Silencing of the UCHL1 gene in human colorectal and ovarian cancers.

Aberrant DNA methylation is associated with many types of human cancers. To identify genes silenced in human colorectal cancers, we performed a microarray analysis for genes whose expression was induced by treatment of HCT116 human colon cancer cells with a demethylating agent, 5-aza-2'-deoxycitidine (5-aza-dC). Seven known genes were identified as being upregulated (> or =8-fold) and expressed at more than twice as high as the average level. Among these was the UCHL1 gene (also known as PGP9.5), which is involved in regulation of cellular ubiquitin levels. A dense CpG island in its promoter region was completely methylated in HCT116 cells, and no mRNA was detected. 5-Aza-dC treatment of HCT116 cells induced dose-dependent demethylation of the CpG island, and restored UCHL1 mRNA and protein expression. UCHL1 silencing was observed in 11 of 12 human colorectal cancer cell lines, and its methylation was detected in 8 of 17 primary colorectal cancers. Further, UCHL1 silencing was observed in 6 of 13 ovarian cancer cell lines, and its methylation was detected in 1 of 17 primary ovarian cancers. These results showed that UCHL1 is inactivated in human colorectal and ovarian cancers by its promoter methylation, and suggest that disturbance of cellular ubiquitin levels is present.

Establishment of a detection system for demethylating agents using an endogenous promoter CpG island.

Disturbances of epigenetic information that result in changes in DNA methylation patterns are involved in carcinogenesis and other human disorders. Detection of agents that can cause epigenetic alterations--i.e. epimutagens--is therefore an important objective. We have developed and now describe the first detection system for demethylating agents that involves an endogenous promoter CpG island (CGI). After screening 10 promoter CGIs of genes silenced in human cancers, a CGI of the FLJ32130 gene was found to respond sensitively to a known demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), by abundantly re-expressing its mRNA. After introducing the Hyg(r)-EGFP fusion gene into exon 3 of the FLJ32130 gene by homologous recombination, we isolated one clone that had the expected recombination outcomes and designated it F117. Two subclones (F117-47 and F117-123) of this original clone that did not share its propensity for leaky expression of the Hyg(r)-EGFP mRNA were then isolated, and methylation of their 5' CGI was confirmed. The addition of 5-aza-dC at doses of 0.1 microM or higher led to their 5' CGI being demethylated, and to Hyg(r)-EGFP being expressed; the anticipated fluorescence was readily confirmed by fluorescence microscopy. We believe that this is the first assay system that detects agents that disturb the methylated status of a CGI that regulates an endogenous promoter.

The absence of Mth1 inactivation and DNA polymerase kappa overexpression in rat mammary carcinomas with frequent A:T to C:G transversions.

Single nucleotide instability (SNI), an increase in spontaneous point mutation rates (MRs) without involvement of microsatellite instability, is present in rat mammary carcinoma cell lines and human breast cancer cell lines. A:T to C:G transversions, which are generally rare, were frequently observed in two rat mammary carcinoma cell lines and in their primary carcinomas, and were considered to be related to the molecular mechanism of SNI. In this study, two known molecular mechanisms that cause increases of A:T to C:G transversions, inactivation of the MutT mammalian homologue (Mth1) gene and overexpression of the DNA polymerase k (Pol k) gene, were analyzed in two rat mammary carcinoma cell lines and 11 rat primary carcinomas. PCR-SSCP analysis revealed no mutations in the entire Mth1 coding region. Quantitative real-time RT-PCR analysis showed that Mth1 mRNA expression was slightly, but significantly, increased in the primary carcinomas (P = 0.001 using GAPDH for normalization, and P = 0.002 using histone H4, t-test), contrary to our expectation, and was decreased to 1 / 2 in the cell lines. The expression of Pol k, which is known to be error-prone with frequent A:T to C:G transversions, was rather decreased in the cell lines and primary carcinomas. Inactivation of Mth1 and overexpression of Pol k were unlikely to have caused SNI in the two rat mammary carcinoma cell lines with a high frequency of A:T to C:G transversions, and searching for other unknown molecular mechanisms is important.