In contrast, only unmethylated DNA bands were evident in SNU-283, SNU-1033, HT-29, and WiDr, in which CD133 gene expression was strong. Both methylated kinase inhibitor 17-DMAG and unmethylated DNA bands were detected in 13 cell lines (SNU-175, SNU-407, SNU-769B, SNU-1040, SNU-1047, SNU-1197, SNU-C1, SNU-C2A, SNU-C5, Colo201, HCT116, LoVo and SW403), which also showed strong expression of the CD133 gene. Three cell lines (Caco-2, Colo205 and DLD-1) contained only methylated DNA bands, even though these cell lines expressed CD133. Methylated or unmethylated DNA bands were not evident in SNU-61 cell line. Figure 3 Methylation analysis of CD133 gene in 32 colorectal cancer cell lines by methylation specific-PCR (MS-PCR). Lanes M and U denote that the product amplified by primer recognizing a methylated sequence and the product amplified by primer recognizing an .

.. Analysis of promoter methylation status of CD133 gene by bisulfite sequencing analysis A study reported that methylation of promoter P1 and exon A1 did not correlate with the CD133 gene transcription level because promoter P1 and exon 1A were hypermethylated with a high (90%-94%) content of methylated CpG dinucleotides in all cell lines examined[11]. Appropriately, we investigated the methylation status of 32 CpG sites in promoter P2 and exon 1B (Figure (Figure1B)1B) relative to the transcription initiation site of CD133 gene by clonal bisulfite sequencing analysis. Representative sequence diagrams of two cell lines are shown in Figure Figure4A.4A. The methylation status of promoter CpG dinucleotides (Figure (Figure4B)4B) was correlated with CD133 expression (Table (Table1).

1). The promoter CpG dinucleotides of CD133 of 13 cell lines (SNU-175, SNU-283, SNU-407, SNU-769B, SNU-1033, SNU-1040, SNU-1197, SNU-C1, SNU-C5, Colo201, Colo205, HT-29, SW403 and WiDr) were mostly unmethylated. Hypermethylation of CpG islands was 64% in the SW480 cell line (undetectable CD133 gene expression), 64%-95% in SNU-503, HCT-15 and LS174T cell lines (low expression of CD133 gene) and 9%-24% in SNU-C4, HCT-8, NCI-H716 and SW1116 cell lines (also low expression of CD133 gene). The distribution of methylated CpG dinucleotides in different clones was not uniform. For example, in SNU-C2A cells the methylation level of CpG in promoter was markedly increased through hypermethylation in two clones, whereas the CpG dinucleotides in other clones were fully unmethylated (Figure (Figure4B).

4B). Similar variations were observed in SNU-1047, DLD-1, HCT116, and LoVo cell lines. The variance was suggestive of the origin of different clones from different alleles of the gene. However, further studies are needed to confirm this possibility. Table 1 Correlation between promoter methylation status and CD133 expression Figure 4 Analysis of methylation status of promoter Entinostat CpG islands of CD133 by clonal sequencing.