BP1 transcriptionally activates bcl-2 and inhibits TNFalpha-induced cell death in MCF7 breast cancer cells.
BP1 transcriptionally activates bcl-2 and inhibits TNFalpha-induced cell death in MCF7 breast cancer cells.
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DOI：
10.1186/bcr1766
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Berg PE
中科院分区：
文献类型：
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作者：
Stevenson HS;Fu SW;Pinzone JJ;Rheey J;Simmens SJ;Berg PE
我们以前已经表明，β蛋白1（BP 1）同源结构域蛋白表达在81%的浸润性导管乳腺癌，BP 1表达增加与肿瘤进展相关。我们目前研究的目的是确定乳腺癌细胞中BP 1水平升高是否与细胞存活率增加相关。通过MTT法和Annexin V法检测稳定过表达BP 1对MCF 7细胞存活率和凋亡的影响，并通过检测caspase的活化来检测。TNFα诱导细胞凋亡。利用实时荧光定量PCR和蛋白质印迹分析研究了BP 1对糖尿病相关基因的潜在调控作用。电泳迁移率变动分析，定点诱变，和瞬时测定进行具体表征的相互作用的BP 1与bcl-2基因的启动子。BP 1的稳定过表达可抑制TNFα诱导的MCF 7乳腺癌细胞凋亡。BP 1的增加导致caspase-7、caspase-8和caspase-9的加工和活化减少，以及caspase底物聚（ADP-核糖）聚合酶（PARP）的失活。全长PARP水平的增加和半胱天冬酶原-8的减少也与BP 1过表达相关。bcl-2基因是BP 1的直接靶标，因为：（i）BP 1蛋白在体外结合到bcl-2 P1启动子上游的共有结合序列。(ii)BP 1过表达的MCF 7细胞bcl-2 mRNA和蛋白水平均升高。(iii)瞬时分析表明，bcl-2启动子活性的增加是由于BP 1蛋白的直接结合和调节。BP 1的表达也阻止了TNFα介导的bcl-2 mRNA和蛋白的下调。这些发现表明，BP 1的增加可能会使乳腺癌细胞具有生存优势，这可能会导致患者对治疗药物的耐药性增加。
We have previously shown that the Beta Protein 1 (BP1) homeodomain protein is expressed in 81% of invasive ductal breast carcinomas, and that increased BP1 expression correlates with tumor progression. The purpose of our current investigation was to determine whether elevated levels of BP1 in breast cancer cells are associated with increased cell survival. Effects on cell viability and apoptosis of MCF7 cells stably overexpressing BP1 were determined using MTT and Annexin V assays, and through examination of caspase activation. TNFα was used to induce apoptosis. The potential regulation of apoptosis-associated genes by BP1 was studied using real-time PCR and western blot analyses. Electrophoretic mobility shift assays, site-directed mutagenesis, and transient assays were performed to specifically characterize the interaction of BP1 with the promoter of the bcl-2 gene. Stable overexpression of BP1 led to inhibition of apoptosis in MCF7 breast cancer cells challenged with TNFα. Increased BP1 resulted in reduced processing and activation of caspase-7, caspase-8, and caspase-9, and inactivation of the caspase substrate Poly(ADP-Ribose) Polymerase (PARP). Increased levels of full-length PARP and a decrease in procaspase-8 were also associated with BP1 overexpression. The bcl-2 gene is a direct target of BP1 since: (i) BP1 protein bound to a consensus binding sequence upstream of the bcl-2 P1 promoter in vitro. (ii) MCF7 cells overexpressing BP1 showed increased levels of bcl-2 mRNA and protein. (iii) Transient assays indicated that increased bcl-2 promoter activity is due to direct binding and modulation by BP1 protein. BP1 expression also prevented TNFα-mediated downregulation of bcl-2 mRNA and protein. These findings suggest mechanisms by which increased BP1 may impart a survival advantage to breast cancer cells, which could lead to increased resistance to therapeutic agents in patients.