M: Methylated site; U: Unmethylated … Re-expression of CD133 after treatment with 5-aza-2��-deoxycytidine To confirm whether the differential CD133 expression was related to DNA methylation, 11 cell lines with undetectable or low expression inhibitor Dorsomorphin of CD133 were treated with 5-aza-2��-deoxycytidine. The treatment recovered CD133 expression in four of the weakly CD133 expressing cell lines (SNU-61, SNU-769A, SNU-C4 and Colo320) and two cell lines without detectable CD133 expression (Figure (Figure5A).5A). However, 5-aza-2��-deoxycytidine treatment did not significantly influence CD133 gene expression in SNU-503, HCT-8, LS174T, NCI-H716 and SW1116 cell lines. Examination of CD133 expression by real- time PCR revealed that CD133 expression was increased after 5-aza-2��-deoxycytidine treatment in SNU-503, HCT-8, LS174T and SW1116 cell lines (Figure (Figure5B),5B), and decreased in the NCI-H716 cell line.
Figure 5 Expression analysis after treatment with 5-aza-2��-deoxycytidine (5-Aza). A: RT-PCR analysis of CD133 expression in 11 cell lines with or without 5-aza-2��-deoxycytidine treatment; B: Representative results of real-time PCR analysis revealing … DISCUSSION It has been postulated that CSCs are able to maintain tumor bulk due to the abilities of self-renewal and differentiation into cells with low potential. CD133 is one of CSC markers in colorectal carcinoma and CD133-positive cells in colon cancer exhibit a high tumorigenic ability in vivo. In a previous study, the CD133-positive cells separated from tumors were able to form tumor bulk in the immuno-compromised mouse models with less numbers than the CD133-negative cells and these cells showed a long-term tumorigenic potential[4].
Furthermore, CD133-positive cells in colon cancer cell lines showed higher levels of proliferation, colony formation and invasive ability in vitro than CD133-negative cells[19]. Presently, CD133 was down-regulated in 11 of the 32 colorectal cancer cell lines. Since abnormal DNA methylation of CD133 gene is related to CD133 expression in colorectal tumors[20], we hypothesize that CD133 expression is regulated by hypermethylation of the promoter region in this gene, and analyzed promoter methylation status of the CD133 gene with a methylation-specific PCR after sodium-bisulfite modification and by clonal sequencing analysis.
A methylation analysis of promoters P1 and P2 in cell lines demonstrated tissue specificity of the promoter P2 methylation and practically no specificity in the methylation of the P1 promoter[11]. Therefore, we designed the primers with promoter P2 sequences for MS-PCR and bisulfite sequencing analysis and sequencing primers containing 32 of CpG dinucleotides (Figure (Figure1B1B). Methylation of the CD133 gene promoter was observed in 13 cell lines (SNU-503, SNU-1047, Carfilzomib SNU-C2A, SNU-C4, Caco-2, DLD-1, HCT-15, HCT116, LoVo, LS174T, NCI-H716, SW403 and SW1116).