Activation of nuclear factor κB by polyamines in breast cancer cells
Activation of nuclear factor κB by polyamines in breast cancer cells
复制标题
DOI：
10.1021/bi991291v
复制
发表时间：
1999-11-09
期刊：
影响因子：
2.9
通讯作者：
Thomas, TJ
中科院分区：
文献类型：
--
作者：
Shah, N;Thomas, T;Thomas, TJ
多胺——腐胺、亚精胺和精胺——参与乳腺癌细胞的生长。多胺作用的一个可能目标是转录因子及其反应元件的智能作用位点。 NF-kappa B 是 rel 转录因子家族的成员，可调节增殖/抗凋亡途径中基因的转录。我们采用电泳迁移率变动分析来研究多胺在 NF-κ B 与 NF-κ B 反应元件 (NRE) 结合中的作用，其共有序列是 GGGGAATTCCCC。使用 MCF-7 乳腺癌细胞的细胞提取物，我们发现在不存在多胺的情况下 NF-kappa B 与 NRE 的结合非常少。添加 1 mM 亚精胺或精胺分别导致 NF-κ B-NRE 结合增加 4 倍和 6 倍。腐胺在 2 mM 浓度下诱导结合增加 2 倍。使用抗体超位移测定，我们确定 NF-kappa B 的 p50 亚基是多胺存在下 NF-kappa B-NRE 复合物形成的主要成分。然而，在抗 p65、c-rel、relB 和 p52 抗体存在下，对应 NF-κ B-NRE 复合物的条带强度降低，表明这些亚基也参与其中。精胺还使用其他乳腺癌细胞系和正常乳腺上皮细胞系的细胞提取物刺激 NF-kappa B-NRE 结合。观察到精胺类似物对 NF-κ B-NRE 结合的不同作用，其中精胺发挥最大作用。含有寡核苷酸的 NRE 的 CD 光谱是不对称的，并且与典型的 B-DNA CD 光谱不同。在多胺存在下，观察到双链体 NRE 的 T-m 呈浓度依赖性增加。在使用 NF-kappa B 驱动的分泌型碱性磷酸酶 (SEAP) 报告基因的瞬时转染实验中，精胺诱导的 NF-kappa B 活性约为对照的 2 倍。精胺诱导的 NF-kappa B 激活也使用 NF-kappa B-EGFP（增强型绿色荧光蛋白）载体在瞬时转染中得到证实，其中绿色荧光蛋白的表达通过荧光显微镜可视化。这些数据显示了多胺的基因调控功能，涉及 NF-kappa B 与 NRE 的增强结合，以及多胺在乳腺癌细胞增殖中作用的可能机制。
Polyamines-putrescine, spermidine, and spermine-are involved in the growth of breast cancer cells. A possible target of polyamine action is at the site of intel action of transcription factors with their response elements. NF-kappa B is a member of the rel family of transcription factors that regulate transcription of genes in the proliferative/anti-apoptotic pathways. We psrformed electrophoretic mobility shift assays to study the role of polyamines in NF-kappa B binding to NF-kappa B response elements (NREs), the consensus sequence of which is GGGGAATTCCCC. Using cellular extract from MCF-7 breast cancer cells, we found very little binding of NF-kappa B to NRE in the absence of polyamines. Addition of 1 mM spermidine or spermine caused a 4- and 6-fold increase in NF-kappa B-NRE binding, respectively. Putrescine induced a 2-fold increase in the binding at 2 mM concentration. Using antibody supershift assays, we identified the p50 subunit of NF-kappa B to be a major component in NF-kappa B-NRE complex formation in the presence of polyamines. However, the decreased intensity of the band corresponding to NF-kappa B-NRE complex in the presence of anti-p65, c-rel, relB and p52 antibodies suggested the participation of these subunits also. Spermine also stimulated NF-kappa B-NRE binding using cellular extracts from other breast cancer cell lines and a normal breast epithelial cell line. A differential effect of spermine analogues on NF-kappa B-NRE binding was observed, with spermine exerting the maximal effect. CD spectra of NRE containing oligonucleotides was asymmetric and distinct from that of a typical B-DNA CD spectrum. A concentration-dependent increase in T-m of the duplex NRE was seen in the presence of polyamines. In transient transfection experiments using an NF-kappa B driven secreted alkaline phosphatase (SEAP) reporter, spermine induced NF-kappa B activity by similar to 2-fold as compared to controls. Spermine induced activation of NF-kappa B was also confirmed using an NF-kappa B-EGFP (enhanced green fluorescent protein) vector in transient transfections in which expression of the green fluorescent protein was visualized by fluorescence microscopy, These data show a gene regulatory function of polyamines involving enhanced binding of NF-kappa B to NRE and a possible mechanism for the action of polyamines in breast cancer cell proliferation.