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【摘要】
Bladder cancer transformation and immortalization require the inactivation of key regulatory genes, including TP53. Genotyping of a large cohort of bladder cancer patients (n = 256) using the TP53 GeneChip showed mutations in 103 cases (40.2%), the majority of them mapping to the DNA-binding core domain. TP53 mutation status was significantly associated with tumor stage (P = 0.0001) and overall survival for patients with advanced disease (P = 0.01). Transcript profiling using oligonucleotide arrays was performed on a subset of these cases (n = 46). Supervised analyses identified genes differentially expressed between invasive bladder tumors with wild-type (n = 24) and mutated TP53 (n = 22). Pathway analyses of top-ranked genes supported the central role of TP53 in the functional network of such gene patterns. A proteomic strategy using reverse phase arrays with protein extracts of bladder cancer cell lines validated the association of identified differentially expressed genes, such as gelsolin, to TP53 status. Immunohistochemistry on tissue microarrays (n = 294) revealed that gelsolin was associated with tumor stage and overall survival, correlating positively with TP53 status in a subset of these patients. This study further reveals that TP53 mutations are frequent events in bladder cancer progression and identified gelsolin related to TP53 status, tumor staging, and clinical outcome by independent high-throughput strategies.
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The nuclear protein Tp53 plays an essential role in the regulation of cell cycle and apoptosis, contributing to transformation and malignancy.1 Tp53 is a DNA-binding protein containing transcription, DNA binding, and oligomerization activation domains, functioning as a tumor suppressor.2,3 Mutants of TP53 that frequently occur in a number of different human cancers, including bladder cancer, fail to bind the consensus DNA-binding site and hence cause the loss of tumor suppressor activity.4 Alterations of the TP53 gene occurs both as germline mutations, such as in cancer-prone families with Li-Fraumeni syndrome, or somatic mutations in diverse human malignancies.5
TP53 is one of the proteins better characterized in cancer research with reported targets, regulators, and binding proteins. For example, targets regulated by TP53 include cell-cycle genes, such as p21, and anti-apoptotic genes, such as bax. Regulators of TP53 include ataxia telangiectasia mutated (ATM) and Chk2, whereas Abl1 and the adenomatous polyposis gene (APC) are among known binding TP53 proteins.6-8 However, little is known of the differential gene expression patterns of human tumors presenting wild-type TP53 compared with those with a mutant protein. With the advent of microarray technologies, characterization of TP53 sequences and gene expression profiles associated with TP53 status are available in a high-throughput manner. Bladder cancer is one of the tumors in which TP53 is altered with a high frequency, mutation rates being 40% in advanced stages of the disease.9-12 The present study was designed to identify targets that would differentiate patients presenting advanced disease with wild-type versus mutant TP53 (Figure 1 ). Gelsolin was selected as one on the genes located to chromosome 9q33, a frequently mutated locus in bladder cancer.9,13 Two proteomic approaches were used to evaluate the link of gelsolin with tumor progression and TP53 status. Immunohistochemical analyses on tissue arrays containing well-annotated bladder tumors and known TP53 status served to associate the expression of gelsolin with TP53, tumor stage, and survival. The differential expression of gelsolin among several bladder cancer cell lines of known TP53 alterations was evaluated by custom-made reverse phase arrays.
Figure 1. Experimental design. TP53 genotyping was performed on 256 bladder tumors using the TP53 sequencing arrays. Gene expression analyses using the U133A array were performed in a subset of 46 bladder tissues to identify targets differentially expressed in bladder cancer regarding their TP53 status (TP53 wild type, n = 24; and TP53 mutated, n = 22). Supervised methods identified 149 probes differentially expressed between those cases with either wild-type or mutated TP53. Two types of validation studies of the association of molecular profiles with TP53 status were performed. Immunohistochemical patterns were analyzed on tissue arrays containing 294 tumors, a subset of them of known TP53 and clinical outcome status. Proteomic reverse phase arrays were also performed on protein extracts of bladder cancer cell lines of known TP53 status.
【关键词】 proteomic profiles association gelsolin progression
Materials and Methods
TP53 Sequencing Analyses
DNA Extraction and Tissue Samples
Total DNA was extracted using a nonorganic method (Oncor, Gaithersburg, MD). Macrodissection of OCT-embedded tissue blocks was performed to ensure a minimum of 75% tumor cells.13 DNA quality was evaluated based on 260/280 ratios of absorbances. Specimens were collected under institutional review board approval. These tumors comprised 10 pTa, 32 pT1, 22 pT2, 175 pT3, and 15 pT4 specimens from patients with bladder cancer.
