OBJECTIVES: To analyze the methylation status and expression profiles of multiple tumor suppressor genes in renal cell carcinoma-derived cell lines. Aberrant promoter methylation is commonly found in human cancers. Nonetheless, it is challenging to demonstrate that methylation of a specific gene results in gene inactivation. METHODS: We simultaneously analyzed methylation and expression profiles of five putative tumor suppressor genes (p15, p16, Rb, BRCA1, and E-cadherin) in 14 different cell lines using bisulfite genomic sequencing and reverse transcriptase-polymerase chain reaction. We also used multiplex polymerase chain reaction to identify homozygous deletions at the p15 and p16 loci. RESULTS: Expression of p16, BRCA1, and E-cadherin was maintained in 4 (29%) of 14 cell lines, regardless of the presence of methylation. Aberrant methylation of p16 was observed in 2 (14%), of BRCA1 in 1 (7%), and of E-cadherin in 9 (64%) of 14 cell lines. Concurrent methylation was observed among p16 and BRCA1 (1 [7%] of 14 cell lines) and among p16 and E-cadherin (1 [7%] of 14 cell lines). We detected homozygous deletion of p16 and p15 in 11 (78%) and 6 (43%) cell lines, respectively. CONCLUSIONS: The present data shows the presence of methylation does not always contribute to the loss of expression of tumor suppressor genes. Therefore, we must be cautious in interpreting the results of methylation assays--in particular, detection of methylation by nonquantitative methods. The data also demonstrated that multiple tumor suppressor genes are simultaneously inactivated in renal cell carcinoma-derived cell lines by distinctive mechanisms.
OBJECTIVES: To analyze the methylation status and expression profiles of multiple tumor suppressor genes in renal cell carcinoma-derived cell lines. Aberrant promoter methylation is commonly found in humancancers. Nonetheless, it is challenging to demonstrate that methylation of a specific gene results in gene inactivation. METHODS: We simultaneously analyzed methylation and expression profiles of five putative tumor suppressor genes (p15, p16, Rb, BRCA1, and E-cadherin) in 14 different cell lines using bisulfite genomic sequencing and reverse transcriptase-polymerase chain reaction. We also used multiplex polymerase chain reaction to identify homozygous deletions at the p15 and p16 loci. RESULTS: Expression of p16, BRCA1, and E-cadherin was maintained in 4 (29%) of 14 cell lines, regardless of the presence of methylation. Aberrant methylation of p16 was observed in 2 (14%), of BRCA1 in 1 (7%), and of E-cadherin in 9 (64%) of 14 cell lines. Concurrent methylation was observed among p16 and BRCA1 (1 [7%] of 14 cell lines) and among p16 and E-cadherin (1 [7%] of 14 cell lines). We detected homozygous deletion of p16 and p15 in 11 (78%) and 6 (43%) cell lines, respectively. CONCLUSIONS: The present data shows the presence of methylation does not always contribute to the loss of expression of tumor suppressor genes. Therefore, we must be cautious in interpreting the results of methylation assays--in particular, detection of methylation by nonquantitative methods. The data also demonstrated that multiple tumor suppressor genes are simultaneously inactivated in renal cell carcinoma-derived cell lines by distinctive mechanisms.
Authors: William C Reinhold; Mark A Reimers; Philip Lorenzi; Jennifer Ho; Uma T Shankavaram; Micah S Ziegler; Kimberly J Bussey; Satoshi Nishizuka; Ogechi Ikediobi; Yves G Pommier; John N Weinstein Journal: Mol Cancer Ther Date: 2010-01-06 Impact factor: 6.261