[Synergistic effect of DNA methylation inhibitor and histone deacetylase inhibitor on RASSF1A gene expression in U266 cells.].
OBJECTIVE
To investigate the effect of DNA methylation in combination with histone deacetylase inhibitor on transcription regulation of Ras associated domain family gene 1(RASSF1A) tumor suppressor gene and the molecular biological behaviors in U266 cells.


METHODS
The U266 cells were treated with different doses of 5-Aza-2'-deoxycytidine (5-Aza-CdR) and Valproate (VPA) each alone or in combination. Methylation-specific PCR (MSP) was used to detect CpG island methylation in RASSF1A promoter. Quantitative real-time reverse transcription polymerase chain reaction (RQ-PCR) was used to examine the expression of RASSF1A gene in U266 cells. MTT was used for cell proliferation. Cell apoptosis and cell cycle were analyzed by flow cytometry.


RESULTS
The methylation of RASSF1A gene promoter was detected in U266 cells, while there was little RASSF1A gene expressing in the control group. The demethylation effect could be detected in the 5-Aza-CdR treated and combined treatment groups but no in the VPA group. The expression level of RASSF1A was induced by 5-Aza-CdR in a concentration-dependent manner while VPA had no such effect. The expression level of RASSF1A mRNA was increased significantly in the combined treatment group. Higher growth inhibition and apoptosis effects were found in 5-Aza-CdR and VPA combination group than that in 5-Aza-CdR or VPA alone group (P < 0.05). After treatment with 5-Aza-CdR or VPA alone for 72 h, more cells were arrested in G(0)/G(1) phase as conpared with control group (P < 0.05), and even more cells were so arrested in combined treatment group (P < 0.05).


CONCLUSIONS
DNA methylation and histone deacetylase inhibitor can synergistically induce demethylation of the RASSF1A gene, re-express RASSF1A gene silenced in U266 cells, inhibit the proliferation of U266 cells and induce cell apoptosis.
Lu
Fei
F
Department of Hematology, Qilu Hospital of Shandong University, Jinan 250012, China.
Liu
Chuan-Fang
CF
Ma
Dao-Xin
DX
Liu
Yan-Ping
YP
Kong
Hai-Li
HL
Zhang
Jing-Jing
JJ
D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D015870:Gene Expression; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D011401:Promoter Regions, Genetic
D056572:Histone Deacetylase Inhibitors