TP53 oligonucleotide array assay (GeneChip p53; Affymetrix, Santa Clara, CA). Purified DNA (100 ng) was subjected to multiplex-polymerase chain reactions (PCRs) amplifying exons 2 to11 simultaneously, using reagents supplied by the manufacturer (Affymetrix). Apart from the DNA, each PCR reaction contained 10 U of AmpliTaq Gold, PCR buffer II, 2.5 mmol/L MgCl2, 5 µl of the primer set, and 0.2 mmol/L each dNTP. The reaction was performed in a final volume of 100 µl. The PCR profile consisted of an initial heating at 95??C for 10 minutes, followed by 35 cycles of 95??C for 30 seconds, 60??C for 30 seconds, and 72??C for 45 seconds, with a final extension step at 72??C for 10 minutes. Forty-five µl of the PCR product was then fragmented by the addition of 0.25 U of fragmentation reagent (DNase I in 10 mmol/L Tris-HCl, pH 7.5, 10 mmol/L CaCl2, 10 mmol/L MgCl2, and 500 ml/L glycerol) along with 2.5 U of calf intestine alkaline phosphatase, 0.4 mmol/L ethylenediaminetetraacetic acid, and 0.5 mol/L Tris-acetate, and incubation at 25??C for 15 minutes, followed by heat inactivation at 95??C for 10 minutes. For labeling, 50 µl of the fragmented DNA was incubated at 37??C for 45 minutes with 10 µmol/L fluorescein-N6-dideoxy-ATP, 25 U of terminal transferase, and TdTase buffer in a total volume of 100 µl, followed by heat inactivation at 95??C for 10 minutes. The sample was hybridized to the chip in a volume of 0.5 ml containing 6x sodium chloride/sodium phosphate/EDTA (SSPE) buffer, 0.5 ml/L Triton X-100, 1 mg of acetylated bovine serum albumin, 2 nmol/L control oligonucleotide, and the labeled DNA sample. Hybridization was done in an oven with constant agitation at 45??C for 30 minutes. The chip was then washed on the wash station four times with 3x SSPE containing 0.05 ml/L Triton X-100. After washing, GeneChips were read using a confocal laser scanner, and data were aligned and analyzed. A reference from the control DNA supplied was also analyzed. This reference belonged to the same PCR round and was measured on the same batch of chips.14,15
Gene Profiling Using U133A GeneChips
Tissue Samples and RNA Extraction
Tumors belonging to patients with invasive bladder cancer (pT2+) were obtained by cystectomy or cystoprostatectomy at Memorial Sloan-Kettering Cancer Center. Specimens were collected under institutional review board approval of this institution. Macrodissection of OCT-embedded tissue blocks was performed to ensure a minimum of 75% tumor cells. Because of the high heterogeneity of muscle-invasive bladder tumors, this conservative cutoff of 75% would guarantee that tumor subpopulations would be representative enough to identify targets associated with TP53 status in cancer cells. Total RNA was extracted using TRIzol (Life Technologies, Rockville, MD) and purification with RNeasy columns (Qiagen, Valencia, CA). RNA quality was evaluated based on 260/280 ratios of absorbances and by gel analysis using an Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA).13 Selection of cases for oligonucleotide arrays focused on balancing numbers of cases with wild-type (n = 24) and mutant TP53 (n = 22), covering the most frequent TP53 mutations in the DNA-binding core domain in cases displaying all advanced disease (PT2+).
Labeling and Hybridization
Complementary DNA of the analyzed specimens was synthesized from 1.5 µg of total RNA using a T7-promoter- tagged oligo-dT primer. RNA target was synthesized by in vitro transcription and labeled with biotinylated nucleotides (Enzo Biochem, Farmingdale, NY). Labeled target was hybridized on GeneChip test 3 arrays (Affymetrix) to assess the quality of the sample before hybridizing onto the human genome U133A arrays including 22,283 probes representing known genes and expressed sequence tags (Affymetrix), as previously reported.13
GeneChip Analysis
Scanned image files were visually inspected for artifacts and analyzed using Affymetrix Microarray Suite 5.0 (MAS 5.0). Expression values of each array were multiplicatively scaled to have an average expression of 500 at least across the central 95% of all genes on the array. Signal was used as the primary measure of expression level, and detection was retained as a complementary measure.13
Immunohistochemical Analyses
Cell Lines, Tissue Arrays, and Immunohistochemistry
Cytospins of bladder cancer cell lines were obtained after centrifugation at low speed, 800 rpm, for 5 minutes.16 Four different bladder cancer microarrays were constructed in the Division of Molecular Pathology and used in this study. These arrays included a total of 294 primary transitional cell carcinomas (TCCs) of the bladder, belonging to patients recruited at Memorial Sloan-Kettering Cancer Center under institutional review board-approved protocols. A total of 93 non-muscle-invasive and 201 invasive TCC tumors were analyzed in these microarrays. These tumors corresponded to 34 grade 1, 69 grade 2, and 191 grade 3 lesions. One of these tissue microarrays comprised a cohort of four non-muscle-invasive lesions and 91 invasive tumors with annotated follow-up and known status of TP53. This array allowed clinical outcome assessment and evaluation of the associations of novel markers with TP53. Protein expression patterns of gelsolin were assessed at the microanatomical level on these tissue microarrays by immunohistochemistry using standard avidin-biotin immunoperoxidase procedures. Western blot assays were performed to address the specificity of the antibodies under study. We used a mouse monoclonal antibody against TP53 (1801) at 1:500 dilution (Calbiochem, San Diego, CA) and gelsolin at 1/1000 (Sigma, St. Louis, MO) on formalin-fixed/paraffin-embedded sections. The avidin-biotin immunoperoxidase technique was the immunohistochemical method applied. For specific epitopes on paraffin sections, we used antigen retrieval methods (0.01% citric acid for 15 minutes under microwave treatment) before incubation with primary antibodies or antiserum overnight at 4??C. Secondary antibodies were biotinylated horse anti-mouse or goat anti-rabbit antibodies (Vector Laboratories, Peterborough, UK), which were used at 1:500 or 1:1000 dilution, respectively. Diaminobenzidine was used as the final chromogen and hematoxylin as the nuclear counterstain. Two independent pathologists (C.C.-C. and N.B.), blinded to the TP53 or clinical status of the samples, reviewed immunohistochemical stainings.
Statistical Analysis
All TCCs (n = 294) were used for the analysis of association among gelsolin with clinicopathological variables and the expression patterns of TP53. The consensus value of the representative cores from each tumor sample arrayed was used for statistical analyses. The association of the expression of the selected targets with histopathological stage and tumor grade was evaluated using the nonparametric Wilcoxon-Mann-Whitney and Kruskall-Wallis tests. There is no consensus on the cutoffs of the immunohistochemical expression of the other markers, and thus they were analyzed as continuous variables.17 Survival analyses were performed taking the cutoffs of 20% for TP53 and 5% for gelsolin.
The associations of the markers identified in the DNA microarray analysis to outcome were also evaluated at the protein level using a subset of 95 TCCs of the bladder cases for which follow up was available. Overall-survival time was defined as the years elapsed between transurethral resection or cystectomy and death from disease (or the last follow-up date). Patients who were alive at the last follow-up or lost to follow-up were censored. For survival analysis, the association of marker expression levels with overall survival was analyzed using the Wald test, and the log-rank test was used to examine their relationship when different cutoffs were applied.17 Survival curves were plotted using the standard Kaplan-Meier methodology. Associations among gelsolin with TP53 were analyzed using Kendall??s b-test.17 Statistical analyses were performed using the SPSS statistical package (version 10.0).
Reverse-Phase Arrays
Bladder Cancer Cell Lines
Nine bladder cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD), grown, and collected under standard tissue culture protocols as previously reported.16 These cell lines were derived from TCCs of the bladder of early stage (RT4), low grade (5637), invasive (T24, J82, UM-UC-3, HT-1376, and HT-1197), and metastatic bladder tumors (TCCSUP), as well as a squamous cell carcinoma cell line (ScaBER). Bladder cancer cell lines were wild type for TP53 (RT4) or presented mutations in TP53 at the following exons: 4 (UM-UC-3, ScaBER), 5 (T24), 7 (HT-1376), 8 (5637 and J82), 10 (TCCSUP), and 11 (HT-1197).16
Protein Lysate Preparation
The bladder cancer cell lines were cultured, and protein extracts were prepared from them as previously described.16 In brief, cells were collected by scraping and washed three times with ice-cold phosphate-buffered saline. The resulting pellets were lysed in buffer containing 9 mol/L urea (Sigma), 4% 3--1-propanesulfonate (CHAPS; Calbiochem), 2%, pH 8.0 to 10.5, Pharmalyte (Amersham Pharmacia Biotech, Piscataway, NJ), and 65 mmol/L dithiothreitol (Amersham Pharmacia Biotech). After lysis, the samples were centrifuged briefly, and the supernatants were stored at C80??C.
