Hu Qun, Zhou Mu-xiang, Liu Shuang-you, Zhang Liu-qing, Liu Ai-quo, Guo Yi-jie, Song Yu
Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Er Ke Za Zhi. 2003 Dec;41(12):921-4.
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-kappaB expression and function in a P53-independent manner.
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 micro g/ml, 7.5 micro g/ml, 5 micro g/ml and 1 micro g/ml on MDM2-transfected EU-4 cells and the parent cells was detected by MTT assay.
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.
(1) MDM2 up-regulated transcriptionally P65 expression. (2) MDM2 increased drug resistance of leukemia cells to ADM. (3) MDM2 elevated NF-kappaB activity in a P53-independent manner in childhood lymphoblastic leukemia cell line.
MDM2因其抑制P53肿瘤抑制功能的能力而被视为原癌基因。但是，有证据表明MDM2在肿瘤发生过程中可能具有不依赖P53的作用。MDM2在人类肉瘤和癌中过度表达。最近的研究表明，MDM2可能作为转录因子来调节参与细胞周期调控和转化的其他基因的表达。在本研究中，研究人员假设MDM2以不依赖P53的方式直接影响NF-κB的表达和功能。
将MDM2转染至缺乏P53表达且MDM2表达水平极低的急性淋巴细胞白血病（ALL）细胞系EU-4细胞中。转染后通过蛋白质印迹法和Northern印迹法检测mRNA水平和蛋白质水平的MDM2和P65表达。由于E-选择素的表达依赖于P65，因此将E-选择素启动子-CAT构建体以及P65和MDM2表达质粒共转染至EU-4细胞中。用ELISA法测定CAT激活情况。通过MTT法检测浓度为15μg/ml、7.5μg/ml、5μg/ml和1μg/ml的阿霉素（ADM）对转染MDM2的EU-4细胞和亲本细胞的影响。
结果显示，MDM2在mRNA和蛋白质水平上均上调P65表达，并且MDM2增加了P65介导的E-选择素启动子的反式激活。没有P65时，MDM2对E-选择素的反式激活没有影响。此外，MDM2反义寡核苷酸不能改变E-选择素的反式激活。MTT结果显示，转染MDM2的EU-4细胞的存活率高于亲本细胞。结果表明，与亲本细胞相比，MDM2转染增加了EU-4细胞对ADM的耐药性。
（1）MDM2转录上调P65表达。（2）MDM2增加白血病细胞对ADM的耐药性。（3）在儿童淋巴细胞白血病细胞系中，MDM2以不依赖P53的方式提高NF-κB活性。
