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• W2154778560 abstract "Gain and loss of chromosomal material is characteristic of bladder cancer, as well as malignant transformation in general. The consequences of these changes at both the transcription and translation levels is at present unknown partly because of technical limitations. Here we have attempted to address this question in pairs of non-invasive and invasive human bladder tumors using a combination of technology that included comparative genomic hybridization, high density oligonucleotide array-based monitoring of transcript levels (5600 genes), and high resolution two-dimensional gel electrophoresis. The results showed that there is a gene dosage effect that in some cases superimposes on other regulatory mechanisms. This effect depended (p < 0.015) on the magnitude of the comparative genomic hybridization change. In general (18 of 23 cases), chromosomal areas with more than 2-fold gain of DNA showed a corresponding increase in mRNA transcripts. Areas with loss of DNA, on the other hand, showed either reduced or unaltered transcript levels. Because most proteins resolved by two-dimensional gels are unknown it was only possible to compare mRNA and protein alterations in relatively few cases of well focused abundant proteins. With few exceptions we found a good correlation (p < 0.005) between transcript alterations and protein levels. The implications, as well as limitations, of the approach are discussed. Gain and loss of chromosomal material is characteristic of bladder cancer, as well as malignant transformation in general. The consequences of these changes at both the transcription and translation levels is at present unknown partly because of technical limitations. Here we have attempted to address this question in pairs of non-invasive and invasive human bladder tumors using a combination of technology that included comparative genomic hybridization, high density oligonucleotide array-based monitoring of transcript levels (5600 genes), and high resolution two-dimensional gel electrophoresis. The results showed that there is a gene dosage effect that in some cases superimposes on other regulatory mechanisms. This effect depended (p < 0.015) on the magnitude of the comparative genomic hybridization change. In general (18 of 23 cases), chromosomal areas with more than 2-fold gain of DNA showed a corresponding increase in mRNA transcripts. Areas with loss of DNA, on the other hand, showed either reduced or unaltered transcript levels. Because most proteins resolved by two-dimensional gels are unknown it was only possible to compare mRNA and protein alterations in relatively few cases of well focused abundant proteins. With few exceptions we found a good correlation (p < 0.005) between transcript alterations and protein levels. The implications, as well as limitations, of the approach are discussed. Aneuploidy is a common feature of most human cancers (1Lengauer C. Kinzler K.W. Vogelstein B. Genetic instabilities in human cancers.Nature. 1998; 17: 643-649Google Scholar), but little is known about the genome-wide effect of this phenomenon at both the transcription and translation levels. High throughput array studies of the breast cancer cell line BT474 has suggested that there is a correlation between DNA copy numbers and gene expression in highly amplified areas (2Pollack J.R. Perou C.M. Alizadeh A.A. Eisen M.B. Pergamenschikov A. Williams C.F. Jeffrey S.S. Botstein D. Brown P.O. Genome-wide analysis of DNA copy-number changes using cDNA microarrays.Nat. Genet. 1999; 23: 41-46Google Scholar), and studies of individual genes in solid tumors have revealed a good correlation between gene dose and mRNA or protein levels in the case of c-erb-B2, cyclin d1, ems1, and N-myc (3de Cremoux P. Martin E.C. Vincent-Salomon A. Dieras V. Barbaroux C. Liva S. Pouillart P. Sastre-Garau X. Magdelenat H. Quantitative PCR analysis of c-erb B-2 (HER2/neu) gene amplification and comparison with p185(HER2/neu) protein expression in breast cancer drill biopsies.Int. J. Cancer. 1999; 83: 157-161Google Scholar, 4Brungier P.P. Tamimi Y. Shuuring E. Schalken J. Expression of cyclin D1 and EMS1 in bladder tumors; relationship with chromosome 11q13 amplifications.Oncogene. 