Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma.

Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (-575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco(2) (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC(3)/c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2'-deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco(2), and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.

Heterogeneous methylation of the O(6)-methylguanine-DNA methyltransferase promoter in immortalized IMR90 cell lines.

Transcriptional silencing of the DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), occurs only in malignant or transformed cell lines, and such MGMT-deficient cells are hypersensitive to chemotherapeutic alkylating agents such as 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide. Previously we demonstrated in a panel of established cell lines that the lack of gene expression correlated with methylation within the CpG island in the MGMT 5' gene flank. Now, we investigated the relationship between CpG methylation, MGMT suppression and drug-sensitivity in normal, diploid MGMT-expressing IMR90 cells and five immortalized sublines (AA, EE, J, KK and Pool), four of which have silenced MGMT. As expected, the MGMT-expressing parental cells were most drug-resistant and free of promoter methylation, whereas the MGMT-silenced immortal sublines were more drug-sensitive and promoter-methylated. Surprisingly, the sole MGMT-positive immortal subline, (AA) showed some promoter methylation although it was relatively drug-resistant; and an apparently MGMT-negative subline, (EE) showed unexpectedly low levels of methylation. We determined if these discrepancies were due to heterogeneity (cellular or allelic) and if this reflected transitional states between expressing and silenced phenotypes. Analysis of the methylation status of CpGs by genomic sequencing of cloned single copy DNA confirmed heterogeneity in both these sublines. With increasing cell culture passage, CpG methylation progressively increased with a concomitant trend to a completely MGMT-silenced phenotype in these sublines.

The effect of dietary fat on malondialdehyde concentrations in Fischer 344 rats.

The effects of dietary fat and age on the level of malondialdehyde (MDA), a product of lipid peroxidation, were investigated in cerebellum, kidney, and liver tissues of female Fischer 344 rats. Groups of rats were fed diets containing various levels of corn oil (3, 5, 10, 15, or 20%), starting at 57 days of age, for a duration of 2, 10, or 20 weeks. High fat diets are thought to promote tumor formation, diabetes, and cardiovascular diseases via induction of oxidation stress, and this can begin early in the lifespan. However, it was observed that rats chronically consuming 3 and 5% corn oil diets yielded significantly higher levels of MDA, as analyzed by high-performance liquid chromatography, compared with those fed higher fat diets. After 20 weeks of feeding, the concentration of MDA in each of the three organs studied showed no significant differences among rats consuming diets containing 10, 15, or 20% corn oil. The levels of MDA were highest in the cerebellum, followed by kidney, and lowest in liver. Over the 20-week feeding period, a decrease in MDA level in both cerebellum and liver was observed.

Methylation of selected CpGs in the human O6-methylguanine-DNA methyltransferase promoter region as a marker of gene silencing.

O6-methylguanine-DNA methyltransferase (MGMT) is a major determinant of susceptibility to methylating carcinogens and of tumor resistance to anticancer methylating and chloroethylating drugs. The silencing of MGMT expression that occurs in 20-30% of human tumor lines is tightly linked to methylation within the MGMTgene 5'CpG island. Previous studies on a very limited number of cell lines showed that such methylation was uneven, with hot-spots where methylation almost invariably occurred and intervening regions with very low incidences of methylation. To ascertain if such hot-spot methylation is in fact a ubiquitous hallmark of MGMT-silenced cells, we determined the methylation status of selected hot-spot CpGs in an extensive panel of MGMT-expressing and -silenced cell lines and xenografts. Using two simple and rapid bisulfite-polymerase chain reaction-based assays, we confirmed that in MGMT-silenced cells, methylation occurred at these sites whereas it was essentially absent in MGMT-expressing cells.

A specific CpG methylation pattern of the MGMT promoter region associated with reduced MGMT expression in primary colorectal cancers.

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) protects cells from the cytotoxic and mutagenic effects of alkylating agents. Approximately 20% of tumor cell lines lack MGMT activity and are highly sensitive to alkylating agents. In established cancer cell lines, MGMT expression appears to be correlated with methylation of residues in both the promoter and the body of the gene. The effect of methylation of the MGMT promoter on gene expression and carcinogenesis in primary tumors is unknown. We investigated methylation of the MGMT promoter region in primary colorectal cancers and normal colonic mucosa. We used five methylation-sensitive restriction enzymes (BssHII, SacII, Eagl, Nael, and Smal) and Southern blot analysis to assess methylation in 46 cancers and 22 controls. Methylation of Eagl and Nael sites was seen in 12 tumors but in none of the 22 normal colorectal mucosa specimens. This difference was statistically significant (P<0.01). Methylation-sensitive single-nucleotide primer extension analysis of four additional cytosine residues confirmed methylation of the promoter region in the tumors identified by Eagl and Nael digestions and served to further quantitate the extent of methylation. Western blot analysis of 21 tumors revealed statistically significant lower MGMT expression in the eight tumors with methylation of the Eagl and Nael sites and nt -128 than in the 13 tumors lacking the methylation pattern (P<0.05). MGMT activity was lower in tumors with methylation than in tumors that were not methylated. The difference was not, however, statistically significant. We conclude that a subset of colorectal tumors is characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression.