027–33. the animal experiments. 21. Yang W, Arii S, Mori A, et al. sFlt-1 gene-transfected fibroblasts: a wound-specific gene therapy inhibits local cancer recurrence. Cancer Res REFERENCES 2001;61:7840–5. 1. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis 22. Kendall RL, Thomas KA. Inhibition of vascular endothelial cell and metastasis: correlation in invasive breast carcinoma. N Engl J Med growth factor activity by an endogenously encoded soluble receptor. Proc 1991;324:1–8. Natl Acad Sci U S A 1993;90:10705–9. 23. Hasumi Y, Mizukami H, Urabe M, et al. Soluble FLT-1 expression 2. Fontanini G, Bigini D, Vignati S, et al. Microvessel count predicts suppresses carcinomatous ascites in nude mice bearing ovarian cancer. metastatic disease and survival in non-small cell lung cancer. J Pathol Cancer Res 2002;62:2019–23. 1995;177:57–63. 24. Anderson H, Yap JT, Wells P, et al. Measurement of renal tumour 3. Freeman MR, Schneck FX, Gagnon ML, et al. Peripheral blood T and normal tissue perfusion using positron emission tomography in a lymphocytes and lymphocytes infiltrating human cancers express phase II clinical trial of razoxane. Br J Cancer 2003;89:262–7. vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res 1995;55:4140–5. 25. Herbst RS, Mullani NA, Davis DW, et al. Development of biologic markers of response and assessment of antiangiostatic activity in a 4. Polverini PJ, Leibovich SJ. Induction of neovascularization in vivo clinical trial of human recombinant endostatin. J Clin Oncol 2002;20: and endothelial cell proliferation in vitro by tumor-associated macro- 3804–14. phages. Lab Invest 1984;51:635–42. 26. Anderson HL, Yap JT, Miller MP, Robbins A, Jones T, Price P. 5. Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992;267:10931–4. Assessment of pharmacodynamic vascular response in a phase I trial of 6. Shibuya M. Role of VEGF-flt receptor system in normal and tumor combretastatin A4 phosphate. J Clin Oncol 2003;21:2823–30. angiogenesis. Adv Cancer Res 1995;67:281–316. 27. Roeckl W, Hecht D, Sztajer H, Waltenberger J, Yayon A, Weich HA. 7. Mahasreshti PJ, Kataram M, Wang MH, et al. Intravenous delivery of Differential binding characteristics and cellular inhibition by soluble adenovirus-mediated soluble FLT-1 results in liver toxicity. Clin Cancer VEGF receptors 1 and 2. Exp Cell Res 1998;241:161–70. Res 2003;9:2701–10. 28. Haberkorn U, Henze M, Altmann A, et al. Transfer of the human 8. Mahasreshti PJ, Navarro JG, Kataram M, et al. Adenovirus-mediated sodium iodide symporter gene enhances iodide uptake in hepatoma cells. soluble FLT-1 gene therapy for ovarian carcinoma. Clin Cancer Res J Nucl Med 2001;42:317–25. 2001;7:2057–66. 29. Haberkorn U, Kinscherf R, Kissel M, et al. Enhanced iodide 9. Kim KJ, Li B, Winer J, et al. Inhibition of vascular endothelial growth transport after transfer of the human sodium iodide symporter gene is factor-induced angiogenesis suppresses tumor growth in vivo. Nature associated with lack of retention and low absorbed dose. Gene Ther 1993;362:841–4. 2003;10:774–80. 10. Skobe M, Rockwell P, Goldstein N, Vosseler S, Fusenig NE. Halting 30. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human angiogenesis suppresses carcinoma cell invasion. Nat Med 1997;3: endothelial cells derived from umbilical veins. Identification by 1222–7. morphologic and immunologic criteria. J Clin Invest 1973;52:2745–56. 11. Saleh M, Stacker SA , Wilks AF. Inhibition of growth of C6 glioma 31. Haberkorn U, Altmann A, Kamencic H, et al. Glucose transport and cells in vivo by expression of antisense vascular endothelial growth apoptosis after gene therapy with HSV thymidine kinase. Eur J Nucl Med factor sequence. Cancer Res 1996;56:393–401. 2001;28:1690–6. 12. Cheng SY, Huang HJS, Nagane M, et al. Suppression of 32. Dimitrakopoulou-Strauss A, Strauss LG, Burger C. Quantitative glioblastoma angiogenicity and tumorigenicity by inhibition of endoge- PET studies in pretreated melanoma patients: a comparison of 6- 18 18 15 neous expression of vascular endothelial growth factor. Proc Natl Acad [ F]Fluoro-L-Dopa with F-FDG and O-water using compartment Sci U S A 1996;93:8502–7. and noncompartment analysis. J Nucl Med 2001;42:248–56. 13. Ramakrishnan S, Olson TA, Bautch VL, Mohanraj D. Vascular 33. Burchert W, Schellong S, van den Hoff J, et al. Oxygen-15-water endothelial growth factor-toxin conjugate specifically inhibits KDR/flk- PET assessment of muscular blood flow in peripheral vascular disease. 1-positive endothelial cell proliferation in vitro and angiogenesis in vivo. J Nucl Med 1997;38:93–8. Cancer Res 1996;56:1324–30. 34. Ohtake T, Kosaka N, Watanabe N, et al. Non-invasive method to 14. Millauer B, Shawver LK, Plate KH, Risau W, Ullrich A. obtain input function for measuring glucose utilization of thoracic and Glioblastoma growth inhibited in vivo by a dominant-negative FLK-1 abdominal organs. J Nucl Med 1991;32:1432–8. mutant. Nature 1994;367:576–9. 35. Sharp PE, LaRegina MC. The laboratory rat. Boca Raton, Boston, 15. Lin P, Sankar S, Shan S, et al. Inhibition of tumor growth by London, New York, Washington: CRC Press; 1998. targeting tumor endothelium using a soluble vascular endothelial growth 36. Kinscherf R, Wagner M, Kamencic H, et al. Characterization of factor receptor. Cell Growth Differ 1998;9:49–58. apoptotic macrophages in atheromatous tissue of humans and heritable 16. Kendall RL, CK Wang G, Thomas KA. Identification of a natural hyperlipidemic rabbits. Atherosclerosis 1999;144:33–9.

37. McDonald DM, Choyke PL. Imaging of angiogenesis: from the LAMg 1 promoter in hepatic stellate cells co-cultured with HepG2 microscope to clinic. Nat Med 2003;9:713–25. cells overexpressing cytochrome P450 2E1. J Biol Chem 2003;278: 38. Yoshimura I, Mizuguchi Y, Miyajima A, et al. Suppression of lung 15360–72. metastasis of renal cell carcinoma by the intramuscular gene transfer of a 52. Collard CD, Montalto MC, Reenstra WR, et al. Endothelial soluble form of vascular endothelial growth factor receptor I. J Urol oxidative stress activates the lectin complement pathway: role of 2004;171:2467–70. cytokeratin 1. Am J Pathol 2001;159:1045–54. 39. Ye C, Feng C, Wang S, et al. sFlt-1 gene therapy of follicular thyroid 53. Davis RJ. Signal transduction by the JNK group of MAP kinases. carcinoma. Endocrinology 2004;145:817–22. Cell 2000;103:239–52. 40. Mittal A, Elmets CA, Katiyar SK. CD11b+ cells are the major source 54. Das DK, Engelman RM, Kimura Y. Molecular adaptation of cellular of oxidative stress in UV radiation-irradiated skin: possible role in photo- defences following preconditioning of the heart by repeated ischaemia. aging and photocarcinogenesis. Photochem Photobiol 2003;77:259–64. Cardiovasc Res 1993;27:578–84. 41. Sluyter R, Halliday GM. Enhanced tumor growth in UV-irradiated 55. Matejka GL. Expression of GH receptor, IGF-I receptor and IGF-I skin is associated with an influx of inflammatory cells into the epidermis. mRNA in the kidney and liver of rats recovering from unilateral renal Carcinogenesis 2000;21:1801–7. ischemia. Growth Horm IGF Res 1998;8:77–82. 