PMC

Distribution of the predicted 200 methylated genes along the chromosomal map by computational approach. Most of the genes are clustered in limited chromosomal regions. No genes were found on chromosome Y.

A, promoter methylation of representative candidate genes. a, methylation of KIF1A and OSMR by conventional methylation-specific PCR in cancer cell lines and primary tissues; M, methylated; U, unmethylated; NBT, normal breast tissues from noncancer patients; BTT, breast tumor tissues; NN, normal colon epithelium from noncancer patients; PN, paired normal colon tissues from colon cancer patients; PT, paired colon cancer tissues. b, representative sequencing results of the PAK3 and NISCH in cancer cell lines, normal, and cancer tissues. Normal tissues were taken from noncancer patients. Arrows, all guanines present after sequencing are complementary to methyl cytosines on the opposite DNA strand. B, re-expression of representative genes analyzed by semiquantitaive RT-PCR or real-time RT-PCR. a and b, reactivated PAK3 and OGDHL were observed by the 5-Aza-dC treatment in H23 and Siha cell lines by semiquantitative RT-PCR. c, overexpression of OSMR was observed by the 5-Aza-dC treatment analyzed by real-time quantitative RT-PCR. Relative fold was calculated by the expression of OSMR mRNA to GAPDH (an internal control). Fold increase of OSMR ranged from 5.2 (HCT116) to 2,868 (DLD-1). Experiments were performed in duplicate; columns, mean; bars, SD. *, P < 0.05. d, the amplification plot of OSMR transcript in DLD-1. A, 5-Aza-dC treated; M, mock treated.

Flowchart for selection of candidate TSG. We used 20 cancer cell lines of 5 different major cancers to screen for candidate TSGs after microarray analysis of cells treated with 5 μmol/L 5-aza-dC treatment (reactivation filter). Coupling the sequence and reactivation filters, we obtained >200 unique genes. We diminished the number of candidates by excluding 25 genes that were previously reported as methylated. By empirical testing, we found 53 genes that harbored promoter hypermethylation in primary tumor tissues by direct sequence analysis or MSP. Twenty-five of these 53 genes were methylated in both normal and tumor tissues. Twenty-eight of these 53 genes showed cancer-specific methylation. QMSP was developed for 8 genes for high throughput analysis in multiple tissue types. CE, cervical cancer; LU, lung cancer; HN, head and neck cancer; TH, thyroid cancer; CO, colon cancer; PA, pancreatic cancer; GA, gastric cancer; ES, esophageal cancer; OV, ovarian cancer; BR, breast cancer; BL, bladder cancer; PR, prostate cancer; KI, kidney cancer. Frequency of methylation for each of the eight genes in each cancer type is shown in .