Effects of Promoter Region 5＇CpG Island Demethylation on the Biological Behavior of Human Colorectal Cancer RKO Cells in Vitro

OBJECTIVE To explore the relationship between the methylation status of the promoter 5＇CpG island region and the biological behavior of human colorectal cancer RKO cells in vitro. METHODS RKO cells were treated with a selective DNA methyltransferase inhibitor-5-aza-2＇-deoxycytidine （5-aza-CdR） for 72 h. Methylationspecific PCR （MSP）, T-A cloning and DNA sequence analysis were used to determinate the 5＇CpG island methylation status of the p16/CDKN2 tumor suppressor gene. Cell growth, morphological changes and apoptosis were analyzed by the MTT assay, flow cytometry, fluorescence staining and electron microscopy. RESULTS The 5＇CpG island of the p16/CDKN2 tumor suppressor gene in RKO cells was a typically hypermethylated. The DNA methyltransferase inhibitor （5-Aza-CdR） effectively reversed the hypermethylation status of the promoter region. With demethylation, RKO cell growth was suppressed, the cells doubling times were prolonged （P〈0.01） and apoptosis was induced, which showed a relationship. CONCLUSION A selective DNA methyltransferase （DNMT） inhibitor can inhibit proliferation by demethylation in 5＇CpG islands, and may be a potential new therapy target for colorectal cancer.

Effects of promoter region 5′CpG island demethylation on the biological behavior of human colorectal cancer RKO cells in vitro

Objective  To explore the relationship between the methylation status of the promoter 5′CpG island region and the biological behavior of human colorectal cancer RKO cells in vitro. Methods  RKO cells were treated with a selective DNA methyltransferase inhibitor-5-aza-2′-deoxycytidine (5-aza-CdR) for 72 h. Methylation-specific PCR (MSP), T-A cloning and DNA sequence analysis were used to determinate the 5′CpG island methylation status of the p16/CDKN2 tumor suppressor gene. Cell growth, morphological changes and apoptosis were analyzed by the MTT assay, flow cytometry, fluorescence staining and electron microscopy. Results  The 5′CpG island of the p16/CDKN2 tumor suppressor gene in RKO cells was a typically hypermethylated. The DNA methyltransferase inhibitor (5-Aza-CdR) effectively reversed the hypermethylation status of the promoter region. With demethylation, RKO cell growth was suppressed, the cells doubling times were prolonged (P<0.01) and apoptosis was induced, which showed a relationship. Conclusion  A selective DNA methyltransferase (DNMT) inhibitor can inhibit proliferation by demethylation in 5′CpG islands, and may be a potential new therapy target for colorectal cancer. This study was supported by grants from the National Key Basic Research Project Foundation (No.G1998051200) and the Science and Technology Development Foundation of Zhejiang Province (No.011110541).