42. Chang SH, Liu CH, Conway R, et al. Role of prostaglandin E2- 56. Weighardt F, Cobianchi F, Cartegni L, et al. A novel hnRNP protein dependent angiogenic switch in cyclooxygenase 2-induced breast cancer (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in progression. Proc Natl Acad Sci U S A 2004;101:591–6. nuclear granules in response to heat shock. J Cell Sci 1999;112:1465–76. 43. Jones MK, Szabo IL, Kawanaka H, et al. von Hippel Lindau tumor 57. Kim BC, Van Gelder H, Kim TA, et al. Activin receptor-like suppressor and HIF-1a: new targets of NSAIDs inhibition of hypoxia- kinase-7 induces apoptosis through activation of MAP kinases in a induced angiogenesis. FASEB J 2002;16:264–6. Smad3-dependent mechanism in hepatoma cells. J Biol Chem 2004; 44. Ren Y, Tsui HT, Poon RT, et al. Macrophage migration inhibitory 279:28458–65. factor: roles in regulating tumor cell migration and expression of angio- 58. Yasuda S, Arii S, Mori A, et al. Hexokinase II and VEGF expression genic factors in hepatocellular carcinoma. Int J Cancer 2003;107:22–9. in liver tumors: correlation with hypoxia-inducible factor 1 a and its 45. Legendre H, Decaestecker C, Nagy N, et al. Prognostic values of significance. J Hepatol 2004;40:117–23. galectin-3 and the macrophage migration inhibitory factor (MIF) in 59. Chen W, Lee J, Cho SY, Fine HA. Proteasome-mediated destruction human colorectal cancers. Mod Pathol 2003;16:491–504. of the cyclin a/cyclin-dependent kinase 2 complex suppresses tumor cell 46. Akbar SM, Abe M, Murakami H, et al. Macrophage migration growth in vitro and in vivo. Cancer Res 2004;64:3949–57. inhibitory factor in hepatocellular carcinoma and liver cirrhosis; 60. Ciafre SA, Barillari G, Bongiorno-Borbone L, Wannenes F, relevance to pathogenesis. Cancer Lett 2001;171:125–32. Izquierdo M, Farace MG. A tricistronic retroviral vector expressing 47. Nguyen MT, Lue H, Kleemann R, et al. The cytokine macrophage natural antiangiogenic factors inhibits angiogenesis in vitro, but is not migration inhibitory factor reduces pro-oxidative stress-induced apoptosis. able to block tumor progression in vivo. Gene Ther 2002;9:297–302. J Immunol 2003;170:3337–47. 61. Lund EL, Bastholm L, Kristjansen PEG. Therapeutic synergy of 48. Chiquet-Ehrismann R, Tucker RP. Connective tissues: signalling by TNP-470 and ionizing radiation: effects on tumor growth, vessel tenascins. Int J Biochem Cell Biol 2004;36:1085–9. morphology and angiogenesis in human glioblastoma multiforme. Clin 49. Latijnhouwers MA, Pfundt R, de Jongh GJ, Schalkwijk J. Tenascin-C Cancer Res 2000;6:971–8. expression in human epidermal keratinocytes is regulated by inflamma- 62. Mishima K, et al. A peptide derived from the non-receptor binding tory cytokines and a stress response pathway. Matrix Biol 1998;17: region of urokinase plasminogen activator inhibits glioblastoma growth 305–16. and angiogenesis in vivo in combination with cisplatin. Proc Natl Acad 50. Latijnhouwers MA, de Jongh GJ, Bergers M, de Rooij MJ, Sci U S A 2000;97:8484–9. Schalkwijk J. Expression of tenascin-C splice variants by human skin 63. Bello L, et al. Low-dose chemotherapy combined with an cells. Arch Dermatol Res 2000;292:446–54. antiangiogenic drug reduces human glioma growth in vivo. Cancer Res 51. Nieto N, Cederbaum AI. Increased Sp1-dependent transactivation of 2001;61:7501–6.

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Kerstin Schmidt, Johannes Hoffend, Annette Altmann, et al.

Clin Cancer Res 2005;11:2132-2140.

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