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• W2155385977 abstract "Caldesmon is a cytoskeleton-associated protein which has not yet been related to neoplastic angiogenesis. In this study we investigated the expression of the caldesmon gene (CALD1) splicing variants and the protein expression level in glioma microvessels versus normal brain microvasculature. To exclude sources of splice variant expression from non-vascular components all possible cellular components present in control and glioma samples were pre-screened by laser-capture microdissection followed by RT-PCR before the cohort study. We discovered differential expression of the splicing variants of CALD1 in the tumor microvessels in contrast to normal brain microvasculature. Missplicing of exons 1, 1 + 4, and 1′ + 4 of the gene is exclusively found in glioma microvessels. To exclude the possibility that this missplicing results from splice-site mutations, mutation scanning was performed by a coupled in vitro transcription/translation assay (IVTT). No premature stop mutations were traced by the IVTT. The transcriptional changes consequently resulted in up-regulation at the protein expression level. The up-regulated expression of caldesmon was coincident with the down-regulated expression of tight junction proteins (occludin and ZO-1). The results support the notion that missplicing of the CALD1 gene in glioma microvasculature is an independent epigenetic event regulated at the transcriptional level. The event coexists with tight junction (TJ) breakdown of the endothelial cells in glioma microvasculature. The data reveal a novel mechanism contributing to dysfunctionality of glioma neovascularization. Caldesmon is a cytoskeleton-associated protein which has not yet been related to neoplastic angiogenesis. In this study we investigated the expression of the caldesmon gene (CALD1) splicing variants and the protein expression level in glioma microvessels versus normal brain microvasculature. To exclude sources of splice variant expression from non-vascular components all possible cellular components present in control and glioma samples were pre-screened by laser-capture microdissection followed by RT-PCR before the cohort study. We discovered differential expression of the splicing variants of CALD1 in the tumor microvessels in contrast to normal brain microvasculature. Missplicing of exons 1, 1 + 4, and 1′ + 4 of the gene is exclusively found in glioma microvessels. To exclude the possibility that this missplicing results from splice-site mutations, mutation scanning was performed by a coupled in vitro transcription/translation assay (IVTT). No premature stop mutations were traced by the IVTT. The transcriptional changes consequently resulted in up-regulation at the protein expression level. The up-regulated expression of caldesmon was coincident with the down-regulated expression of tight junction proteins (occludin and ZO-1). The results support the notion that missplicing of the CALD1 gene in glioma microvasculature is an independent epigenetic event regulated at the transcriptional level. The event coexists with tight junction (TJ) breakdown of the endothelial cells in glioma microvasculature. The data reveal a novel mechanism contributing to dysfunctionality of glioma neovascularization. Genome-wide analyses have revealed that 40 to 60% of human genes undergo alternative splicing.1Modrek B Lee C A genomic view of alternative splicing.Nat Genet. 2002; 30: 13-19Crossref PubMed Scopus (1045) Google Scholar Alternative splicing, therefore, seems to contribute considerably to enable the highly complex and diverse functions encoded by the human genome. Alternative splicing permits vertebrate pre-mRNA to be processed into multiple mRNAs differing in their precise combination of exon sequences, resulting in the encoding of different protein isoforms.2Grabowski PJ Black DL Alternative RNA splicing in the nervous system.Prog Neurobiol. 2001; 65: 289-308Crossref PubMed Scopus (280) Google Scholar Multiple modes of alternative splicing exist, such as alternative 5′ or 3′splice-site usage, differential inclusion or skipping of particular exons, mutually exclusion of exons, and more.3Smith CW Valcarcel J Alternative pre-mRNA splicing: the logic of combinatorial control.Trends Biochem Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar Importantly, alternative splicing is often tightly regulated in a cell type- or developmental stage-specific mode.3Smith CW Valcarcel J Alternative pre-mRNA splicing: the logic of combinatorial control.Trends Biochem Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar The essential nature of this process is underscored by the fact that misregulation (missplicing events) is often related to human disease.4Philips AV Cooper TA RNA processing and human disease.Cell Mol Life Sci. 2000; 57: 235-249Crossref PubMed Scopus (108) Google Scholar, 5Stoss O Stoilov O Daoud R Hartmann AM Olbrich M Stamm S Misregulation of pre-mRNA splicing that causes human diseases.Gene Ther Mol Biol. 2000; : 9-28Google Scholar, 6Stoilov P Meshorer E Gencheva M Glick D Soreq H Stamm S Defects in pre-mRNA processing as causes of and predisposition to diseases.DNA Cell Biol. 2002; 21: 803-818Crossref PubMed Scopus (70) Google Scholar The caldesmon gene (CALD1) is a single gene with transcriptional variance characterized by the recombination of different alternative splicing modes regulated by specific promoter activities.7Hayashi K Yano H Hashida T Takeuchi R Takeda O Asada K Takahashi E Kato I Sobue K Genomic structure of the human caldesmon gene.Proc Natl Acad Sci USA. 1992; 89: 12122-12126Crossref PubMed Scopus (73) Google Scholar The human CALD1 shares common structural and expressional properties through mammals.8Payne AM Yue P Pritchard K Marston SB Caldesmon mRNA splicing and isoform expression in mammalian smooth-muscle and non-muscle tissues.Biochem J. 1995; 305: 445-450Crossref PubMed Scopus (20) Google Scholar, 9Haruna M Hayashi K Yano H Takeuchi O Sobue K Common structural and expressional properties of vertebrate caldesmon genes.Biochem Biophys Res Commun. 1993; 197: 145-153Crossref PubMed Scopus (14) Google Scholar The gene is located on chromosome 7q33–34, consists of at least 15 exons and gives rise to two major classes of protein isoforms, ie, high molecular weight caldesmon (120 to 150 kd, h-CaD) and low molecular weight caldesmon (70 to 80 kd, l-CaD).7Hayashi K Yano H Hashida T Takeuchi R Takeda O Asada K Takahashi E Kato I Sobue K Genomic structure of the human caldesmon gene.Proc Natl Acad Sci USA. 1992; 89: 12122-12126Crossref PubMed Scopus (73) Google Scholar, 10Huber PA Caldesmon.Int J Biochem Cell Biol. 1997; 29: 1047-1051Crossref PubMed Scopus (99) Google Scholar The conserved regions of all isoforms encoded by exon 2, 3a, and 5 to 15 contain caldesmons’ capacity to bind to actin, tropomyosin, Ca (2+)-calmodulin, myosin, and phospholipids.11Bryan J Caldesmon: fragments, sequence, and domain mapping.Ann NY Acad Sci. 1990; 599: 100-110Crossref PubMed Scopus (12) Google Scholar The exons 1, 3b, and 4 are alternatively spliced. Exon 3b encodes the central α helix which is absent from l-CaD. h-CaD isoforms are restricted to fully differentiated smooth muscle cells (SMCs) and regulate the smooth muscle tone. l-CaD consists of at least four splicing variants (WI-38 l-CaDs I and II, Hela l-CaDs I and II) which are expressed via differential inclusion of the variable alternative spliced exons 1, 1′ and 4 of the gene.7Hayashi K Yano H Hashida T Takeuchi R Takeda O Asada K Takahashi E Kato I Sobue K Genomic structure of the human caldesmon gene.Proc Natl Acad Sci USA. 1992; 89: 12122-12126Crossref PubMed Scopus (73) Google Scholar The exons 1 and 1′ encode the short amino terminus specific for Hela l-CaDs and WI-38 l-CaDs or h-CaD, respectively.7Hayashi K Yano H Hashida T Takeuchi R Takeda O Asada K Takahashi E Kato I Sobue K Genomic structure of the human caldesmon gene.Proc Natl Acad Sci USA. 1992; 89: 12122-12126Crossref PubMed Scopus (73) Google Scholar The l-CaD isoforms are ubiquitously distributed in various cells and dedifferentiated SMCs. They play roles in the regulation of cell contractility, adhesion-dependent signaling, and cytoskeletal organization, influencing granule movement, hormone secretion, and reorganization of microfilaments during mitosis via mitosis-specific phosphorylation by cdc2 protein kinase.10Huber PA Caldesmon.Int J Biochem Cell Biol. 1997; 29: 1047-1051Crossref PubMed Scopus (99) Google Scholar, 11Bryan J Caldesmon: fragments, sequence, and domain mapping.Ann NY Acad Sci. 1990; 599: 100-110Crossref PubMed Scopus (12) Google Scholar, 12Yamakita Y Yamashiro S Matsumura F Characterization of mitotically phosphorylated caldesmon.J Biol Chem. 1992; 267: 12022-12029Abstract Full Text PDF PubMed Google Scholar The distinct functions of different cell types must involve different isoforms of caldesmon. However, the expression of the various CALD1 splicing variants and protein isoforms has only been investigated in a limited selection of normal human tissues.8Payne AM Yue P Pritchard K Marston SB Caldesmon mRNA splicing and isoform expression in mammalian smooth-muscle and non-muscle tissues.Biochem J. 1995; 305: 445-450Crossref PubMed Scopus (20) Google Scholar In human aorta, all splicing variants of the gene have been investigated. The expression was restricted to h-CaD (exon 1, 3b, and 4) and WI-38 l-CaD II (exon 1′).8Payne AM Yue P Pritchard K Marston SB Caldesmon mRNA splicing and isoform expression in mammalian smooth-muscle and non-muscle tissues.Biochem J. 1995; 305: 445-450Crossref PubMed Scopus (20) Google Scholar In glioma, microvascular proliferation or hyperplasia is a notorious event. Microvascular architecture and density in low-grade gliomas are similar to that in normal brain tissue. In anaplastic gliomas and glioblastomas however, microvascular hyperplasia such as glomeruloid and branching or sprouting proliferation, is a common event. Leakage of these vessels leads to perivascular edema and shows in neuroradiologic presentations of high-grade gliomas. The proliferated or hyperplastic vessels are dysfunctional in that there is a disruption of the blood-brain barrier. In a previous study we found the low molecular isoform of caldesmon (l-CaD) in the cerebrospinal fluid (CSF) of glioma patients.13Zheng PP Luider TM Rob P Avezaat CJJ van den Bent MJ Sillevis Smitt PAE Kros JM Identification of tumor-related proteins by proteomic analysis of cerebrospinal fluid from patients with primary brain tumors.J Neuropathol Exp Neurol. 2003; 62: 855-862PubMed Google Scholar It was noticed by immunohistochemistry on tissue sections of the very gliomas that the expression of caldesmon was restricted to the blood vessels while no immunopositivity was obtained in glial cells. In the present study, we further investigated the CALD1 splicing variants, focusing on l-CaD in tissue samples of 68 patients with gliomas. In order to localize the caldesmon protein in the tumors, immunohistochemistry was performed on tissue sections of the gliomas of the same patients from whom the CSF samples were used. The expression of caldesmon appeared to be restricted to the blood vessels and was not seen in glial cells. In addition, any possible or minor cellular components present in the normal controls and glioma samples were pre-screened by LCM/RT-PCR and immunohistochemistry. Missplicings in glioma microvessels are revealed by RT-PCR. The transcriptional changes consequently result in an up-regulated protein expression level. Alterations of splicing patterns could result from splice-site mutations via activation of cryptic splice-site usage.16Krawczak M Reiss J Cooper DN The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences.Hum Genet. 1992; 90: 41-54Crossref PubMed Scopus (1118) Google Scholar, 17Stamm S Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome.Hum Mol Genet. 2002; 11: 2409-2416Crossref PubMed Scopus (174) Google Scholar The phenotypic effects of such mutations on mRNA splicing often cause codon frame-shifts or single base substitution consequently resulting in premature termination codons.18Cooper TA Mattox W The regulation of splice-site selection, and its role in human disease.Am J Hum Genet. 1997; 61: 259-266Abstract Full Text PDF PubMed Scopus (230) Google Scholar, 19van der Luijt RB Khan PM Vasen HF Tops CM van Leeuwen-Cornelisse IS Wijnen JT van der Klift HM Plug RJ Griffioen G Fodde R Molecular analysis of the APC gene in 105 Dutch kindreds with familial adenomatous polyposis: 67 germline mutations identified by DGGE, PTT, and southern analysis.Hum Mutat. 1997; 9: 7-16Crossref PubMed Scopus (117) Google Scholar Such splice-site mutations account for at least 15% of point mutations causing disease in humans.16Krawczak M Reiss J Cooper DN The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences.Hum Genet. 1992; 90: 41-54Crossref PubMed Scopus (1118) Google Scholar, 20Nakai K Sakamoto H Construction of a novel database containing aberrant splicing mutations of mammalian genes.Gene. 1994; 141: 171-177Crossref PubMed Scopus (255) Google Scholar To rule out the presence of splice-site mutations resulting in the missplicing events of the CALD1 gene in our tumor cases, the samples were scanned by coupled in vitro transcription/translation assay (IVTT, also known as the protein truncation test (PTT)). The principle of IVTT is based on targeting mutations that generate truncated proteins induced by premature translation termination.21Bateman JF Freddi S Lamande SR Byers P Nasioulas S Douglas J Otway R Kohonen-Corish M Edkins E Forrest S Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsense-mediated mRNA instability.Hum Mutat. 1999; 13: 311-317Crossref PubMed Scopus (55) Google Scholar IVTT enables to pinpoint the site of a mutation, offers good sensitivity, and a low false-positive rate.22Den Dunnen JT Van Ommen GJ The protein truncation test: a review.Hum Mutat. 1999; 14: 95-102Crossref PubMed Scopus (45) Google Scholar Further tight junction proteins (occludin and ZO-1) were co-investigated in this study. Interestingly, the up-regulated expression of l-CaD resulting from CALD1 missplicing was coincident with the down-regulation of occludin and ZO-1, causing tight junction (TJ) breakdown of the endothelial cells in glioma microvasculature. The study was conducted on 68 snap-frozen specimens of glioma stored in the archives of the Department of Pathology, Erasmus Medical Center, Rotterdam, the Netherlands. Histopathological typing and grading of the tumors was performed on the corresponding paraffin sections based on the latest World Health Organization classification of tumors of the central nervous system.23Kleihues P Louis DN Scheithauer BW Rorke LB Reifenberger G Burger PC Cavenee WK The WHO classification of tumors of the nervous system.J Neuropathol Exp Neurol. 2002; 61: 215-225226–219Crossref PubMed Scopus (1550) Google Scholar Frozen section screening was to get rid of those samples with massive necrosis, hemorrhage, and contaminating normal tissues. Finally, the tumors analyzed included 26 glioblastomas, 23 oligodendrogliomas (among which 18 anaplastic oligodendrogliomas), and 19 pilocytic astrocytomas. Six samples of white matter obtained from patients without neurological or systemic disease served as controls. To determine the site and distribution of the l-CaD protein expression in the tumors and control samples, tissue sections were immunohistochemically stained with a monoclonal anti-l-CaD antibody at a 1:40 dilution (BD Biosciences). The immunohistochemical procedure was described previously.13Zheng PP Luider TM Rob P Avezaat CJJ van den Bent MJ Sillevis Smitt PAE Kros JM Identification of tumor-related proteins by proteomic analysis of cerebrospinal fluid from patients with primary brain tumors.J Neuropathol Exp Neurol. 2003; 62: 855-862PubMed Google Scholar The main purpose for the pre-screening experiments was to determine whether unfractional samples can be used for analysis. The white matter controls predominantly consist of glial cells and blood vessels with minor blood cell components. In glioma, the major cellular components are neoplastic glial cells (glioma cells) and hyperplastic or proliferated microvessels with possible or minor contaminating cells such as inflammatory cells, fibroblasts, and leptomeningeal cells. Normal glial cells and normal vessels were captured from the control samples (4 cases), while glioma cells and glioma vessels were captured from glioma cases (10 cases), respectively. Fibroblasts and leptomeningeal cells were captured from normal dura and arachnoid (two each from autopsy cases), respectively. Since all kinds of inflammatory cells are derived from transmigration of leukocytes from blood vessels into the brain tissue, 20 normal blood samples were used for