Cytogenetic Analysis Of Urinary Bladder Cancer Combining Comparative Genomic Hybridization And Chromosome-specific Painting

【Objective】Urinary bladder cancer is one of the most common malignant neoplasmsin China. Development and progression of bladder cancer is driven by the malfunction ofspecific genes (i.e., overexpression of oncogenes or inactivation of tumor suppressor genes).Previous studies revealed multiple genetic and epigenetic alterations. However, the eventsinvolved in bladder cancer remain unknown. A number of technical difficulties limited thelarge-scale karyotypic analyses of bladder cancer. To survey entire chromosome alterations,we performed comparative genomic hybridization (CGH) in 26 urinary bladder cancers andone cancer cell line of the urinary bladder, and then further analyzed the aberrations of thecell line by multiplex fluorescence in situ hybridization. 【Methods】1) Tumor DNAand normal reference DNAwere isolated by Proteinase Kdigestion and phenol/chloroform extraction, tumor DNA was labeled by nick translationwith Biotin-16-dUTP, and reference placental DNA, with digoxigenin-11-dUTP. Equalamounts of tumor and reference DNA probes were mixed and precipitated with 20μgunlabeled Cot-1 DNA. The hybridization mixture was applied on slides and hybridized for48～72 hours at 37℃ in a moist chamber. After hybridization, the tumor DNA probes weredetected by avidin-FITC, biotinylated goat-anti-avidin, and a second layer of avidin-FITC.The reference DNA probes were detected by mouse anti-digoxigenin, rabbit anti-mouseantibody-TRITC, and sheep anti-rabbit antibody-TRITC. The chromosomes were II第三军医大学硕士学位论文counterstained with 4,6-diamidino-2-phenylindole (DAPI). Regions of gain or loss of DNAsequences were observed as changes in the ratio of the intensities of the two fluorochromesalong with the chromosome target. 0.8 and 1.2 were chosen as thresholds as theidentification of DNA copy number decreases (below 0.8) and increases (above 1.2). 2)Whole chromosome painting probes were labeled by PCR with Biotin-16-dUTP,digoxigenin-11-dUTP, or both Biotin-16-dUTP and digoxigenin-11-dUTP, separately.Equal amounts of painting probes were mixed and precipitated with 20μg unlabeled Cot-1DNA. Hybridization and post-hybridization were treated as described above.【Result】 All of the 26 urinary bladder cancer showed DNA-sequence copy numberchanges involving one or more regions of the genome. Recurrent genetic alterations includegains of chromosome 19q(12/26), 1p(11/26), 20q(10/26), 5p(9/26), 17q(9/26),3p(8/26), 16q(8/26), 21q(8/26), 3q(7/26), 8q(7/26), 9q(7/26), 11q(7/26),and losses of chromosome 9q (10/26), 9p (9/26), 17p(6/26), 18q(6/26) and 19p(6/26).High-level amplifications were on chromosomes 3p, 5p, 3q and 17q in some cases. Theincidence of chromosome DNA losses was lower than previously reported. Translocationsof chromosomes 1, 3, 5, 7, 8, 13, 16, 17 and 20 were observed in bladder cancer cell lineBIU87.【Conclusion】 Nonrandom chromosome DNAgains and losses occurred Chinese bladdercancer. The aberrant chromosome sites probably contain candidate oncogenes or tumorsuppressor genes associated with the development and the progression of bladder cancer.