[The study of 5-Aza-2'-deoxycytidine on transcription regulation of p16/CDKN2 gene demethylation in RKO human colorectal cell line].
OBJECTIVE
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.


METHODS
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.


RESULTS
(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.


CONCLUSIONS
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.
Fang
Xiao-ming
XM
Cancer Institute, Second Affiliated Hospital, Medical School of Zhejiang University, Hangzhou 310009, China.
Sun
Li-feng
LF
Peng
Jia-ping
JP
Dong
Qi
Q
Zheng
Shu
S
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors*; D000077209:Decitabine; D019942:Genes, p16; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic / Q000187:drug effects*
D000964:Antimetabolites, Antineoplastic; D012333:RNA, Messenger; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine