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• W46378411 abstract "Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified ∼100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades. Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified ∼100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades. Low-grade, noninvasive urothelial tumors rarely progress to muscle invasive disease but ∼70% of these tumors recur.1Harnden P Parkinson MC Transitional cell carcinoma of the bladder: diagnosis and prognosis.Curr Diagnostic Pathol. 1996; 3: 109-121Abstract Full Text PDF Google Scholar This has led to the development of rigorous protocols for cystoscopic follow-up, because noninvasive urine-screening tests such as cytology have limited sensitivity and specificity and are observer-dependent. Diagnostic kits have been devised to overcome this problem, but perform only slightly better than cytology. For instance, the average published sensitivities and specificities are 60% and 77%, respectively, for the Bard bladder tumor antigen (BTA) test, which is based on the detection of human factor H-related protein, and 67% and 72%, respectively, for the NMP22 test, which relies on changes in nuclear matrix proteins.2Ross JS Cohen MB Biomarkers for the detection of bladder cancer.Adv Anat Pathol. 2001; 8: 37-45Crossref PubMed Scopus (13) Google Scholar The results are even poorer for low-grade tumors with sensitivities of less than 50% for both the Bard test and cytology.3Gutierrez Banos JL Henar Rebollo RM Antolin Juarez FM Garcia BM Usefulness of the BTA STAT test for the diagnosis of bladder cancer.Urology. 2001; 57: 685-689Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar A better understanding of the events associated with early tumor development may lead to the development of more sensitive and specific tests. One approach to identifying the genes involved would be to compare gene expression of normal urothelium and tumor tissue. However, most gene expression studies of clinical material have compared tumors of different stages and grades,4Gromova I Gromov P Celis JE bc10: a novel human bladder cancer-associated protein with a conserved genomic structure downregulated in invasive cancer.Int J Cancer. 2002; 98: 539-546Crossref PubMed Scopus (42) Google Scholar, 5Gromova I Gromov P Celis JE A novel member of the glycosyltransferase family, beta 3 Gn-T2, highly downregulated in invasive human bladder transitional cell carcinomas.Mol Carcinog. 2001; 32: 61-72Crossref PubMed Scopus (13) Google Scholar, 6Gromova I Gromov P Celis JE Identification of true differentially expressed mRNAs in a pair of human bladder transitional cell carcinomas using an improved differential display procedure.Electrophoresis. 1999; 20: 241-248Crossref PubMed Scopus (44) Google Scholar therefore concentrating on genes involved in tumor progression rather than initiation. One of the reasons for this may be that there is generally sufficient tumor mRNA for direct comparisons, whereas normal urothelium is composed of only a few cell layers, with a poor yield of mRNA. Cell lines have been used to circumvent the problem of mRNA abundance.7Li C Chen CC Yang TH Tzai TS Huang CH Liou JT Su IJ Tzeng CC Chiu AW Shieh B Establishment of a mini-gene expression database for bladder tumor.J Formos Med Assoc. 2002; 101: 104-109PubMed Google Scholar, 8Lee YG Macoska JA Korenchuk S Pienta KJ MIM, a potential metastasis suppressor gene in bladder cancer.Neoplasia. 2002; 4: 291-294Abstract Full Text PDF PubMed Scopus (140) Google Scholar Although findings from tumor cell lines can subsequently be validated on tissue samples and microarrays as elegantly shown by Sanchez-Carbayo and colleagues,9Sanchez-Carbayo M Socci ND Charytonowicz E Lu M Prystowsky M Childs G Cordon-Cardo C Molecular profiling of bladder cancer using cDNA microarrays: defining histogenesis and biological phenotypes.Cancer Res. 2002; 62: 6973-6980PubMed Google Scholar this strategy cannot be used for direct comparisons with normal cells, as many of the genes they express de novo in culture are also expressed by tumor cells.10Smith BA Kennedy WJ Harnden P Selby PJ Trejdosiewicz LK Southgate J Identification of genes involved in human urothelial cell-matrix interactions: implications for the progression pathways of malignant urothelium.Cancer Res. 2001; 61: 1678-1685PubMed Google Scholar A further difficulty in the use of fresh clinical material is that the morphological detail on frozen sections may be too poor to confidently distinguish between normal urothelium and low-grade dysplasia, a distinction that is difficult even on optimally fixed material.1Harnden P Parkinson MC Transitional cell carcinoma of the bladder: diagnosis and prognosis.Curr Diagnostic Pathol. 1996; 3: 109-121Abstract Full Text PDF Google Scholar, 11Nagy GK Frable WJ Murphy WM Classification of premalignant urothelial abnormalities. A Delphi study of the National Bladder Cancer Collaborative Group A.Pathol Annu. 1982; 17: 219-233PubMed Google Scholar, 12Richards B Parmar MK Anderson CK Ansell ID Grigor K Hall RR Morley AR Mostofi FK Risdon RA Uscinska BM Interpretation of biopsies of “normal” urothelium in patients with superficial bladder cancer. MRC Superficial Bladder Cancer Sub Group.Br J Urol. 1991; 67: 369-375Crossref PubMed Scopus (48) Google Scholar, 13Robertson AJ Beck JS Burnett RA Howatson SR Lee FD Lessells AM McLaren KM Moss SM Simpson JG Smith GD Observer variability in histopathological reporting of transitional cell carcinoma and epithelial dysplasia in bladders.J Clin Pathol. 1990; 43: 17-21Crossref PubMed Scopus (47) Google Scholar Finally, normal and dysplastic urothelium are often intermingled, hence the need for selective dissection. We therefore sought to develop a strategy to overcome these difficulties. We exploited the differential expression of cytokeratin 20 (CK20) in normal versus dysplastic urothelium14Harnden P Allam A Joyce AD Patel A Selby P Southgate J Cytokeratin 20 expression by non-invasive transitional cell carcinomas: potential for distinguishing recurrent from non-recurrent disease.Histopathology. 1995; 27: 169-174Crossref PubMed Scopus (73) Google Scholar, 15Harnden P Eardley I Joyce AD Southgate J Cytokeratin 20 as an objective marker of urothelial dysplasia.Br J Urol. 1996; 78: 870-875Crossref PubMed Google Scholar, 16Harnden P Mahmood N Southgate J Expression of cytokeratin 20 redefines urothelial papillomas of the bladder.Lancet. 1999; 353: 974-977Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar to objectively define areas of normal urothelial organization and maturation. Although CK20, a marker of terminal differentiation, can be negative in highly inflamed or regenerative urothelium, its pattern of expression in stable urothelium is consistent.15Harnden P Eardley I Joyce AD Southgate J Cytokeratin 20 as an objective marker of urothelial dysplasia.Br J Urol. 1996; 78: 870-875Crossref PubMed Google Scholar Superficial umbrella cells are positive and expression is also seen in occasional cells located in the intermediate layers. The latter are tear-shaped, tapering into a thin CK20-positive process extending toward the basement membrane, as is seen for superficially located umbrella cells (personal observation). This contrasts with the full-thickness expression seen in dysplasia/carcinoma in situ and in a high proportion of tumors,14Harnden P Allam A Joyce AD Patel A Selby P Southgate J Cytokeratin 20 expression by non-invasive transitional cell carcinomas: potential for distinguishing recurrent from non-recurrent disease.Histopathology. 1995; 27: 169-174Crossref PubMed Scopus (73) Google Scholar, 15Harnden P Eardley I Joyce AD Southgate J Cytokeratin 20 as an objective marker of urothelial dysplasia.Br J Urol. 1996; 78: 870-875Crossref PubMed Google Scholar although a small proportion of papillary tumors may be negative.14Harnden P Allam A Joyce AD Patel A Selby P Southgate J Cytokeratin 20 expression by non-invasive transitional cell carcinomas: potential for distinguishing recurrent from non-recurrent disease.Histopathology. 1995; 27: 169-174Crossref PubMed Scopus (73) Google Scholar, 16Harnden P Mahmood N Southgate J Expression of cytokeratin 20 redefines urothelial papillomas of the bladder.Lancet. 1999; 353: 974-977Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Using CK20-labeled slides as a reference, normal urothelium was isolated from the adjacent hematoxylin and eosin (H&E)-stained frozen sections using laser capture microdissection.17Best CJ Emmert-Buck MR Molecular profiling of tissue samples using laser capture microdissection.Expert Rev Mol Diagn. 2001; 1: 53-60Crossref PubMed Scopus (42) Google Scholar Tumor was identified by its papillary architecture as well as full-thickness CK20 expression. RNA was extracted and amplified and subtractive cDNA libraries were generated. Laser capture microdissection has been used to select tissues for analysis by DNA and cDNA microarrays,18Kitahara O Furukawa Y Tanaka T Kihara C Ono K Yanagawa R Nita ME Takagi T Nakamura Y Tsunoda T Alterations of gene expression during colorectal carcinogenesis revealed by cDNA microarrays after laser-capture microdissection of tumor tissues and normal epithelia.Cancer Res. 2001; 61: 3544-3549PubMed Google Scholar, 19Lin YM Furukawa Y Tsunoda T Yue CT Yang KC Nakamura Y Molecular diagnosis of colorectal tumors by expression profiles of 50 genes expressed differentially in adenomas and carcinomas.Oncogene. 2002; 21: 4120-4128Crossref PubMed Scopus (166) Google Scholar, 20Crnogorac-Jurcevic T Efthimiou E Nielsen T Loader J Terris B Stamp G Baron A Scarpa A Lemoine NR Expression profiling of microdissected pancreatic adenocarcinomas.Oncogene. 2002; 21: 4587-4594Crossref PubMed Scopus (191) Google Scholar, 21Ernst T Hergenhahn M Kenzelmann M Cohen CD Bonrouhi M Weninger A Klaren R Grone EF Wiesel M Gudemann C Kuster J Schott W Staehler G Kretzler M Hollstein M Grone HJ Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue.Am J Pathol. 2002; 160: 2169-2180Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 22Miura K Bowman ED Simon R Peng AC Robles AI Jones RT Katagiri T He P Mizukami H Charboneau L Kikuchi T Liotta LA Nakamura Y Harris CC Laser capture microdissection and microarray expression analysis of lung adenocarcinoma reveals tobacco smoking- and prognosis-related molecular profiles.Cancer Res. 2002; 62: 3244-3250PubMed Google Scholar or high-density oligonucleotide arrays23Luzzi 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 in other tumor types but we believe that this is the first report combining subtractive hybridization with laser capture microdissection. The expression of two of the genes that we identified by this method, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), was investigated at the protein level in our test case to validate our approach. We also examined a panel of urothelial tumors of different grades and stages for patterns of tissue expression. Finally we compared tissue expression of TATI to its level in urine, to further explore its potential as a noninvasive marker of disease. Human bladder specimens were obtained from 10 patients after informed consent. In theater, the specimens were orientated and mounted on metal disks in cryogenic embedding compound (Leica, Milton Keynes, UK), frozen on dry ice and stored in liquid nitrogen. Serial sections (9 μm) were prepared using a cryostat with a clean steel blade onto Superfrost plus glass slides. The slides were transferred immediately to −20°C, where they were kept desiccated and used for laser capture microdissection within a few days. The sample selected for subsequent analysis was from a patient who was presenting for the first time, and had therefore not received any previous intravesical treatment, and whose sample contained the greatest amount of normal urothelium and a well-defined World Health Organization (1999 classification24Epstein JI Amin MB Reuter VR Mostofi FK The World Health Organization/International Society of Urological Pathology Consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee.Am J Surg Pathol. 1998; 22: 1435-1448Crossref PubMed Scopus (1385) Google Scholar) low-grade, World Health Organization/International Society of Urologic Pathology (ISUP) (1998 classification25Mostofi FK Davis CJ Sesterhenn IA World Health Organization.Histological Typing of Urinary Bladder Tumours. ed 2. Springer-Verlag, Berlin1999Crossref Google Scholar) grade 1 papillary tumor. CK20 expression was restricted to the superficial cells in the normal urothelium, but the tumor showed full-thickness expression. Rapid immunostaining protocols for use with laser capture microdissection have been developed.26Fend F Emmert-Buck MR Chuaqui R Cole K Lee J Liotta LA Raffeld M Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.Am J Pathol. 1999; 154: 61-66Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar However because of the anticipated low yield of mRNA from normal urothelium and the concern about compromising its quality, sections for dissection were stained by a rapid method for H&E only, but every fifth section was labeled for CK20 to distinguish areas of normal and dysplastic urothelium. For H&E staining, all solutions were prepared using autoclaved diethyl pyrocarbonate-treated (0.1% v/v) double-distilled water. Thawed sections were immersed in 70% ethanol for 30 seconds, rinsed in diethyl pyrocarbonate-treated water, stained in Meyer's hematoxylin for 2 minutes, washed in diethyl pyrocarbonate-treated water for 30 seconds and blued in Scott's solution [2% (w/v) MgSO4 and 0.35% (w/v) Na2H2CO3] for 1 minute. Tissues were dehydrated through 75% and 95% ethanol (30 seconds each) and counterstained in eosin for 2 minutes. Finally, sections were washed for 1 minute in two changes each of 95% ethanol, absolute ethanol, and xylene, and air-dried. Frozen sections were labeled for CK20 expression. Briefly, sections were fixed in acetone for 2 minutes and air-dried. Endogenous avidin-binding activity was quenched using an avidin-biotin blocking kit (Vector Laboratories, Peterborough, UK), according to the manufacturer's instructions. Sections were incubated for 10 minutes with normal rabbit serum (10% v/v in Tris-buffered saline) to block nonspecific secondary antibody binding. Sections were incubated with primary antibody (clone CK20.3; LabVision, Newmarket, UK) for 60 minutes, followed by incubation with biotinylated rabbit anti-mouse immunoglobulins (DAKO, High Wycombe, UK) for 30 minutes. Finally, sections were incubated for a further 30 minutes with the streptavidin-biotin-horseradish peroxidase complex (ABC kit, DAKO), prepared according to the manufacturer's instructions. Sections were washed with Tris-buffered saline, pH 7.6, after each step and all incubations were performed at ambient temperature in a humidified atmosphere. Antibody binding was visualized after a peroxidase-activated 3,3′-diaminobenzidine (DAB; Sigma, Gillingham, UK) substrate reaction for 15 minutes and counterstained with hematoxylin, dehydrated, cleared, and mounted in DePeX. Microdissection was performed using a PixCell II laser capture microscope equipped with an infrared diode laser (Arcturus Engineering, Mountain View, CA), as described by others.26Fend F Emmert-Buck MR Chuaqui R Cole K Lee J Liotta LA Raffeld M Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.Am J Pathol. 1999; 154: 61-66Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 27Curran S McKay JA McLeod HL Murray GI Laser capture microscopy.Mol Pathol. 2000; 53: 64-68Crossref PubMed Scopus (129) Google Scholar, 28Emmert-Buck MR Bonner RF Smith PD Chuaqui RF Zhuang Z Goldstein SR Weiss RA Liotta LA Laser capture microdissection.Science. 1996; 274: 998-1001Crossref PubMed Scopus (2121) Google Scholar The laser power was 60 mW, the pulse width was 50 ms, the spot size was 30 μm, and in total ∼70,000 laser shots were needed to collect the RNA from 20 slides. Two strategies were followed. Either the selected areas were microdissected from the tissue, or where the area of interest made up the majority of the section, it was found most efficient to dissect and discard areas that were not of interest. In the latter case, sterile 25 μl “Easiseal” frames and coverslips (Hybaid Ltd., Ashford, UK) were used to encapsulate the tissue sections from which the RNA could then be extracted. The RNA from laser-captured material was extracted using the Purescript RNA isolation kit by pipetting the lysis solution directly on to the caps, pipetting up and down, transferring the product to a microfuge tube, and then following the manufacturer's recommended protocol (Flowgen, Lichfield, UK). RNA quality was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers for β2 microglobulin, which amplify a fragment of 123 bp (forward 5′-CTCGCGCTACTC-TCTCTTTCT-3′ and reverse 5′-TGTCGGATTGATGAAA-CCCAG-3′). Each RT-PCR reaction was performed using RNA from an individual tissue section. Whole ureter RNA was used as a positive control. To prepare cDNA, 120 ng of tumor and normal RNA was heated in diethyl pyrocarbonate-treated water containing oligo dT and RNAsin at 65°C for 5 minutes, 37°C for 10 minutes, and cooled on ice. A reaction buffer containing PCR buffer II (Perkin Elmer, Warrington, UK), 8 mmol/L MgCl2, 0.5 mmol/L dNTP, 1 pmol each of forward and reverse primers and 5 U MMLV reverse transcriptase (Pharmacia Biotech., St. Albans, UK) was added to the mix, which was incubated at 37°C for 60 minutes before denaturation at 75°C for 5 minutes. The conditions for the β2-microglobulin PCR were 96°C for 10 minutes, followed by 33 cycles of 96°C for 30 seconds, 60°C for 1 minute, 74°C for 1 minute, and a final extension of 74°C for 7 minutes, in the presence of 1.25 U Taq polymerase (Perkin Elmer). RT-negative controls, from which the RT was omitted, were included in all reactions. Quantification of RNA was performed by Northern blot analysis because this was highly sensitive and allowed conservation of the small samples. Approximately one-tenth of the total RNA from the laser-dissected tumor samples was electrophoresed in 1% (w/v) agarose gels containing 1.8% (v/v) formaldehyde and examined on a UV transilluminator to check for RNA degradation. As a standard, RNA prepared from a cell line was quantified on a spectrophotometer and serial dilutions were analyzed on the same agarose gel as the laser-dissected RNA. The RNA was transferred to a nitrocellulose membrane (Hybond N+ membrane; Amersham, Slough, UK) by capillary transfer and hybridized with a riboprobe complementary to 18S ribosomal RNA (Ambion, distributed by AMS Biotechnology Ltd., Abingdon, UK), which had been labeled with [α32P] CTP (Amersham) using the T7 labeling kit (Promega, Southampton, UK). The Rapid-hyb system (Amersham) was used for hybridization according to the manufacturer's instructions and the amount of hybridized probe was quantified on a phosphorimager (Molecular Imager FX System; Bio-Rad Laboratories, Hemel Hempstead, UK). One hundred ng of total RNA was used to prepare cDNA using the SMART cDNA synthesis amplification kit (Clontech, Basingstoke, UK). The RNA was not DNase-treated before the production of cDNA to minimize RNA loss and as the SMART cDNA method utilizes primers specific for mRNA. Subtractive hybridization was performed using the PCR Select Subtractive Hybridization kit (Clontech) based on the method of suppression subtractive hybridization developed by Diatchenko and colleagues.29Diatchenko L Lau YF Campbell AP Chenchik A Moqadam F Huang B Lukyanov S Lukyanov K Gurskaya N Sverdlov ED Siebert PD Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.Proc Natl Acad Sci USA. 1996; 93: 6025-6030Crossref PubMed Scopus (2730) Google Scholar This method selectively amplifies differentially expressed sequences, and the generation of high- and low-abundance sequences is equalized during the first hybridization. The PCR allows amplification of equalized differentially expressed sequences. Each step of the cDNA synthesis and subtractive hybridization procedure was monitored using the positive control samples provided by the manufacturer. We verified the efficiency of subtraction by PCR analysis by comparing GAPDH levels in subtracted and unsubtracted cDNA using the method and GAPDH primers provided by the manufacturer. cDNAs generated were cloned into the pGEM-T Easy vector using a ligation kit (Promega) and transformed into JM109 competent bacteria. Colonies selected at random were grown overnight and used to prepare plasmid DNA (Qiagen, Crawley, UK). The presence of an insert within the plasmid DNA was assessed by digestion using EcoRI and analyzed in 1% agarose gels. Sequencing of plasmid DNA insert was performed on an ABI Prism 377 sequencer using Big Dye terminator sequencing (Perkin Elmer). Each sequence was compared to the EMBL database. Twenty-eight tumors from 26 patients were investigated for the expression of 67LR and TATI. These patients had also given consent for urine testing. There were 18 noninvasive tumors (pTa grade 1, n = 4; grade 2, n = 11; grade 3, n = 3), 6 tumors invading the lamina propria (pT1 grade 2, n = 2; grade 3, n = 4), and 4 muscle invasive tumors, all of grade 3. Seven of the patients with noninvasive tumors and all of the patients with invasive tumors had no previous history of bladder cancer. Biopsies from 11 patients with no history of bladder cancer were also investigated. These patients had presented either with microscopic hematuria or symptoms of bladder dysfunction (we did not have ethical approval to perform biopsies in healthy volunteers). Four had histologically normal bladder mucosa, and the remainder had varying degrees of inflammation with Von Brunn's nest formation, cystitis cystica, or nonkeratinizing squamous epithelium. The monoclonal antibody clone MluC5 recognizes 67LR.30Buto S Ghirelli C Aiello P Tagliabue E Ardini E Magnifico A Montuori N Sobel ME Colnaghi MI Menard S Production and characterization of monoclonal antibodies directed against the laminin receptor precursor.Int J Biol Markers. 1997; 12: 1-5PubMed Google Scholar The monoclonal antibody against TATI does not cross-react with epidermal growth factor, with which TATI shares genomic and sequence homology.31Marchbank T Chinery R Hanby AM Poulsom R Elia G Playford RJ Distribution and expression of pancreatic secretory trypsin inhibitor and its possible role in epithelial restitution.Am J Pathol. 1996; 148: 715-722PubMed Google Scholar Immunostaining for CK20 (monoclonal clone 20.8, 1:20; DAKO) was performed to identify areas of normal urothelial differentiation in the flat mucosa as previously described.14Harnden P Allam A Joyce AD Patel A Selby P Southgate J Cytokeratin 20 expression by non-invasive transitional cell carcinomas: potential for distinguishing recurrent from non-recurrent disease.Histopathology. 1995; 27: 169-174Crossref PubMed Scopus (73) Google Scholar, 15Harnden P Eardley I Joyce AD Southgate J Cytokeratin 20 as an objective marker of urothelial dysplasia.Br J Urol. 1996; 78: 870-875Crossref PubMed Google Scholar, 16Harnden P Mahmood N Southgate J Expression of cytokeratin 20 redefines urothelial papillomas of the bladder.Lancet. 1999; 353: 974-977Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar As these were paraffin sections, trypsinization (10 minutes) was required for antigen retrieval. Immunohistochemistry on paraffin-embedded tissue was performed as previously described.32Martignone S Pellegrini R Villa E Tandon NN Mastroianni A Tagliabue E Menard S Colnaghi MI Characterization of two monoclonal antibodies directed against the 67 kDa high affinity laminin receptor and application for the study of breast carcinoma progression.Clin Exp Metastasis. 1992; 10: 379-386Crossref PubMed Scopus (59) Google Scholar, 33Playford RJ Hanby AM Quinn C Calam J Influence of inflammation and atrophy on pancreatic secretory trypsin inhibitor levels within the gastric mucosa.Gastroenterology. 1994; 106: 735-741Abstract PubMed Scopus (15) Google Scholar Briefly, 5-μm sections were dewaxed and rehydrated. Antigen retrieval was not required for MluC5 but was achieved by microwaving in 10 mmol/L of citrate buffer, pH 6.0, for 10 minutes for TATI. This was followed by blocking endogenous avidin-binding sites using an avidin/biotin blocking kit according to the manufacturer's instructions (Vector Laboratories). Sections were incubated sequentially in primary antibody (MluC5, 1:2000; TATI, 1:1000) biotinylated rabbit anti-mouse IgG secondary antibodies, followed by streptavidin/horseradish peroxidase ABC complex according to the manufacturer's instructions (DAKO), with washing between each step. Negative controls were performed by omission of the primary antibody. Positive controls were a malignant melanoma for MluC5 and duodenal mucosa for TATI. However, normal ureter was subsequently used (as for CK20), once the consistency of TATI expression in normal urothelium was established. Urine was collected before cystoscopy and tumor resection in 24 of the 26 patients. Urine samples were also collected from seven healthy volunteers and four patients with urinary tract infections or stones. Samples were placed on ice and a Complete Protease Inhibitor Cocktail tablet added. After sieving through a 100-μm nylon filter to remove cell debris, samples were centrifuged for 10 minutes at 800 × g at 4°C and the supernatants aliquoted and stored at −80°C until assayed. Soluble TATI concentrations were determined using a commercially available radioimmunoassay accordingto the manufacturer's instructions (Orion Diagnostica, Espoo, F" @default.
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• W46378411 date "2003-08-01" @default.
• W46378411 modified "2023-10-16" @default.
• W46378411 title "Identification of Genes Up-Regulated in Urothelial Tumors" @default.
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