The study of 5-Aza-2'-deoxycytidine on transcription regulation of p16/CDKN2 gene demethylation in RKO human colorectal cell line
Fang, X.-m.; Sun, L.-f.; Peng, J.-p.; Dong, Q.; Zheng, S.
Zhonghua Yi Xue Za Zhi 83(23): 2077-2082
2003
ISSN/ISBN: 0376-2491 PMID: 14703421 Document Number: 563198
To explore the transcription regulation of DNA 5'CpG island demethylation on p16/CDKN2 tumor suppressor gene and effects of growth on RKO human colorectal cancer cell line. RKO cell line was exposed to the specific demethylating agent, 5-Aza-2'-deoxycytidine, for seventy-two hours to detect whether the silencing of p16/CDKN2 cell cycle regulatory gene could be reversed. Methylation-specific PCR (MSP), T-A cloning and sequence analysis were evaluated for methylation status. Growth speed, expression of DNA methyltransferase mRNA, p16/CDKN2 mRNA and protein were determined by MTT assay, reverse transcription polymerase chain reaction (RT-PCR), Real-time PCR, immunohistochemistry and western blot. (1) All cytosines in CpG dinucleotides in untreated RKO cells with 5-Aza-2'-deoxycytidine remain as C, while all cytosines in treated RKO cells have been converted to thymidine. (2) RKO cell line after treatment with three different concentration 5-Aza-2'-deoxycytidine grew slowly and double time increased to 1.49, 1.64, 1.87-times respectively. (3) The expression of RKO cell p16/CDNK2 gene mRNA treated with 5-Aza-2'-deoxycytidine increased to 4.89, 16.91, 19.97-times respectively, but the expression of DNA methyltransferase mRNA was inhibited. (4) Immunohistochemistry and western blot indicated that 5-Aza-2'-deoxycytidine could increase the p16/CDKN2 gene protein expression. DNA promoter hypermethylation is associated with p16/CDKN2 gene silence in RKO human colorectal cancer cell line. 5-Aza-2'-deoxycytidine may effectively cause demethylation and inhibit the growth of tumor cell by reactivating the gene transcription silenced by aberrant hypermethylation.