E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation.
E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation.
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DOI：
10.1186/1471-2407-7-24
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发表时间：
2007-01-30
期刊：
影响因子：
3.8
通讯作者：
McMasters KM
中科院分区：
文献类型：
--
作者：
Hao H;Dong Y;Bowling MT;Gomez-Gutierrez JG;Zhou HS;McMasters KM
彪马是一种促凋亡的Bcl2家族成员，已被证明参与多种细胞类型的凋亡。我们试图确定PUMA的诱导在E2F-1诱导黑色素瘤细胞凋亡中是否起关键作用。用实时荧光定量聚合酶链式反应和Western印迹法检测重组病毒感染SK-MEL-2和HCT116细胞后PUMA基因和蛋白的表达水平。用双荧光素酶报告基因检测E2F-1过表达对PUMA启动子的激活作用。免疫细胞化学显示E2F-1诱导Bax易位。用caspase-9比色试剂盒检测caspase-9活性的诱导。实时定量聚合酶链式反应和Western印迹分析检测到E2F-1过表达对SK-MEL-2黑色素瘤细胞PUMA基因和蛋白表达的上调作用。为了支持这一发现，我们在PUMA启动子中发现了六个推测的E2F-1结合位点。随后的双荧光素酶报告实验表明，E2F-1的表达可使SK-MEL-2细胞中PUMA基因启动子的活性提高9.3倍。在PUMA基因敲除细胞系中，进一步研究了PUMA在E2F-1诱导细胞凋亡中的作用。细胞存活率分析表明，HCT116 PUMA-/-细胞对Ad-E2F-1介导的细胞死亡的抵抗力高于HCT116 PUMA+/+细胞。此外，在感染Ad-E2F-1的HCT116 PUMA+/+结肠癌细胞系中也发现PUMA启动子的诱导倍数为2.2倍。缺乏反式激活结构域的截短E2F-1蛋白的过表达未能上调PUMA启动子，提示PUMA可能是E2F-1的转录靶点。E2F-1诱导的癌细胞凋亡伴随着Bax从胞浆向线粒体的移位和caspase-9活性的诱导，提示E2F-1通过细胞色素C/APAF-1依赖的途径由PUMA介导。我们的研究有力地证明了E2F-1通过上调PUMA和Bax易位来诱导黑色素瘤细胞的凋亡。本文提供的信号通路将进一步加深对E2F-1诱导癌细胞凋亡作为癌症治疗策略的机制的深入了解。
PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling pathways provided here will further enhance insights on the mechanisms of E2F-1-induced cancer cell apoptosis as a strategy for cancer therapy.
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DOI：
10.1073/pnas.0401002101
发表时间：
2004-05-11
影响因子：
11.1
作者：
Iyer, NG;Chin, SF;Caldas, C
通讯作者：
Caldas, C
影响因子：
4.8
作者：
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影响因子：
56.9
作者：
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通讯作者：
Strasser, A
影响因子：
6.4
作者：
Dong, YB;Yang, HL;McMasters, KM
通讯作者：
McMasters, KM
影响因子：
56.9
作者：
Wei, MC;Zong, WX;Korsmeyer, SJ
通讯作者：
Korsmeyer, SJ