Protein Lysate Array Design and Production
Arrays were prepared on nitrocellulose-coated glass slides (FAST Slides; Schleicher & Schuell, Keene, NH) by using a pin-in-ring format GMS 417 arrayer (Affymetrix) with four 500-µm-diameter pins. Because the samples were viscous, the pin-in-ring format was used to avoid problems because of clogging of quills. Five twofold serial dilutions were made from each lysate. Four 384-well microtiter plates (Genetix, New Milton, UK) were used to array 180 spots (plus eight spatial registration marks for use in image processing) on a 21 x 35-mm area of nitrocellulose membrane. The first dilution (fourfold) was made with buffer containing 5 mol/L urea, 2% Pharmalyte, pH 8 to 10.5, and 65 mmol/L dithiothreitol. The remaining dilutions were then made with buffer containing 6 mol/L urea, 1% CHAPS, 2% Pharmalyte, pH 8 to 10.5, and 65 mmol/L dithiothreitol. Hence, only the lysate concentration changed along each dilution series. The urea concentration was thus kept at 6 mol/L and the CHAPS concentration at 2%, to keep proteins in their denatured forms. To avoid evaporation in the microtiter plate during spotting, humidity in the array chamber was kept at 70 to 90% with a Vicks ultrasonic humidifier (Kaz, Hudson, NY).18
Detection of Specific and Total Protein on Microarrays
Each array was incubated with a specific primary antibody, which was detected by using the catalyzed signal amplification system (DAKO, Carpinteria, CA). Briefly, each slide was washed manually with deionized water to remove urea. Then, in an Autostainer universal staining system (DAKO), it was blocked with I-block (Tropix, Bedford, MA) and incubated with primary and secondary antibodies. Also in the Autostainer, it was then incubated with streptavidin-biotin complex, biotinyl tyramide (for amplification) for 15 minutes, streptavidin-peroxidase for 15 minutes, and 3,3'-diaminobenzidine tetrahydrochloride chromogen for 5 minutes. Between steps, the slide was washed with catalyzed signal amplification buffer. The signal was scanned with a Perfection 1200S scanner (Epson America, Long Beach, CA) with 256-shade gray scale at 600 dots per inch. For detection of total protein, arrays were stained with SYPRO ruby protein blot stain (Molecular Probes, Eugene, OR) and scanned with a FluorImager SI (Amersham Pharmacia Biotech) at 100-µm resolution. Gelsolin expression was quantified at a 1/1000 dilution using a mouse monoclonal antibody (Sigma), whereas mutated TP53 was measured using a mouse monoclonal at 1/500 (Calbiochem, Darmstadt, Germany). Spot images were converted to raw pixel values by a modified version of the P-SCAN (Peak Quantification with Statistical Comparative Analysis) software.18,19
Western Blotting
Murine monoclonal antibodies were screened for specificity by Western blotting with 20 µg of lysate protein per lane. Western blotting of gelsolin was performed at a 1/500 dilution using a mouse monoclonal antibody (Sigma). The running buffer contained 62.5 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, and 2.5% 2-mercaptoethanol. We used a 4 to 15% sodium dodecyl sulfate-polyacrylamide linear gradient gel (Tris?
