title: DNA methylation alterations in urothelial carcinoma.
abstract: In urothelial cancer, hypermethylation of specific genes and genome-wide hypomethylation, reflected in decreased methylation of LINE-1 retrotransposons, have both been reported, but were never investigated in the same specimens. We analyzed hypermethylation of six genes by methylation-specific PCR and LINE-1 hypomethylation by Southern blotting in 96 carcinoma tissues. Hypermethylation frequencies were: SFRP1 (55%), APC (45%), RASSF1A (35%), DAPK1 (29%), RARB2 (19%), and CDKN2A (2%). Three groups of cancers could be discerned, with escalating hypermethylation. Hypermethylation increased with tumor stage, particularly at the transition to invasive cancers, and RARB2 hypermethylation was indicative of lymph node involvement. A comparison to a previous study on prostate cancer using the same techniques suggests that hypermethylation in urothelial carcinoma occurs in a random rather than coordinated manner. LINE-1 hypomethylation was present in 90% of specimens, largely independent of hypermethylation. Lack of hypomethylation indicated a significantly better clinical prognosis. Bisulfite sequencing of SFRP1 demonstrated dense or patchy hypermethylation in tumor tissues that likely accounts for discrepant reported frequencies. In urothelial carcinoma cell lines, the same genes as in tissues were frequently hypermethylated. SFRP1 hypermethylation was concordant with lack of expression. 5-Aza-deoxycytidine induced its reexpression in some lines, whereas additional treatment with a histone deacetylase inhibitor was required in others. Thus, epigenetic SFRP1 inactivation occurs in a graduated manner. In conclusion, markers of genome-wide hypomethylation seem optimally suited for urothelial carcinoma detection, whereas combinations of hypermethylation and hypomethylation assays hold promise for classification.
authors.affiliation: Urologische Klinik, Heinrich-Heine-Universität, Düsseldorf, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Neuhausen
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Florl
authors.forename: Marc-Oliver
authors.initials: MO
authors.lastname: Grimm
authors.forename: Wolfgang A
authors.initials: WA
authors.lastname: Schulz
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015139:Blotting, Southern; D018899:CpG Islands; D015653:Cystectomy; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008875:Middle Aged; D009361:Neoplasm Invasiveness / Q000473:pathology; D016133:Polymerase Chain Reaction; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D018626:Retroelements / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000473:pathology / Q000601:surgery; D014571:Urologic Neoplasms / Q000235:genetics* / Q000473:pathology; D000094742:Secreted Frizzled-Related Proteins
chemical_list: D004273:DNA, Neoplasm; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D018626:Retroelements; D025521:Tumor Suppressor Proteins; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins