5-Aza-2'-deoxycitydine induces demethylation and up-regulates transcription of p16INK4A gene in human gastric cancer cell lines.
BACKGROUND
To investigate the effects of DNA methylation on the expression of tumor-associated genes and the cell cycle in human gastric cancer cells.


METHODS
Two gastric cancer cell lines (MKN-45 and HGC-27) were treated with DNA methyltransferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC). The expressions of p16INK4A, p21WAF1, p53, p73, c-Ha-ras and c-myc genes mRNA were detected by using reverse transcription PCR (RT-PCR). DNA methylation status of p16INK4A gene promoter was assayed by bisulfite modification and sequencing. The cell cycle was analyzed by using flow cytometry (FCM).


RESULTS
5-aza-dC induced the demethylation of p16INK4A gene promoter. The expression of p16INK4A mRNA was obviously up-regulated by treatment with 10 micro mol/L (MKN-45 cells) or 5 micro mol/L (HGC-27 cells) of 5-aza-dC for 24 hours. However, 5-aza-dC treatment failed to regulate the expressions of p21WAF1, p53, p73, c-Ha-ras and c-myc genes in MKN-45 and HGC-27 cells. Furthermore, 5-aza-dC induced the cell cycle arrest in G1 phase in HGC-27 cell, but not in MKN-45 cell.


CONCLUSIONS
DNA methylation regulates the transcription of p16INK4A but not p21WAF1 and proto-oncogenes in human gastric cancer cell lines MKN-45 and HGC-27.
Fang
Jing-yuan
JY
Renji Hospital, Shanghai Institute of Digestive Disease, Shanghai Second Medical University, Shanghai 200001, China. jingyuanfang@yahoo.com.
Yang
Li
L
Zhu
Hong-yin
HY
Chen
Ying-xuan
YX
Lu
Juan
J
Lu
Rong
R
Cheng
Zhong-hua
ZH
Xiao
Shu-dong
SD
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D015870:Gene Expression; D016147:Genes, Tumor Suppressor / Q000502:physiology; D019942:Genes, p16 / Q000502:physiology*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics*; D014158:Transcription, Genetic; D015854:Up-Regulation
D004791:Enzyme Inhibitors; D000077209:Decitabine; D001374:Azacitidine