细胞凋亡过程中bcl-2基因的甲基化

Methylation of bcl2 gene in the apoptosis of mousefibroblast and MCF7 cell lines

为探讨凋亡过程中，ｂｃｌ－２基因下调与该基因甲基化状态的关系，用５－氟尿嘧啶（５－Ｆｕ）诱导小鼠成纤维细胞ＮＣ３Ｈ１０，ＴＣ３Ｈ１０及人乳腺癌细胞ＭＣＦ－７的凋亡，分别检测了这三种细胞凋亡过程中ｂｃｌ－２的表达变化，与其调控区及编码区的甲基化状况．我们曾观察到５－Ｆｕ作用２４～４８ｈ出现细胞存活率下降，ＤＮＡ梯状断裂及细胞周期凋亡峰显现等典型凋亡现象．Ｎｏｒｔｈｅｒｎ杂交显示，在５－Ｆｕ作用１２ｈ时ｂｃｌ－２ｍＲＮＡ水平已明显降低．由此，我们用小鼠ｂｃｌ－２（ｍｂｃｌ－２）及人ｂｃｌ－２（ｈｂｃｌ－２）基因调控区ＰＣＲ扩增片段及ｂｃｌ－２编码区（ｃＤＮＡ）片段作为探针，与５－Ｆｕ作用１２ｈ的细胞ＤＮＡ的ＭｓｐⅠ／ＨｐａⅡ酶切产物进行Ｓｏｕｔｈｅｒｎ杂交，以未作用的细胞ＤＮＡ同样酶切杂交为对照．通过杂交带谱的变化，分析ｂｃｌ－２基因的甲基化状况．结果显示：ｍｂｃｌ－２及ｈｂｃｌ－２在５－Ｆｕ作用１２ｈ后调控区甲基化水平增高，但其编码区甲基化状态皆未出现可检出的变化．

The cause of downregulation of bcl2 expression in apoptosis is not fully understood.To investigate whether downregulation of the gene might be associated with its DNA methylation,the apoptosis of cells from three cell lines were induced by 5fluorouracil(5Fu).Our results indicate that the methylation of bcl2 gene regulatory region,but not coding region,is associated with the down regulation in the process.The apoptosis of mouse embryo fibroblasts C3H10 T1/2 C1/8 (designated as NC3H10),its tritium transformed counterpart (designated as TC3H10)and human breast cancer(MCF7) cells was induced by 5Fu (10 mmol/L).Our previous Northern blotting results showed that P53 mRNA level increased significantly after exposing these cells to 5Fu for 6 h,while bcl2 mRNA level decreased significantly after 5Fu treatment for 12 h.Then DNA ladder appeared after exposing these cell lines to 5Fu for 24-48 h.Apoptotic peak appeared meantime as shown by flow cytometry analysis.At the same time the surival rate of these cells also declined.Therefore,the cells were treated by 5Fu for 12 h.Then their genomic DNAs were extracted and cut with MspⅠ/HpaⅡ.Southern blotting was carried out by using the PCR products of regulatory regions of mbcl2 or hbcl2 as probes,and by using the bcl2 cDNA fragment as probes of coding region of bcl2.The DNA methylation state was analysed by the change of hybridized band of Southern blotting.The results showed that the methylation level of mbcl2 and hbcl2's regulatory regions increased after exposing NC3H10,TC3H10 and MCF7 cells to 5Fu for 12 h.While the methylation of coding regions of mbcl2 and hbcl2 did not change at the same time.The data suggest that the downregulation of bcl2 in the apoptotic process induced by 5Fu is associated with the methylation increasing of regulatory region of bcl2.