screening of possible CALD1 expression in leukocytes. LCM was performed by using a Robot Microbeam laser microscope as the manufacturer instructed (P.A.L.M, Microlaser Technologies, Bernried, Germany). Frozen sections for LCM were prepared by using Rnase-free conditions. The used frozen tissue blocks were sectioned at 5 μm in cryostat, mounted on non-coated clean glass sliders, and stored at −80°C until use. The staining procedures of the sections were mainly based on the published protocol at http://pathbox.wustl.edu/∼tisscore/protocols.htm.24Luzzi V Holtschlag V Watson MA Expression profiling of ductal carcinoma in situ by laser-capture microdissection and high-density oligonucleotide arrays.Am J Pathol. 2001; 158: 2005-2010Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar A slight modification of the protocol was made by skipping “automation buffer” and using the stainer (HisGene, Arcturus) instead of Mayer's hemotoxylin and eosin. For Robot Microbeam laser microdissection, the tissue area of interest was selected and positioned (Figure 1, A, C, and E), and cut out using a focused, pulsed laser beam. The dissected areas were collected in the cap of a microcentrifuge tube containing 18 μl Trizol (Invitrogen) via laser pressure catapulting. The object of interest was catapulted off the slide using a high-energy, defocused, short-duration laser pulse (Figure 1, B, D, and F). The cap with the procured tissues was immediately placed on a microcentrifuge tube containing 200 μl Trizol (Invitrogen) and lysed by mixing for further RNA isolation. Total cellular RNA was extracted from the selected specimens using Trizol per the manufacturer's protocol (Invitrogen). First-strand complementary DNA (cDNA) was generated with an oligo (dT)23 primer and DuraScript reverse transcriptase (Sigma). The resulting cDNA was amplified by PCR using CALD1 specific primer sets spanning the splice sites of this gene toward all of the four splicing variants of l-CaD as described elsewhere.7Hayashi K Yano H Hashida T Takeuchi R Takeda O Asada K Takahashi E Kato I Sobue K Genomic structure of the human caldesmon gene.Proc Natl Acad Sci USA. 1992; 89: 12122-12126Crossref PubMed Scopus (73) Google Scholar A primer set for the CALD1, designed to amplify all of the splicing variants,25Yamamura H Yoshikawa H Tatsuta M Akedo H Takahashi K Expression of the smooth muscle calponin gene in human osteosarcoma and its possible association with prognosis.Int J Cancer. 1998; 79: 245-250Crossref PubMed Scopus (41) Google Scholar was used for initial examining the microdissected samples (the product size for l-CaD isoforms is 744 bp). Glyceraldehyde 3-phosphate dehydrogenase (GAPD) fragment26Shibuta K Begum NA Mori M Shimoda K Akiyoshi T Barnard GF Reduced expression of the CXC chemokine hIRH/SDF-1α mRNA in hepatoma and digestive tract cancer.Int J Cancer. 1997; 73: 656-662Crossref PubMed Scopus (49) Google Scholar was prepared by PCR as an internal control. All of the primers used in this study were commercially synthesized (Invitrogen). The amount of each RNA sample used was selected on the basis of identical amounts of GAPD cDNA amplified from each sample. At a 2-minute initial denaturation at 94°C, amplification conditions were as follows: 94°C for 15 seconds, 66°C (60°C for GAPD) for 30 seconds and 68°C for 1 minute for 36 cycles (40 cycles for the microdissected samples), and final extension at 68°C for 5 minutes. GAPD transcripts were amplified by 30 cycles (36 cycles for the microdissected samples). Negative controls, consisting of one sample without reverse transcriptase and one without template, were included in each experiment. The PCR products were resolved by electrophoresis on a 1% agarose gel containing ethidium bromide, and viewed under ultraviolet illumination. At least three experiments were performed for reproducibility. Total cellular protein was co-isolated and lysed during RNA isolation by Trizol according to the manufacturer's protocol (Invitrogen). Protein concentrations in the extracts were determined by the BCA protein assay (Pierce Chemical Co). Protein samples (15 μg/lane) were separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose. Electroblotting was performed on a nitrocellulose membrane in 25 mmol/L Tris and 192 mmol/L glycine containing 20% methanol. The membrane was then pre-treated with 5% skim milk in Tris-buffered saline with Tween (TBST) overnight at 4°C. The membrane was incubated with monoclonal anti-l-CaD antibody (BD Biosciences, dilution 1:1000) in TBST or anti-α,β-tubulin cocktail (Lab Vision, dilution 1:1000) in TBST for 1 hour at room temperature. The membrane was further incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Zymed Laboratory Inc., dilution 1:10,000) for 1 hour at room temperature. The peroxidase was finally activated with enhanced chemiluminescence (ECL kit, Amersham) and the immunoreactivity was visualized with Kodak X-Omat AR X-ray film. For quantitative analysis of l-CaD, the bands on films were scanned by using a UMAX PowerLook II scanner (Genomic Solutions, Huntingdon, UK) at 750 dpi. Densitometric analysis was performed with the ONE-Dscan software (Scanalytics, Billerica). Each experiment was repeated at least twice. The average change in band intensities was normalized against tubulin. The specimens used by a coupled in vitro transcription/translation assay (IVTT) were pre-treated by puromycin (200 μg/ml) (Sigma) for 6 hours at room temperature before RNA isolation. Puromycin is a translation inhibitor known to suppress nonsense-mediated mRNA decay (NMD), thus allowing mutation screening at the RNA level.27Andreutti-Zaugg C Scott RJ Iggo R Inhibition of nonsense-mediated messenger RNA decay in clinical samples facilitates detection of human MSH2 mutations with an in vivo fusion protein assay and conventional techniques.Cancer Res. 1997; 57: 3288-3293PubMed Google Scholar The PCR products suitable for IVTT analysis were generated with sense primers containing a T7 RNA polymerase promoter, a start condon, and the Kozak translation initiation sequence.28Hogervorst FB Cornelis RS Bout M van Vliet M Oosterwijk JC Olmer R Bakker B Klijn JG Vasen HF Meijers-Heijboer H Menko FH Cornelisce CJ de Dunnen JT Devilee P Van Ommen G-JB Rapid detection of BRCA1 mutations by the protein truncation test.Nat Genet. 1995; 10: 208-212Crossref PubMed Scopus (279) Google Scholar The transcripts were amplified by 40 cycles. Since the normal transcripts containing exons 1, 1 + 4, and 1′ + 4 were not detected in the control white matter, the corresponding transcripts used in IVTT were retrieved from normal stomach, prostate, and thyroid, respectively. The PCR product (2.5 μl) amplified from each transcript was incubated in the TNT/T7 non-radioactive coupled transcription and translation system (TNT Quick for PCR DNA, Promega) for 90 minutes at 30°C in a total volume of 50 μl in the presence of biotinylated lysine residues. The biotinylated lysine residues are incorporated into nascent protein during translation, eliminating the need for labeling with [35S] methionine or other radioactive amino acids, and allowing non-radioactive detection of protein synthesized in vitro by binding either streptavidin-alkaline phosphatase (streptavidin-AP) or streptavidin-horseradish peroxidase (streptavidin-HRP). In our experiments, the in vitro synthesized proteins were size-fractionated on 12% SDS-polyacrylamide mini-gels. The fractionated proteins were electroblotted onto nitrocellulose at a constant voltage of 100V for 60 minutes. Streptavidin-HRP binding (dilution 1:20,000) was used for visualization with enhanced chemiluminescence (ECL kit, Amersham) as the protocol (Promega). Cryostat sections (5 μm) were cut onto non-coated microscope slides (Menzel-Glaser). Slides were fixed in 100% ice-acetone at room temperature for 10 minutes and air-dried. After washes with phosphate-buffered saline (PBS), indirect immunofluorescence was carried out by using the anti-occludin polyclonal antibody (Zymed Laboratories Inc., dilution 1:100) and anti-ZO-1 polyclonal antibody (Zymed Laboratories Inc., dilution 1:100) with incubation for 1 hour at room temperature. The FITC-conjugated swine anti-rabbit IgG (Dako, dilution 1:100) was used for 1 hour at room temperature for visualization. Slides were washed with PBS, mounted in imselmount (Klinipath), and covered with Pertex and covering glass. A CCD video camera (Leica) mounted on a Leica (DMRXA) fluorescence microscope was used to capture digital images on a Leica computer (Q550 CW) running the software (Leica CW 4000 FISH Version V 1.0). For immunohistochemistry, 5-μm paraffin sections were cut onto aminopropyltriethoxysilane (APES)-coated glass slides (Knittel Glaser). Dewaxed sections were pre-treated in 0.1% pronase (Sigma) for 10 minutes at 37°C. After the pre-treatment, the slides were washed with PBS and incubated with anti-occludin polyclonal antibody (Zymed Laboratories Inc., dilution 1:100) for 2 hours at room temperature. The biotinylated goat anti-rabbit (Dako, dilution 1:200) was used for 1 hour at room temperature for visualization. Again the slides were washed in PBS and incubated with StreptABC complex/AP (Dako, dilution 1:100) for 1 hour at room temperature. After the slides were washed with PBS, enzyme detection was performed by using a solution of Tris-HCl (pH 8.0) containing 1% new fuchsin, 1% natriumnitriet, 0.03% napthol AS-MX phosphate (Sigma), and 0.025% levamisol (Acros) for 1 hour at room temperature. The slides were washed again with PBS, mounted in imselmount (Klininpath), and covered with Pertex and covering glass for examination under a light microscope. Immunohistochemistry showed that the expression of the caldesmon protein was restricted to the blood vessels. The immunoreactivity for the caldesmon protein was stronger in the hyperplastic microvessels of the gliomas as compared to normal brain microvasculature (Figure 2, A to E). The enhanced immunoreactivity was confirmed by immunoblotting analysis showing a higher expression of total l-CaD in the tumor samples (see below). At the light microscopical level no immunopositivity was observed in neoplastic and normal glial cells in the specimens. Neither the neoplastic, nor the normal, glial cells captured by LCM showed expression of CALD1 transcripts, in contrast to the positive controls (tumor vessels) (Figure 1, Figure 3).Figure 3Analysis of CALD1 transcript expression in purified glial cells by LCM. The used primer set was designed to amplify all of the splicing variants. Lane 1 represents the positive control (tumor vessels; Figure 1, E and F) showing CALD1 transcript expression (744 bp). Lane 2 (normal glial cells) and lane 3 (neoplastic glial cells) show no expression of CALD1 transcripts. The housekeeping gene (GAPD) was identically amplified in all of the samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The microdissected microvessels from the normal controls showed expression of exon 1′(WI-38 II) (Figure 4). Fibroblasts express CALD1 with restriction to exon 1′ and with immunopositivity of caldesmon (Figure 5). Further, no CALD1 expression was found in leptomeningeal cells by LCM/RT-PCR and immunohistochemistry (Figure 6). No CALD1 expression in leukocytes or inflammatory cells was detected (Figure 7).Figure 5Laser-capture microdissection of fibroblasts from normal dura. A: Normal dura before microdissection. B: The microdissected target cell population from A. C: Positive immunoreactivity for caldesmon is shown in the fibroblasts. D: The RT-PCR results from the micodissected fibroblasts. The fibroblasts show CALD1 expression with restriction to exon 1′.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Laser-capture microdissection of normal leptomeningeal cells from the arachnoid. A: Normal arachnoid before microdissection. B: The microdissected target leptomeningeal cell population from A. C: No immunoreactivity for caldesmon is seen in the leptomeningeal cells. D: The RT-PCR results from the micodissected leptomeningeal cells. PC, positive control; LC, leptomingeal cells. The housekeeping gene (GAPD) was identically amplified in the PC and LC. No expression of CALD1 transcript in leptomeningeal cells was found.View Large Image" @default.
• W2155385977 created "2016-06-24" @default.
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• W2155385977 date "2004-06-01" @default.
• W2155385977 modified "2023-10-18" @default.
• W2155385977 title "Differential Expression of Splicing Variants of the Human Caldesmon Gene (CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature" @default.
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