title: Methylation of tumour suppressor genes APAF-1 and DAPK-1 and in vitro effects of demethylating agents in bladder and kidney cancer.
abstract: To examine the significance of the methylation level of the p53 target and tumour suppressor genes apoptotic protease activating factor-1 (APAF-1) and death-associated protein kinase-1 (DAPK-1) in 80 microdissected tumour samples from transitional cell carcinoma (TCC) of the bladder and 80 tumour samples from clear-cell renal cell carcinoma (RCC) as well as from non-tumourous bladder and kidney tissue. Growth-inhibitory effects of the demethylating agents 5-Aza-2'-deoxycytidine (5-Aza-CdR) and zebularine were investigated in TCC and RCC cell lines. The methylation frequency of APAF-1 (DAPK-1) was 100% (77%) in TCC and 100% (33%) in RCC. The methylation levels of APAF-1 could differentiate between the individual tumour stages in TCC as well as in RCC. The APAF-1 methylation levels in RCC were significantly higher in tumours larger than 4 cm and in high-grade tumours. The methylation frequencies in normal tissue for APAF-1 (DAPK-1) were 11% (8%) in bladder tissue and 9% (5%) in kidney tissue. The growth-inhibitory effect of the demethylating agents in TCC (RT4, T24) and RCC (A498, ClearCa-5) cell lines resulted in a 17-132% prolongation of the doubling time (DT). In RCC cell lines, zebularine was superior to 5-Aza-CdR in achieving a DT prolongation. Quantitative real time RT-PCR detected a re-expression of mRNA transcripts of APAF-1 or DAPK-1. In conclusion, demethylating agents effectively retard growth of TCC and RCC cell lines. Methylation level analysis of specific genes has the potential for further tumour characterisation in TCC and RCC.
authors.affiliation: Department of Urology, Charité - Campus Benjamin Franklin, Universitätsmedizin Berlin, Hindenburgdamm 30, Berlin 12200, and Institute of Pathoogy, University Hospital Hamburg-Eppendorf, Germany. frank.christoph@charite.de
authors.forename: F
authors.initials: F
authors.lastname: Christoph
authors.forename: C
authors.initials: C
authors.lastname: Kempkensteffen
authors.forename: S
authors.initials: S
authors.lastname: Weikert
authors.forename: J
authors.initials: J
authors.lastname: Köllermann
authors.forename: H
authors.initials: H
authors.lastname: Krause
authors.forename: K
authors.initials: K
authors.lastname: Miller
authors.forename: M
authors.initials: M
authors.lastname: Schostak
authors.forename: M
authors.initials: M
authors.lastname: Schrader
mesh_terms: D000368:Aged; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics* / Q000378:metabolism; D001374:Azacitidine / Q000737:chemistry / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D002292:Carcinoma, Renal Cell / Q000235:genetics; D002295:Carcinoma, Transitional Cell / Q000235:genetics; D002478:Cells, Cultured; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019175:DNA Methylation; D064550:Death-Associated Protein Kinases; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007680:Kidney Neoplasms / Q000235:genetics*; D008297:Male; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001743:Urinary Bladder / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000235:genetics*
chemical_list: C506592:APAF1 protein, human; D000964:Antimetabolites, Antineoplastic; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; D012333:RNA, Messenger; D003562:Cytidine; C009131:pyrimidin-2-one beta-ribofuranoside; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D001374:Azacitidine