[5-aza-2'-deoxycytidine induces changes of histone H3-lysine 9 methylation in bladder tumor cells].
OBJECTIVE
To investigate the effect of DNA methyltransferases inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) on the histone H3-lysine 9 methylation status and gene expression of RUNX3 in human bladder tumor cells.


METHODS
Human bladder tumor cells of the line EJ were cultured and treated with 5-Aza-CdR for 24 h. MTT test was used to observe the proliferation and growth of the EJ cells. Other EJ cells were cultured and treated with 5-Aza-CdR and then chromatin immunoprecipitation assay was used to analyze the histone H3-lysine 9 methylation status of RUNX3 promoter and the second exon. The expression of RUNX3 was measured by RT-PCR.


RESULTS
The survival rate of the EJ cells treated with 5-Aza-CdR of the concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0 micromol/L were 98.1%, 95.3%, 75.9%, 52.3%, 16.2%, and 7.7% respectively. And the survival rates of the EJ cells treated with 5-Aza-CdR for 12, 24, 36, 48, 72, and 96 hours were 89.4%, 85.2%, 78.6%, 37.1%, 8.9%, and 7.1% respectively. The survival rates of the 1.0, 2.0, 5.0, and 10.0 micromol/L group were significantly lower than that of the control group (all P < 0.05). Before the intervention, the amplified bands of the histone H3-lysine 9 methylation status of RUNX3 gene promoter and the second exon zone were obvious, but both disappeared after the treatment with 2.0 and 5.0 micromol/L 5-Aza-CdR. Before the intervention, RUNX3 gene was not expressed, and was expressed after treatment with 2.0 micromol/L 5-Aza-CdR.


CONCLUSIONS
5-Aza-CdR not only obviously inhibits the proliferation of human bladder cancer cells but also reactivates RUNX3 gene through demethylation of histone H3-lysine9 at RUNX3 promoter and the second exon. H3-lysine 9 trimethylation may be one of the most important reasons for gene inactivation of the RUNX3 gene.
Yang
Na
N
Department of Clinical Laboratory Medicine, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, China.
Li
Zhi
Z
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D004305:Dose-Response Relationship, Drug; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008745:Methylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D013997:Time Factors; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology
D000964:Antimetabolites, Antineoplastic; D050677:Core Binding Factor Alpha 3 Subunit; D006657:Histones; C493729:Runx3 protein, human; D000077209:Decitabine; D008239:Lysine; D001374:Azacitidine