摘要
目的 MDM2是一种原癌基因 ,可与P5 3结合使P5 3失去抑癌功能。有证据表明 ,MDM2可能有P5 3不依赖的癌基因活性 ,作为一个转录因子直接调节细胞周期调控和转化过程中的其他基因的表达。研究显示 ,NF κB的P6 5亚型是一个肿瘤药物诱导的细胞凋亡的重要抑制因子 ,在细胞转化及凋亡的调控中起着重要的作用。探讨儿童急性淋巴细胞白血病MDM 2 ,NF κB及P5 3相互作用的机制。方法 应用儿童急性淋巴细胞白血病细胞EU 4细胞株作为模型 ,转染MDM2进入EU 4细胞 ,用免疫印迹及Northern杂交法检测转染前后P6 5的表达。因为E Selectin的表达是P6 5依赖性的 ,共转染MDM 2 ,P6 5及E Selectin启动子CAT报告基因进入EU 4细胞 ,用ELISA测CAT活性。同时分别将不同浓度的阿霉素 (15 μg/ml,7 5 μg/ml,5 μg/ml,及 1μg/ml)加入转染前后的EU 4细胞 ,用MTT法检测细胞的药物敏感性。结果 MDM2的转染能上调P6 5的表达并增加了NF κB的活性 ,该作用为P5 3不依赖性。而在缺乏P6 5的情况下 ,MDM2对CAT活性没有影响。MDM2反义寡核苷酸处理对CAT活性没有影响。MTT检测表明 ,稳定转染MDM2的细胞对阿霉素(ADM )耐药。结论 (1)MDM2上调了儿童白血病细胞P6 5的表达。 (2 )MDM2 增加了白血病细胞对阿霉素的抗药性。 (3)在P5
Objective MDM2 is considered a proto-oncogene due to its ability to inhibit P53 tumor-suppressor function. But, evidence showed that MDM2 might have a P53-independent role in tumorigenesis. MDM2 is over-expressed in human sarcoma and carcinoma. Recent studies showed that MDM2 might act as a transcriptional factor to modulate expressions of other genes involved in cell cycle regulation and transformation. In the present study, the investigators hypothesized that MDM2 directly affected NF-κB expression and function in a P53-independent manner.Methods MDM2 was transfected to acute lymphoblastic leukemia (ALL) line EU-4 cells lacking P53 expression and expressing very low levels of MDM2.MDM2 and P65 expression in mRNA level and protein level were detected by Western blot and Northern blot after transfection. Since the expression of E-selectin is P65 dependent, E-selectin promoter-CAT construct and P65 and MDM2 expression plasmids were co-transfected to EU-4 cells. CAT activation was determined with ELISA. The effect of adriamycin (ADM) at the concentrations of 15 μg/ml, 7.5 μg/ml, 5 μg/ml and 1 μg/ml on MDM2-transfected EU-4 cells and the parent cells was detected by MTT assay.Results The results showed that MDM2 up-regulated P65 expression at both mRNA and protein levels, and MDM2 increased P65-mediated transactivation of E-selectin promoter. Without P65, MDM2 had no effect on the transactivation of E-selectin. Moreover, MDM2 antisense could not change the transactivation of E-selectin. MTT results showed that the survival rate of MDM2 transfected EU-4 cells was higher than that of parental cells. The results suggested that MDM2 transfection increase drug resistance of EU-4 cells to ADM compared with parent cells.Conclusion ①MDM2 up-regulated transcriptionally P65 expression. ②MDM2 increased drug resistance of leukemia cells to ADM. ③MDM2 elevated NF-κB activity in a P53-independent manner in childhood lymphoblastic leukemia cell line.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2003年第12期921-924,共4页
Chinese Journal of Pediatrics
基金
国家自然科学基金资助 (3 9970 778)