【参考文献】
Levine AJ: The p53 tumor-suppressor gene. N Engl J Med 1992, 326:1350-1352
Levine AJ: P53, the cellular gatekeeper for growth and division. Cell 1997, 88:323-331
Sengupta S, Harris CC: p53: traffic cop at the crossroads of DNA repair and recombination. Nat Rev Mol Cell Biol 2005, 6:44-55
Wolff EM, Liang G, Jones PA: Mechanisms of disease: genetic and epigenetic alterations that drive bladder cancer. Nat Clin Pract Urol 2005, 2:502-510
Soussi T, Lozano G: p53 mutation heterogeneity in cancer. Biochem Biophys Res Commun 2005, 331:834-842
Cordon-Cardo C, Dalbagni G, Saez GT, Oliva MR, Zhang ZF, Rosai J, Reuter VE, Pellicer A: p53 mutations in human bladder cancer: genotypic versus phenotypic patterns. Int J Cancer 1994, 56:347-353
Markl IDC, Jones PA: Presence and location of TP53 mutation determines pattern of CDKN2A/ARF pathway inactivation in bladder cancer. Cancer Res 1998, 58:5348-5353
Zhao R, Gish K, Murphy M, Yin Y, Notterman D, Hoffman WH, Tom E, Mack DH, Levine AJ: Analysis of p53-regulated gene expression patterns using oligonucleotide arrays. Genes Dev 2000, 14:981-993
Dalbagni G, Presti J, Reuter V, Fair WR, Cordon-Cardo C: Genetic alterations in bladder cancer. Lancet 1993, 342:469-471
Lianes P, Orlow I, Zhang ZF, Oliva MR, Sarkis AS, Reuter VE, Cordon-Cardo C: Altered patterns of MDM2 and TP53 expression in human bladder cancer. J Natl Cancer Inst 1994, 86:1325-1330
Cordon-Cardo C, Sheinfeld J, Dalbagni G: Genetic studies and molecular markers of bladder cancer. Semin Surg Oncol 1997, 13:319-327
Cordon-Cardo C: p53 and RB: simple interesting correlates or tumor markers of critical predictive nature? J Clin Oncol 2004, 22:975-977
Sanchez-Carbayo M, Socci ND, Lozano J, Saint F, Cordon-Cardo C: Defining molecular profiles of poor outcome in patients with invasive bladder cancer using oligonucleotide microarrays. J Clin Oncol 2006, 24:778-789
Wikman FP, Lu ML, Thykjaer T, Olesen SH, Andersen LD, Cordon-Cardo C, Orntoft TF: Evaluation of the performance of a p53 sequencing microarray chip using 140 previously sequenced bladder tumor samples. Clin Chem 2000, 46:1555-1561
Lu ML, Wikman F, Orntoft TF, Charytonowicz E, Rabbani F, Zhang Z, Dalbagni G, Pohar KS, Yu G, Cordon-Cardo C: Impact of alterations affecting the p53 pathway in bladder cancer on clinical outcome, assessed by conventional and array-based methods. Clin Cancer Res 2002, 8:171-179
Sanchez-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, 62:6973-6980
Dawson-Saunders B, Trapp RG: Basic and Clinical Biostatistics, ed 2. 1994 Appleton and Lange, Norwalk
Carlisle AJ, Prabhu VV, Elkahloun A, Hudson J, Trent JM, Linehan WM, Williams ED, Emmert-Buck MR, Liotta LA, Munson PJ, Krizman DB: Development of a prostate cDNA microarray and statistical gene expression analysis package. Mol Carcinog 2000, 28:12-22
Nishizuka S, Charboneau L, Young L, Major S, Reinhold WC, Waltham M, Kouros-Mehr H, Bussey KJ, Lee JK, Espina V, Munson PJ, Petricoin E, III, Liotta LA, Weinstein JN: Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays. Proc Natl Acad Sci USA 2003, 100:14229-14234
Tanaka M, Mullauer L, Ogiso Y, Fujita H, Moriya S, Furuuchi K, Harabayashi T, Shinohara N, Koyanagi T, Kuzumaki N: Gelsolin: a candidate for suppressor of human bladder cancer. Cancer Res 1995, 55:3228-3232
Sakai N, Ohtsu M, Fujita H, Koike T, Kuzumaki N: Enhancement of G2 checkpoint function by gelsolin transfection in human cancer cells. Exp Cell Res 1999, 251:224-233
Celis A, Rasmussen HH, Celis P, Basse B, Lauridsen JB, Ratz G, Hein B, Ostergaard M, Wolf H, Orntoft T, Celis JE: Short-term culturing of low-grade superficial bladder transitional cell carcinomas leads to changes in the expression levels of several proteins involved in key cellular activities. Electrophoresis 1999, 20:355-361
Rao J, Seligson D, Visapaa H, Horvath S, Eeva M, Michel K, Pantuck A, Belldegrun A, Palotie A: Tissue microarray analysis of cytoskeletal actin-associated biomarkers gelsolin and E-cadherin in urothelial carcinoma. Cancer 2002, 95:1247-1257
Spinardi L, Rietdorf J, Nitsch L, Bono M, Tacchetti C, Way M, Marchisio PC: A dynamic podosome-like structure of epithelial cells. Exp Cell Res 2004, 295:360-374
作者单位：From the Tumor Markers Group,* Spanish National Cancer Center, the Division of Molecular Pathology, the Computational Biology Center, and the Department of Urology,¶ Memorial Sloan-Kettering Cancer Center, New York, New York; and the Center for Applied Proteomics and Molecular Medicine, George