MAPK／ERK信号通路调节K562细胞中mdr1基因的诱导性表达

The mitogen-activated protein kinase pathway regulates the induced expression of mdrl gene in K562 cells

目的观察多柔比星（doxombicin，DOX）诱导K562细胞mdr1基因表达过程中丝裂原活化蛋白激酶（mitogen—activated protein kinase，MAPK）／细胞外信号调节激酶（ERK）信号通路的作用，探讨mdr1基因的转录调控机制。方法多柔比星初始浓度为0．01μg/ml，诱导K562细胞24h后撤药继续培养至细胞状态恢复，加入多柔比星继续诱导24h，浓度增加为0．02μg/ml。依上述方法多柔比星浓度逐渐增加，直至0．05μg／ml。收集DOX浓度为0．01、0．03和0．05μg/ml时的细胞。RT—PCR检测mdrl基因表达，流式细胞仪检测mdrl基因编码的P糖蛋白（P—gP）的表达，Western blot检测ERK活化情况。MAPK的抑制剂PD98059预处理K562细胞1h后与多柔比星共同作用，逆转录（RT）-PCR和流式细胞仪分别检测mdr1基因和P—gP的表达情况。结果经多柔比星作用后K562细胞中ERK磷酸化增强，同时mdr1基因转录上调，及其蛋白产物P—gP的表达也增加，当多柔比星浓度为0．05μg／ml时两者的表达均增加了5倍之多。而用PD98059预处理细胞后能明显抑制多柔比星诱导mdr1基因转录和蛋白表达，多柔比星浓度为0．03μg/ml时PD98059对mdr1基因表达的抑制率为（74．1±0．11）％，多柔比星浓度为0．05μg/ml时抑制率为（70．2±0．14）％。结论多柔比星能够诱导K562细胞mdr1基因表达，同时激活MAPK／ERK信号通路，阻断ERK的活化能抑制mdr1基因的诱导性表达。

Objective To investigate the effect of mitogen-activated protein kinase（MAPK） pathway on the transcriptional expression of mdrl gene induced by doxornbicin （ DOX ） and study the transcription regulation of mdrl gene. Methods K562 cells were treated with DOX （ 0. 01 μg/ml ） with the initial concentration of 0. 01μg/ml for 24 hours, then change the culture media without DOX. K562 cells were cultured until the its status was recovered. Subsequently the cells were treated with DOX （0. 02μg/ml） for 24 hours again. The concentration of DOX was increased until 0. 05μg/ml by following the protocol above. K562 cells were collected at the concentration of 0. 01μg/ml, 0. 03μg/ml and 0. 05μg/ml DOX. Expression of mdrl gene were examined by reverse transcription-polymerase chain reaction （RT-PCR）. Pglycoprotein （P-gp） was detected by flow cytometry. Western blot was performed to detect ERK and P-ERK. K562 cells were pretreated with MAPK inhibitor PD98059 for 1 hour, and then DOX was added. RT-PCR and FCM were used to detect the expression of rnclrl mRNA and P-gp. Results When K562 cells were exposured to DOX, the phosphorylation of ERK was increased, the mdrl gene was highly expressed as well as its corresponding protein P-gp. When the concentration of DOX was 0. 05μg/ml, the expression of mdrl gene and P-gp were increased over 5 fold. When K562 cells were pretreated with MAPK inhibitor PD98059, the expression of mdrl gene induced by DOX （ the concentration was 0. 03 μg/ml and 0. 05μg/ml ） was effectively inhibited by （ 74. 1±0. 11 ） % and （ 70. 2±0. 14 ） % respectively. Conclusions DOX could induce the expression of mdrl gene in K562 cells accompanied by the activation of MAPK/ERK pathway. The block of activation of ERK could inhibit the induced expression of mdrl gene.