Methylation of tumour suppressor genes APAF-1 and DAPK-1 and in vitro effects of demethylating agents in bladder and kidney cancer.
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.
Christoph
F
F
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
Kempkensteffen
C
C
Weikert
S
S
Köllermann
J
J
Krause
H
H
Miller
K
K
Schostak
M
M
Schrader
M
M
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*
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