1996; 12: 1747-1753Google Scholar, 5Slavc I. Ellenbogen R. Jung W.H. Vawter G.F. Kretschmar C. Grier H. Korf B.R. myc gene amplification and expression in primary human neuroblastoma.Cancer Res. 1990; 50: 1459-1463Google Scholar). However, a high cyclin D1 protein expression has been observed without simultaneous amplification (4Brungier P.P. Tamimi Y. Shuuring E. Schalken J. Expression of cyclin D1 and EMS1 in bladder tumors; relationship with chromosome 11q13 amplifications.Oncogene. 1996; 12: 1747-1753Google Scholar), and a low level of c-myc copy number increase was observed without concomitant c-myc protein overexpression (6Sauter G. Carroll P. Moch H. Kallioniemi A. Kerschmann R. Narayan P. Mihatsch M.J. Waldman F.M. c-myc copy number gains in bladder cancer detected by fluorescence in situ hybridization.Am J. Pathol. 1995; 146: 1131-1139Google Scholar). In human bladder tumors, karyotyping, fluorescent in situ hybridization, and comparative genomic hybridization (CGH) 1The abbreviations used are: CGH, comparative genomic hybridization; TCC, transitional cell carcinoma; LOH, loss of heterozygosity; PA-FABP, psoriasis-associated fatty acid-binding protein; 2D, two-dimensional. 1The abbreviations used are: CGH, comparative genomic hybridization; TCC, transitional cell carcinoma; LOH, loss of heterozygosity; PA-FABP, psoriasis-associated fatty acid-binding protein; 2D, two-dimensional. have revealed chromosomal aberrations that seem to be characteristic of certain stages of disease progression. In the case of non-invasive pTa transitional cell carcinomas (TCCs), this includes loss of chromosome 9 or parts of it, as well as loss of Y in males. In minimally invasive pT1 TCCs, the following alterations have been reported: 2q−, 11p−, 1q+, 11q13+, 17q+, and 20q+ (7Richter J. Jiang F. Gorog J.P. Sartorious G. Egenter C. Gasser T.C. Moch H. Mihatsch M.J. Sauter G. Marked genetic differences between stage pTa and stage pT1 papillary bladder cancer detected by comparative genomic hybridization.Cancer Res. 1997; 57: 2860-2864Google Scholar, 8Richter J. Beffa L. Wagner U. Schraml P. Gasser T.C. Moch H. Mihatsch M.J. Sauter G. Patterns of chromosomal imbalances in advanced urinary bladder cancer detected by comparative genomic hybridization.Am. J. Pathol. 1998; 153: 1615-1621Google Scholar, 9Bruch J. Wohr G. Hautmann R. Mattfeldt T. Bruderlein S. Moller P. Sauter S. Hameister H. Vogel W. Paiss T. Chromosomal changes during progression of transitional cell carcinoma of the bladder and delineation of the amplified interval on chromosome arm 8q.Genes Chromosomes Cancer. 1998; 23: 167-174Google Scholar, 10Hovey R.M. Chu L. Balazs M. De Vries S. Moore D. Sauter G. Carroll P.R. Waldman F.M. Genetic alterations in primary bladder cancers and their metastases.Cancer Res. 1998; 15: 3555-3560Google Scholar, 11Simon R. Burger H. Brinkschmidt C. Bocker W. Hertle L. Terpe H.J. Chromosomal aberrations associated with invasion in papillary superficial bladder cancer.J. Pathol. 1998; 185: 345-351Google Scholar, 12Koo S.H. Kwon K.C. Ihm C.H. Jeon Y.M. Park J.W. Sul C.K. Detection of genetic alterations in bladder tumors by comparative genomic hybridization and cytogenetic analysis.Cancer Genet. Cytogenet. 1999; 110: 87-93Google Scholar). It has been suggested that these regions harbor tumor suppressor genes and oncogenes; however, the large chromosomal areas involved often contain many genes, making meaningful predictions of the functional consequences of losses and gains very difficult. In this investigation we have combined genome-wide technology for detecting genomic gains and losses (CGH) with gene expression profiling techniques (microarrays and proteomics) to determine the effect of gene copy number on transcript and protein levels in pairs of non-invasive and invasive human bladder TCCs. Bladder tumor biopsies were sampled after informed consent was obtained and after removal of tissue for routine pathology examination. By light microscopy tumors 335 and 532 were staged by an experienced pathologist as pTa (superficial papillary), grade I and II, respectively, tumors 733 and 827 were staged as pT1 (invasive into submucosa), 733 was staged as solid, and 827 was staged as papillary, both grade III. Tissue biopsies, obtained fresh from surgery, were embedded immediately in a sodium-guanidinium thiocyanate solution and stored at −80 °C. Total RNA was isolated using the RNAzol B RNA isolation method (WAK-Chemie Medical GMBH). poly(A)+ RNA was isolated by an oligo(dT) selection step (Oligotex mRNA kit; Qiagen). 1 μg of mRNA was used as starting material. The first and second strand cDNA synthesis was performed using the SuperScript® choice system (Invitrogen) according to the manufacturer's instructions but using an oligo(dT) primer containing a T7 RNA polymerase binding site. Labeled cRNA was prepared using the MEGAscrip® in vitro transcription kit (Ambion). Biotin-labeled CTP and UTP (Enzo) was used, together with unlabeled NTPs in the reaction. Following the in vitrotranscription reaction, the unincorporated nucleotides were removed using RNeasy columns (Qiagen). Array hybridization and scanning was modified from a previous method (13Wodicka L. Dong H. Mittmann M. Ho M.H. Lockhart D.J. Genome-wide expression monitoring in Saccharomyces cerevisiae.Nat. Biotechnol. 1997; 15: 1359-1367Google Scholar). 10 μg of cRNA was fragmented at 94 °C for 35 min in buffer containing 40 mm Tris acetate, pH 8.1, 100 mm KOAc, 30 mm MgOAc. Prior to hybridization, the fragmented cRNA in a 6× SSPE-T hybridization buffer (1 m NaCl, 10 mm Tris, pH 7.6, 0.005% Triton), was heated to 95 °C for 5 min, subsequently cooled to 40 °C, and loaded onto the Affymetrix probe array cartridge. The probe array was then incubated for 16 h at 40 °C at constant rotation (60 rpm). The probe array was exposed to 10 washes in 6× SSPE-T at 25 °C followed by 4 washes in 0.5× SSPE-T at 50 °C. The biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate, 10 μg/ml (Molecular Probes) in 6× SSPE-T for 30 min at 25 °C followed by 10 washes in 6× SSPE-T at 25 °C. The probe arrays were scanned at 560 nm using a confocal laser scanning microscope (made for Affymetrix by Hewlett-Packard). The readings from the quantitative scanning were analyzed by Affymetrix gene expression analysis software. Microsatellite Analysis was performed as described previously (14Christensen M. Sunde L. Bolund L. Orntoft T.F. Comparison of three methods of microsatellite detection.Scand. J. Clin. Lab. Invest. 1999; 59: 167-177Google Scholar). Microsatellites were selected by use of www.ncbi.nlm.nih.gov/genemap98, and primer sequences were obtained from the genome data base at www.gdb.org. DNA was extracted from tumor and blood and amplified by PCR in a volume of 20 μl for 35 cycles. The amplicons were denatured and electrophoresed for 3 h in an ABI Prism 377. Data were collected in the Gene Scan program for fragment analysis. Loss of heterozygosity was defined as less than 33% of one allele detected in tumor amplicons compared with blood. TCCs were minced into small pieces and homogenized in a small glass homogenizer in 0.5 ml of lysis solution. Samples were stored at −20 °C until use. The procedure for 2D gel electrophoresis has been described in detail elsewhere (15Celis J.E. Ostergaard M. Basse B. Celis A. Lauridsen J.B. Ratz G.P. Andersen I. Hein B. Wolf H. Orntoft T.F. Rasmussen H.H. Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas.Cancer Res. 1996; 56: 4782-4790Google Scholar, 16Celis J.E. Ratz G. Basse B. Lauridsen J.B. Celis A. Celis J.E. Cell Biology: A Laboratory Handbook. Vol. 3. Academic Press, Orlando, FL1994: 222-230Google Scholar). Gels were stained with silver nitrate and/or Coomassie Brilliant Blue. Proteins were identified by a combination of procedures that included microsequencing, mass spectrometry, two-dimensional gel Western immunoblotting, and comparison with the master two-dimensional gel image of human keratinocyte proteins; see biobase.dk/cgi-bin/celis. Hybridization of differentially labeled tumor and normal DNA to normal metaphase chromosomes was performed as described previously (10Hovey R.M. Chu L. Balazs M. De Vries S. Moore D. Sauter G. Carroll P.R. Waldman F.M. Genetic alterations in primary bladder cancers and their metastases.Cancer Res. 1998; 15: 3555-3560Google Scholar). Fluorescein-labeled tumor DNA (200 ng), Texas Red-labeled reference DNA (200 ng), and human Cot-1 DNA (20 μg) were denatured at 37 °C for 5 min and applied to denatured normal metaphase slides. Hybridization was at 37 °C for 2 days. After washing, the slides were counterstained with 0.15 μg/ml 4,6-diamidino-2-phenylindole in an anti-fade solution. A second hybridization was performed for all tumor samples using fluorescein-labeled reference DNA and Texas Red-labeled tumor DNA (inverse labeling) to confirm the aberrations detected during the initial hybridization. Each CGH experiment also included a normal control hybridization using fluorescein- and Texas Red-labeled normal DNA. Digital image analysis was used to identify chromosomal regions with abnormal fluorescence ratios, indicating regions of DNA gains and losses. The average green:red fluorescence intensity ratio profiles were calculated using four images of each chromosome (eight chromosomes total) with normalization of the green:red fluorescence intensity ratio for the entire metaphase and background correction. Chromosome identification was performed based on 4,6-diamidino-2-phenylindole banding patterns. Only images showing uniform high intensity fluorescence with minimal background staining were analyzed. All centromeres, p arms of acrocentric chromosomes, and heterochromatic regions were excluded from the analysis. The CGH analysis identified a number of chromosomal gains and losses in the two invasive tumors (stage pT1, TCCs 733 and 827), whereas the two non-invasive papillomas (stage pTa, TCCs 335 and 532) showed only 9p−, 9q22-q33−, and X−, and 7+, 9q−, and Y−, respectively. Both invasive tumors showed changes (1q22–24+, 2q14.1-qter−, 3q12-q13.3−, 6q12-q22−, 9q34+, 11q12-q13+, 17+, and 20q11.2-q12+) that are typical for their disease stage, as well as additional alterations, some of which are shown in Fig. 1. Areas with gains and losses deviated from the normal copy number to some extent, and the average numerical deviation from normal was 0.4-fold in the case of TCC 733 and 0.3-fold for TCC 827. The largest changes, amounting to at least a doubling of chromosomal content, were observed at 1q23 in TCC 733 (Fig. 1A) and 20q12 in TCC 827 (Fig. 1B).Fig. 1DNA copy number and mRNA expression level. Shown from left to right are chromosome (Chr.), CGH profiles, gene location and expression level of specific genes, and overall expression level along the chromosome. A, expression of mRNA in invasive tumor 733 as compared with the non-invasive counterpart tumor 335. B, expression of mRNA in invasive tumor 827 compared with the non-invasive counterpart tumor 532. The average fluorescent signal ratio between tumor DNA and normal DNA is shown along the length of the chromosome (left). The bold curve in the ratio profile represents a mean of four chromosomes and is surrounded by thin curves indicating one standard deviation. The central vertical line (broken) indicates a ratio value of 1 (no change), and the vertical lines next to it (dotted) indicate a ratio of 0.5 (left) and 2.0 (right). In chromosomes where the non-invasive tumor 335 used for comparison showed alterations in DNA content, the ratio profile of that chromosome is shown to the right of the invasive tumor profile. The colored bars represents one gene each, identified by the running numbers above the bars (the name of the gene can be seen at www.MDL.DK/sdata.html). The bars indicate the purported location of the gene, and the colors indicate the expression level of the gene in the invasive tumor compared with the non-invasive counterpart; >2-fold increase (black), >2-fold decrease (blue), no significant change (orange). The bar to the far right, entitled Expression shows the resulting change in expression along the chromosome; the colors indicate that at least half of the genes were up-regulated (black), at least half of the genes down-regulated (blue), or more than half of the genes are unchanged (orange). If a gene was absent in one of the samples and present in another, it was regarded as more than a 2-fold change. A 2-fold level was chosen as this corresponded to one standard deviation in a double determination of ∼1800 genes. Centromeres and heterochromatic regions were excluded from data analysis.View Large Image Figure ViewerDownload (PPT) The mRNA levels from the two invasive tumors (TCCs 827 and 733) were compared with the two non-invasive counterparts (TCCs 532 and 335). This was done in two separate experiments in which we compared TCCs 733 to 335 and 827 to 532, respectively, using two different scaling settings for the arrays to rule out scaling as a confounding parameter. Approximately 1,800 genes that yielded a signal on the arrays were searched in the Unigene and Genemap data bases for chromosomal location, and those with a known location (1096) were plotted as bars covering their purported locus. In that way it was possible to construct a graphic presentation of DNA copy number and relative mRNA levels along the individual chromosomes (Fig. 1). For each mRNA a ratio was calculated between the level in the invasive versus the non-invasive counterpart. Bars, which represent chromosomal location of a gene, were color-coded according to the expression ratio, and only differences larger than 2-fold were regarded as informative (Fig. 1). The density of genes along the chromosomes varied, and areas containing only one gene were excluded from the calculations. The resolution of the CGH method is very low, and some of the outlier data may be because of the fact that the boundaries of the chromosomal aberrations are not known at high resolution. Two sets of calculations were made from the data. For the first set we used CGH alterations as the independent variable and estimated the frequency of expression alterations in these chromosomal areas. In general, areas with a strong gain of chromosomal material contained a cluster of genes having increased mRNA expression. For example, both chromosomes 1q21-q25, 2p and 9q, showed a relative gain of more than 100% in DNA copy number that was accompanied by increased mRNA expression levels in the two tumor pairs (Fig. 1). In most cases, chromosomal gains detected by CGH were accompanied by an increased level of transcripts in both TCCs 733 (77%) and 827 (80%) (Table I, top). Chromosomal losses, on the other hand, were not accompanied by decreased expression in several cases, and were often registered as having unaltered RNA levels (Table I, top). The inability to detect RNA expression changes in these cases was not because of fewer genes mapping to the lost regions (data not shown).TABLE ICorrelation between alterations detected by CGH and by expression monitoringCGH alterationsTumor 733 vs. 335ConcordanceCGH alterationsTumor 827 vs. 532ConcordanceExpression change clustersExpression change clusters13 Gain10 Up-regulation77%10 Gain8 Up-regulation80%0 Down-regulation0 Down-regulation3 No change2 No change10 Loss1 Up-regulation50%12 Loss3 Up-regulation17%5 Down-regulation2 Down regulation4 No change7 No changeExpression change clustersTumor 733 vs. 335ConcordanceExpression change clustersTumor 827 vs. 532ConcordanceCGH alterationsCGH alterations16 Up-regulation11 Gain69%17 Up-regulation10 Gain59%2 Loss5 Loss3 No change2 No change21 Down-regulation1 Gain38%9 Down-regulation0 Gain33%8 Loss3 Loss12 No change6 No change15 No change3 Gain60%21 No change1 Gain81%3 Loss3 Loss9 No change17 No change Open table in a new tab In the second set of calculations we selected expression alterations above 2-fold as the independent variable and estimated the frequency of CGH alterations in these areas. As above, we found that increased transcript expression correlated with gain of chromosomal material (TCC 733, 69% and TCC 827, 59%), whereas reduced expression was often detected in areas with unaltered CGH ratios (Table I, bottom). Furthermore, as a control we looked at areas with no alteration in expression. No alteration was detected by CGH in most of these areas (TCC 733, 60% and TCC 827, 81%; see Table I, bottom). Because the ability to observe reduced or increased mRNA expression clustering to a certain chromosomal area clearly reflected the extent of copy number changes, we plotted the maximum CGH aberrations in the regions showing CGH changes against the ability to detect a change in mRNA expression as monitored by the oligonucleotide arrays (Fig. 2). For both tumors TCC 733 (p < 0.015) and TCC 827 (p < 0.00003) a highly significant correlation was observed between the level of CGH ratio change (reflecting the DNA copy number) and alterations detected by the array based technology (Fig. 2). Similar data were obtained when areas with altered expression were used as independent variables. These areas correlated best with CGH when the CGH ratio deviated 1.6- to 2.0-fold (Table I, bottom) but mostly did not at lower CGH deviations. These data probably reflect that loss of an allele may only lead to a 50% reduction in expression level, which is at the cut-off point for detection of expression alterations. Gain of chromosomal material can occur to a much larger extent. In TCC 733, several chromosomal areas exhibiting DNA amplification were preceded or followed by areas with a normal CGH but reduced mRNA expression (see Fig. 1, TCC 733 chromosome 1q32, 2p21, and 7q21 and q32, 9q34, and 10q22). To determine whether these results were because of undetected loss of chromosomal material in these regions or because of other non-structural mechanisms regulating transcription, we examined two microsatellites positioned at chromosome 1q25–32 and two at chromosome 2p22. Loss of heterozygosity (LOH) was found at both 1q25 and at 2p22 indicating that minor deleted areas were not detected with the resolution of CGH (Fig. 3). Additionally, chromosome 2p in TCC 733 showed a CGH pattern of gain/no change/gain of DNA that correlated with transcript increase/decrease/increase. Thus, for the areas showing increased expression there was a correlation with the DNA copy number alterations (Fig. 1A). As indicated above, the mRNA decrease observed in the middle of the chromosomal gain was because of LOH, implying that one of the mechanisms for mRNA down-regulation may be regions that have undergone smaller losses of chromosomal material. However, this cannot be detected with the resolution of the CGH method. In both TCC 733 and TCC 827, the telomeric end of chromosome 11p showed a normal ratio in the CGH analysis; however, clusters of five and three genes, respectively, lost their expression. Two microsatellites (D11S1760, D11S922) positioned close to MUC2, IGF2, and cathepsin D indicated LOH as the most likely mechanism behind the loss of expression (data not shown). A reduced expression of mRNA observed in TCC 733 at chromosomes 3q24, 11p11, 12p12.2, 12q21.1, and 16q24 and in TCC 827 at chromosome 11p15.5, 12p11, 15q11.2, and 18q12 was also examined for chromosomal losses using microsatellites positioned as close as possible to the gene loci showing reduced mRNA transcripts. Only the microsatellite positioned at 18q12 showed LOH (Fig. 3), suggesting that transcriptional down-regulation of genes in the other regions may be controlled by other mechanisms. 2D-PAGE analysis, in combination with Coomassie Brilliant Blue and/or silver staining, was carried out on all four tumors using fresh biopsy material. 40 well resolved abundant known proteins migrating in areas away from the edges of the pH gradient, and having a known chromosomal location, were selected for analysis in the TCC pair 827/532. Proteins were identified by a combination of methods (see “Experimental Procedures”). In general there was a highly significant correlation (p < 0.005) between mRNA and protein alterations (Fig. 4). Only one gene showed disagreement between transcript alteration and protein alteration. Except for a group of cytokeratins encoded by genes on chromosome 17 (Fig. 5) the analyzed proteins did not belong to a particular family. 26 well focused proteins whose genes had a know chromosomal location were detected in TCCs 733 and 335, and of these 19 correlated (p < 0.005) with the mRNA changes detected using the arrays (Fig. 4). For example, PA-FABP was highly expressed in the non-invasive TCC 335 but lost in the invasive counterpart (TCC 733; see Fig. 5). The smaller number of proteins detected in both 733 and 335 was because of the smaller size of the biopsies that were available.Fig. 5Comparison of protein and transcript levels in invasive and non-invasive TCCs. The upper part of the figure shows a 2D gel (left) and the oligonucleotide array (right) of TCC 532. The red rectangles on the upper gel highlight the areas that are compared below. Identical areas of 2D gels of TCCs 532 and 827 are shown below. Clearly, cytokeratins 13 and 15 are strongly down-regulated in TCC 827 (red annotation). The tile on the array containing probes for cytokeratin 15 is enlarged below the array (red arrow) from TCC 532 and is compared with TCC 827. The upper row of squares in each tile corresponds to perfect match probes; the lower row corresponds to mismatch probes containing a mutation (used for correction for unspecific binding). Absence of signal is depicted as black, and the higher the signal the lighter the color. A high transcript level was detected in TCC 532 (6151 units) whereas a much lower level was detected in TCC 827 (absence of signals). For cytokeratin 13, a high transcript level was also present in TCC 532 (15659 units), and a much lower level was present in TCC 827 (623 units). The 2D gels at the bottom of the figure (left) show levels of PA-FABP and adipocyte-FABP in TCCs 335 and 733 (invasive), respectively. Both proteins are down-regulated in the invasive tumor. To the right we show the array tiles for the PA-FABP transcript. A medium transcript level was detected in the case of TCC 335 (1277 units) whereas very low levels were detected in TCC 733 (166 units). IEF, isoelectric focusing.View Large Image Figure ViewerDownload (PPT) 11 chromosomal regions where CGH showed aberrations that corresponded to the changes in transcript levels also showed corresponding changes in the protein level (Table II). These regions included genes that encode proteins that are found to be frequently altered in bladder cancer, namely cytokeratins 17 and 20, annexins II and IV, and the fatty acid-binding proteins PA-FABP and FBP1. Four of these proteins were encoded by genes in chromosome 17q, a frequently amplified chromosomal area in invasive bladder cancers.TABLE IIProteins whose expression level correlates with both mRNA and gene dose changesProteinChromosomal locationTumor TCCCGH alterationTranscript alterationaAbs, absent; Pres, present.Protein alterationAnnexin II1q21733GainAbs to PresaAbs, absent; Pres, present.IncreaseAnnexin IV2p13733Gain3.9-Fold upIncreaseCytokeratin 1717q12-q21827Gain3.8-Fold upIncreaseCytokeratin 2017q21.1827Gain5.6-Fold upIncrease(PA-)FABP8q21.2827Loss10-Fold downDecreaseFBP19q22827Gain2.3-Fold upIncreasePlasma gelsolin9q31827GainAbs to PresIncreaseHeat shock protein 2815q12-q13827Loss2.5-Fold upDecreaseProhibitin17q21827/733Gain3.7-/2.5-Fold upbIn cases where the corresponding alterations were found in both TCCs 827 and 733 these are shown as 827/733.IncreaseProlyl-4-hydroxyl17q25827/733Gain5.7-/1.6-Fold upIncreasehnRNPB17p15827Loss2.5-Fold downDecreasea Abs, absent; Pres, present.b In cases where the corresponding alterations were found in both TCCs 827 and 733 these are shown as 827/733. Open table in a new tab Most human cancers have abnormal DNA content, having lost some chromosomal parts and gained others. The present study provides some evidence as to the effect of these gains and losses on gene expression in two pairs of non-invasive and invasive TCCs using high throughput expression arrays and proteomics, in combination with CGH. In general, the results showed that there is a clear individual regulation of the mRNA expression of single genes, which in some cases was superimposed by a DNA copy number effect. In most cases, genes located in chromosomal areas with gains often exhibited increased mRNA expression, whereas areas showing losses showed either no change or a reduced mRNA expression. The latter might be because of the fact that losses most often are restricted to loss of one allele, and the cut-off point for detection of expression alterations was a 2-fold change, thus being at the border of detection. In several cases, however, an increase or decrease in DNA copy number was associated with de novo occurrence or complete loss of transcript, respectively. Some of these transcripts could not be detected in the non-invasive tumor but were present at relatively high levels in areas with DNA amplifications in the invasive tumors (e.g. in TCC 733 transcript from cellular ligand of annexin II gene (chromosome 1q21) from absent to 2670 arbitrary units; in TCC 8" @default.
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• W2154778560 date "2002-01-01" @default.
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• W2154778560 title "Genome-wide Study of Gene Copy Numbers, Transcripts, and Protein Levels in Pairs of Non-invasive and Invasive Human Transitional Cell Carcinomas" @default.
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• W2154778560 doi "https://doi.org/10.1074/mcp.m100019-mcp200" @default.
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