The Experimental Research On The Promoter Methylation Of Estrogen Receptor Isforms In Leukemia Cells

Many types of tumors have a tumor-specific methylation pattern.Methylation of gene promotor can be used to diagnose tumor and predict curative effect or duration of patient survival.There were many studys on tumor-specific methylation patter of estrogen receptor isoforms(ERa,ERβ) in several human cancers such as breast,endometrial and prostate etc, but few on leukemia in our country.This experiment was designed to detect the promotor methylation and the mRNA and protein expression of ER isoforms in leukemia cell lines by MSP(methylation specific polymerase chain reaction),RT-PCR(reverse transcription PCR) and Immunohistochemistry.On the other hand,we treated the leukemia cell lines by the demethylation reagent-Hydralazine,then detected changes in the promotor methylation and the mRNA and protein reexpression of ERα,and clarified the significance of promoter methylation ststus of ER isoforms on altered ER expression in leukemia,and investigated the potentional possibility of Hydralazine for the treatment of leukemia.Methods:1.Leukemia cell lines HL-60,MOLT-4 and Jurkat were cultured and treated with Hydralazine.2.The CpG island methylation status of ERa(ERα-A,ERα-B) and ERβin Leukemia cell lines and normal human peripheral mononuclear cells were detected by MSP.3.RT-PCR and Immunohistochemistry technology were used to analyze the expression level of ERαand ERβin HL-60,MOLT-4 and Jurkat.4.MSP,RT-PCR and Immunohistochemistry technology were used to analyze the CpG island methylation status of ERα(A,B) and expression level of ERαin the leukemia cell lines after Hydralazine treatment.Results:1.MSP analysis showed that CpG islands of ERα(A,B) were methylated and ERβwas unmethylated in the three leukemia cell lines(HL-60,MOLT-4 and Jurkat).In normal peripheral mononuclear cell samples,only unmethylated bands were observed.2.RT-PCR and Immunohistochemistry technology analysis showed that ERαwas inactived but ERβwas expressed in the three leukemia lines.3.CpG islands of ERα(ERα-A,ERα-B)were demethylated and reexpression of mRNA and protein of ERαcould be found after Hydralazine treatment.Conclusion1.Aberrant promoter methylation of ERαand ERβwas found in the three leukemia lines:HL-60,MOLT-4 and Jurkat,but was not found in normal peripheral mononuclear cell samples,which indicated that DNA methylation of ER isoforms may be one of molecule mechanisms in leukemia pathogenesis.2.After Hydralazine treatment,the CpG islands of ERαpromoter were demethylated, and the expression of mRNA and protein was restored,which demonstrated that promotor methylation may contribute to the loss of demethylated ERαexpression and Hydralazine may be noval treatment strategy for leukemia.3.Aberrant expression of ERβwas not found in the three leukemia lines: HL-60,MOLT-4 and Jurkat.