| European Urology - Gene Expression in Normal Urothelium Depends on Location within the Bladder: A Possible Link to Bladder Carcinogenesis |
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| Wednesday, 19 July 2006 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Volume 50, Issue 2, Pages 290-301 (August 2006) 1. Introduction: Transformation of the normal bladder epithelium is accompanied by a plethora of molecular and biochemical changes [1], [2], [3]; the first of these genetic alterations may precede the diagnosis of cancer by several decades. Early studies of the bladder have revealed that more than 70% of primary bladder tumours arise in the area around the ureteric orifice; the remaining 30% occur in other regions of the bladder [4], [5]. Embryology of the bladder is complex; even though the trigone that encompasses the ureteric orifices has developed from the mesodermal Wolffian ducts, the remainder of the bladder is of endodermal origin [6]. In the resting state, the urothelium has an extremely low mitotic index, and remains quiescent until the need arises for proliferation. Previous research has shown deficiency in the nucleotide salvage pathway enzyme thymidine kinase (TK1) in the ureteric orifice compared to the dome (most distant); this could increase susceptibility to carcinogenesis [7]. To date, however, there has been no comprehensive explanation for this phenomenon.
In this study, microarray analysis was used on paired samples from normal ureteric orifice and dome bladder mucosa samples. Differences in genes or pathways between these two anatomically distinct sites could provide important clues about the mechanism that governs carcinogenesis in the bladder and possibly in other epithelial tissues. 2. Methods2.1. Tissue procurementEthical approval was granted by the University of Ulster, Northern Ireland, UK (project no. 98/3). The consenting patient group consisted of males aged 26–81 who were referred to the Belfast City Hospital Haematuria clinic. On cytoscopic examination, when no observable lesions were found, small samples of normal ureteric orifice and dome tissue were obtained by the cold cup method. Samples were stored in RNALater at 4°C until collection. 2.2. RNA extractionRNeasy Mini-kit (Qiagen, UK) was used according to the manufacturer's instruction to isolate total RNA from tissue specimens. The integrity of the RNA was verified by agarose gel electrophoresis and quantified with spectrophotometry. Subsequently, to minimise individual variation and obtain a “biological average”, RNA extracted from the ureteric orifice and dome biopsies of the 33 male patients was pooled and concentrated to 5μg/μl in a final volume of 20μl. Of this total RNA, 1μg was retained for further analysis by RT-PCR. 2.3. Microarray hybridisation and scanningThe hybridisation to the human UniGEM 2.0 microarray was performed by Incyte Genomics (St. Louis, Missouri, USA). Briefly, for this analysis, 100μg of RNA from each pooled ureteric orifice and dome samples were transcribed into cDNA labelled with Cy3 and Cy5 fluorescence, respectively. The samples were hybridised to the array slide, which consisted of 10,176 probes. These included 96 control probes (a variety of control elements arrayed in quadruplicate, including yeast fragments added to the two-probe labelling reactions in known amounts and increasing concentrations to control for signal sensitivity). Background was subtracted based on fluorescence from the control probes, and the ratio of Cy3:Cy5 was determined. Data from the UniGEM array were initially analysed with the commercially available GEMTools™ Software package (Incyte, Palo Alto, California, USA). Results were corroborated by the data normalisation/analysis methodology described here. 2.4. Data analysisAlthough only one cDNA microarray was conducted, collecting a pooled sample per bladder location from a cohort of 33 patients reduced interindividual variability and data noise. Additionally, applying normalisation of differential expression levels ensured the usability of the data. A normalisation factor was calculated as follows:   and  are both measures of hybridisation intensity for an observed ith gene. The Cy5 channel is scaled by the normalisation factor, so the normalised differential expression ratio can be denoted as:
  ) approaches zero, we can define Z-scores for each gene as  , where the differentially expressed genes, at a 95% confidence level, would be those with |Zi|≥1.96.
We used the Gene Ontology Consortium (www.geneontology.org/) to gather information on function, mapping, and expression of individual genes and ESTs and to identify mechanistic patterns. Genes with multiple roles were included in more than one category. 2.5. Complementary DNA synthesisTotal RNA (1μg) was reverse transcribed by Superscript II enzyme with 0.5μg oligo (dT)18 (Invitrogen, Paisley, UK). The reaction mixture was incubated at 42°C for 2h, then incubated at 72°C for 10min. The resulting cDNA was diluted tenfold. 2.6. Semiquantitative PCREach gene-specific primer set (Cdc25B, PKM, and TK1) was optimized to determine the exponential phase of PCR. PCR was performed in a final volume of 50μl that contained 1μl of cDNA, 0.4μM of each primer (Table 1), 0.2mM dNTPs, 1.5mM MgCl2, 1.25U Taq, 5μl of 10× PCR buffer (Invitrogen, Paisley, UK), and the remainder molecular-grade water. To ensure the fidelity of mRNA extraction and reverse transcription, we subjected samples to PCR amplification with oligonucleotide primers specific for the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and normalized.
2.7. Real-time PCRReal-time RT-PCR was performed on a LightCycler Thermal cycling and continuous monitoring of PCR products were performed with the Fast Start DNA Master SYBR Green I kit (Roche Diagnostics Ltd, Lewes, UK) that contained Taq-DNA polymerase, reaction buffer, dNTP mix, SYBR® Green I dye, and MgCl2. In a final volume of 20μl of master mix that contained 0.25μM of each primer (Table 1), 2μl cDNA was added. Seladin-1, PDGFra, and r28S amplification were carried out in triplicate for each sample. The cycling conditions were as follows: initial denaturation at 95°C for 15min, followed by 50 cycles at 95°C for 15s, 60°C for 30s, and 72°C for 30s. The emitted fluorescence was captured and analysed with the Lightcycler analysis software version 3.5. The crossing points (Ct), were determined by the second derivative algorithm and arithmetic baseline adjustment. Melting curve analysis enabled differentiation of the PCR products. Standard curves were constructed from tenfold serial dilutions of cDNA prepared from the Seladin-1, PDGFra, and r28S expressing T24 bladder cancer line by plotting the relation between the Ct and the logarithm of copy numbers of amplified products. Four standards (serial dilutions) and a negative control without template were included in each run. 3. Results3.1. Genes exhibit differential expression profiles in ureteric orifice versus dome cellsA UniGEM 2.0 microarray that contained 10,176 cDNA clones was used to examine the relative gene expression between ureteric orifice and dome mRNAs that were extracted from the bladder. We used a ratio-to-intensity (R-I) plot to assess the quality of our normalised gene expression distributions by inspecting the symmetry of the intensity-dependent effects. The hybridisation scatter plot and R-I plot with the values of Cy3-ureteric orifice and Cy5-dome show a very tight distribution pattern (Fig. 1a and b). The between-channels symmetry observed in both plots visually indicates the sufficiency of the applied normalisation method and ensured high quality and more balanced measures of differential-expression between the two bladder locations. This is indicated by the good balance correlation between the quantities of mRNA and the intensity in Cy3-Cy5 labelling.  Fig. 1. (a) The R-I plot depict log2-transformed normalized differential-expression levels (  ) as a function of  . (b) Scatter plots for Cy3-ureteric orifice and Cy5-hybridisation. Plots show the variation in fluorescent signal intensities between the ureteric orifice and dome. The parallel lines flanking the centre diagonal line highlight changes in expression of 2-, 5-, and 10-fold.Most points (Fig. 1a) are close to a differential expression value of 1 (ureteric orifice and dome show equal signal intensity) and are generally within the 1.5-fold range. A much smaller proportion of clones exhibits a ratio of Cy3:Cy5 of greater than 1.5-fold (Fig. 1b). In fact, when either a ratio of 2.0 or |Zi|≥1.96 was used as cut-off criteria there was a high similarity in expression and sensitivity with a very similar list of differentially expressed genes generated. The exception was only two genes different at the bottom end of the scored list. Therefore, the following criteria were used to define candidate genes that are differentially expressed between the ureteric orifice and the dome: (1) a ratio between Cy3 and Cy5 greater than 2.0; (2) signal intensity greater than 100; (3) signal to background ratio >2.5; and (4) signal size >40% of the spotting area. The application of these criteria revealed that 96.9% of the clones display a ureteric orifice to dome ratio, or dome to ureteric orifice ratio, of <2.0; the remaining clones show a ratio >2.0. Analysis of these genes revealed that 211 genes were upregulated and 101 downregulated (Table 2, Table 3, Table 4, Table 5). As seen in Table 4, most of the “increased” genes are involved in “metabolism” and “growth and development.” In contrast, most of the “decreased” genes are involved in “signal transduction” (Table 2).
3.2. RT-PCR analysis of differentially expressed genesWe selected five genes that were differentially expressed between the ureteric orifice and dome for RT-PCR analysis on the pooled sample to confirm the expression data from our microarray studies. Three of these genes—Cdc25B, TK1, and PKM—were elevated in cancer; PDGFra is often downregulated. In addition, Seladin-1 (aliases KIAA0018, 24-dehydrocholesterol reductase, DHCR24), the most highly upregulated gene on the array, was also selected even though it had no known function at the time of this study. Based on this information, we selected these genes for confirmation by RT-PCR. Consistent with the microarray data, Cdc25B, PKM, TK1, and Seladin-1 were all found to be elevated and PDGFra was confirmed to be downregulated in the ureteric orifice compared to the dome (Fig. 2). Therefore, the RT-PCR results obtained for these clones were comparable to the array result (Table 6) and confirmed the direction of fold change for all the samples where the microarray fold change was >2.0.  Fig. 2. A strong correlation between the levels of expression of five transcripts in the ureteric orifice versus the dome discovered by microarray analysis and confirmed by RT-PCR. Confirmation analysis by RT-PCR was carried out on five differentially expressed clones uncovered by microarray analysis. The results of RT-PCR were normalised to the expression of r28S or GAPDH in each sample, and expressed as fold change of the ureteric orifice over the expression of the dome.
4. DiscussionOne of the major applications of microarray is comparative gene expression analysis of cells that have various types of biological behaviour. Studies that involve normal tissue are extremely rare because obtaining normal, well-preserved surgical samples is difficult. To our knowledge this is the first report to examine two normal anatomically distinct regions of the bladder. In this study we obtained small-paired samples of bladder from the ureteric orifice and dome of 33 male volunteers (see Methods section for selection criteria). Equal amounts of isolated RNA from each sample were pooled to obtain two samples (ureteric orifice and dome). The main advantages of this approach were that it obviated the need to amplify signal from minute samples, which could create bias in the samples and lead to inaccuracies in expression differences; and it gave a biological average by removing noise that was generated by the normal genetic variation of individuals and left the most affected genes increased or decreased in the two samples. A recent study suggests that pooling samples can, in a single assay, provide results that summarize the expression of individual samples, and that independently derived pools can provide nearly identical measures of gene expression [8]. The disadvantage of this approach is the loss of information on individuals; however, since we were interested in the principle of predisposing genes in a population rather than in a single patient, we felt this approach was justified.This study provided a unique opportunity to make a comprehensive evaluation of the genetic background (10,176 genes) in these two areas that clearly illustrate differential sensitivity to carcinogenesis. Notably, 96.9% of all the genes on the array did not differ between the two regions of the bladder. Analysis revealed differential expression of 312 genes: 211 were upregulated in the ureteric orifice; 101 were downregulated. The genes were organised according to known cellular function and the main groups upregulated in the ureteric orifice were involved in “metabolism” and “growth and development.” This suggests that the ureteric orifice may have a faster turnover rate that may influence cancer predisposition by increasing the probability of mutations. Conversely, previous research that involves the bladder epithelium has reported a very low turnover rate [9], [10], which is consistent with the reported life span of normal urothelium of 200 days [11], [12]. However, these studies do not detail the precise region of the bladder that was examined, so any regional differences could have gone undetected. Five of the transcripts that showed differential expression were selected for further evaluation: four upregulated genes, Cdc25B, TK1, PKM, Seladin-1, and one downregulated gene, PDGFra. All genes showed good correlation between the microarray result and the evaluation by PCR (Table 6). Oncomine analysis of expression data indicated that Cdc25B is elevated in primary bladder tumours compared to other tumour types (p=6.1E-4) [13]. Cdc25B is a rate-limiting component of mitosis, and its overexpression can result in cells bypassing the S-phase replication checkpoint and entering mitosis prematurely regardless of the state of their DNA [14]. Cdc25B was overexpressed in the ureteric orifice compared to the dome, and Cdc25B could be an early marker for predisposition to bladder carcinogenesis. Human cytosolic TK1 is a salvage pathway enzyme for 2′-deoxythymidine 5′-triphosphate (dTTP) formation. Microarray analysis of TK1 levels indicated a sixfold increase in the ureteric orifice compared to the dome; RT-PCR confirmed this with a 7.5-fold elevation. Interestingly, analysis of microarray data with Oncomine revealed that TK1 is overexpressed in primary bladder tumours relative to other tumour types (p=0.003) [13]. Overexpression of TK1 promotes proliferation by providing dTTP for DNA replication and by attenuating the function of p21Waf1 in the presence of DNA damage [15]. Evidence suggests that some of the signals that link metabolism to cell proliferation may be provided by phosphometabolites [16], [17]. PKM, an isoform of pyruvate kinase [18], was upregulated in the ureteric orifice compared to the dome by sixfold. Shifts towards K-type isozyme, which reverses the development process, have been observed in neoplastic tissues such as brain tumour [19], breast cancer [20] and hepatoma [21]. The mechanism that causes the switch from the M-type to the K-type isoenzyme is currently unknown. PDGFraα is involved in migration, proliferation, cell contractile function, and alteration of cellular metabolic activities. PDGFra was downregulated in the ureteric orifice by 2.7-fold, and was confirmed by real time RT-PCR (2.5-fold). Oncomine analysis showed that PDGFra is decreased in tumours relative to normal bladder tissue (p=0.009) [22]. Cdc25B, TK1, PKM, and PDGFra have all been implicated in the pathogenesis of various cancers, unlike Seladin-1, whose role is yet to be defined. Seladin-1 was upregulated in the ureteric orifice samples on the array by 8.3-fold RT-PCR and 11.8-fold by RT-PCR. This gene was first discovered in patients with Alzheimer's disease, where decreased expression levels were associated with apoptosis and neuronal degeneration [23]. Its role in bladder epithelium is unknown. However, Seladin-1 maps to chromosome 1p33-p31.1 [23], [24], [25], a region that is noted for amplification in early stage bladder cancer [26]. Recent evidence suggests Seladin-1 is a regulator of cellular response to oncogenic and oxidative stress [23], and ablation of this gene in the presence of these stresses results in cellular transformation [27]. In contrast, elevated levels of Seladin-1 expression have been found in adenomas from patients who suffer from Cushing syndrome [28] as well as breast [29] and prostate [30] tumours. The significance of Seladin-1 upregulation in the ureteric orifice of the bladder is yet to be established; however, hypothesising that it may facilitate inhibition of apoptosis is tempting. This, coupled with the proposed increase in proliferation, could increase the probability of mutation and accumulation of aberrant cells. Identification of the early molecular alterations that contribute to carcinogenesis is a major goal in cancer research. Although a number of genetic alterations have been reported in urological cancers, there is still a lack of knowledge as to the initial molecular changes [31], [32]. This study has provided the first evidence of differential gene expression in two normal areas of human bladder mucosa that exist in the same physiological environment yet illustrate differential sensitivity to carcinogenesis. Of particular note is Seladin-1, whose significance in cancer is yet to be clarified and whose expression was significantly higher in the area of the bladder that is more prone to tumour development. Recent literature has implicated altered Seladin-1 expression levels in carcinogenesis [28], [29], [30]. Further work that investigates the levels of Seladin-1 in primary tumours may also be informative. In addition to the limited number of genes discussed here, our analysis identified a number of poorly characterised ESTs that are likely to represent novel genes of unknown function, much like Seladin-1, which was novel at the time of this study. Additional study of these genes may provide new insights into bladder cancer biology as well as other epithelial cancers types. References1. . Two molecular pathways to transitional cell carcinoma of the bladder. Cancer Res. 1994;54:784–788. 2. . Morphological and immunocytochemical characteristics of human tumor cell lines for use in a disease-oriented anticancer drug screen. Anticancer Res. 1992;12:1035–1053. 3. . Analysis of genetic alterations in normal bladder urothelium. Urology. 2003;62:1134–1138. 4. Melicow MM. Tumors of the bladder: a multifaceted problem. J Urol. 1974;112:467–478. 5. . The site of recurrence of non-infiltrating bladder tumours. Br J Urol. 1978;50:237–242. 6. Hutch J. Anatomy and physiology of the bladder, trigone and urethra. Butterworth; 1972;. 7. . Why do most primary bladder neoplasms first appear around the ureteric orifices?. Br J Urol. 1993;71:34–37. 8. Agrawal D, Chen T, Irby R, et al.. Osteopontin identified as lead marker of colon cancer progression, using pooled sample expression profiling. J Natl Cancer Inst. 2002;94:513–521. 9. . A light and electron microscopic study of the effects of a single dose of cyclophosphamide on various organs in the rat. I. The urinary bladder. Lab Invest. 1967;16:44–65. 10. . Growth factors in bladder wound healing. J Urol. 1997;157:2388–2395. 11. . The biology of bladder cancer. Ann R Coll Surg Engl. 1972;51:1–16. 12. . Histology and cell proliferation in human bladder tumors. An autoradiographic study. Cancer. 1974;33:115–126. 13. . Identifying distinct classes of bladder carcinoma using microarrays. Nat Gen. 2003;33:90–96. 14. . Cdc25B, Cdc25C differ markedly in their properties as initiators of mitosis. J Cell Biol. 1999;146:573–584. 15. . Interaction of human thymidine kinase 1 with p21(Waf1). Biochem J. 2001;356:829–834. 16. . The role of phosphometabolites in cell proliferation, energy metabolism, and tumor therapy. J Bioenerg Biomembr. 1997;29:315–330. 17. Kaplan O, Navon G, 18. Imamura K, Tanaka T. Pyruvate kinase isozymes from rat. Methods Enzymol. 1982;90 Pt E:150–165. 19. Van Veelen CW, Rijksen G, Van Ketel BA, Staal GE. The pyruvate kinase isoenzyme shift in human gliomas: a potential marker in the treatment of gliomas. Br J Neurosurg. 1988;2:257–263. 20. . Quantitative determination of the degree of differentiation of mammary tumors by pyruvate kinase kinetic analysis. Cancer Res. 1981;41:2019–2020. 21. . Isozymes of pyruvate kinase in liver and hepatomas of the rat. Cancer Res. 1974;34:1439–1446. 22. . Multiclass cancer diagnosis using tumour gene expression signatures. PNAS. 2001;98:15149–15154. 23. . The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer's disease-associated neurodegeneration and oxidative stress. J Neurosci. 2000;20:7345–7352. 24. . Prediction of the coding sequences of unidentified human genes. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1 (supplement). DNA Res. 1994;1:47–56. 25. . Mutations in the 3beta-hydroxysterol Delta24-reductase gene cause desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis. Am J Hum Genet. 2001;69:685–694. 26. . Detection of genetic alterations in bladder tumors by comparative genomic hybridization and cytogenetic analysis. Cancer Genet and Cytogenetics. 1999;110:87–93. 27. . Regulation of cellular response to oncogenic and oxidative stress by Seladin-1. Nature. 2004;432:640–645. 28. . The human homolog of Diminuto/Dwarf1 gene (hDiminuto): a novel ACTH-responsive gene overexpressed in benign cortisol-producing adrenocortical adenomas. J Clin Endocrinol Metab. 2001;86:5130–5137. 29. . Differentially expressed genes and estrogen receptor status in breast cancer. Int J Oncol. 2003;23:1425–1430. 30. . Identifying immunotherapeutic targets for prostate carcinoma through the analysis of gene expression profiles. Ann N Y Acad Sci. 2002;975:232–246. 31. Porkka KP, Visakorpi T. Molecular mechanisms of prostate cancer. Eur Urol. 2004;45:683–691. 32. Kim WJ. Genetic and epigenetic aspects of bladder cancer. J Cell Biochem. 2005;95:24–33Review. Sharon C. Dohertya, Stephanie R. McKeowna, Jesus A. Lopezb, Ian K. Walshc, Valerie J. McKelvey-Martina a Cancer and Ageing Research Group, Biomedical Sciences Research Institute, University of Ulster, Coleraine, Northern Ireland, UK BT52 1SA Accepted 10 January 2006 published online 25 January 2006.
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