Lewis Joan S, Vijayanathan Veena, Thomas T J, Pestell Richard G, Albanese Chris, Gallo Michael A, Thomas Thresia
Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey, NJ 08903, USA.
Oncol Res. 2005;15(3):113-28. doi: 10.3727/096504005776367924.
Estradiol (E2) and the naturally occurring polyamines (putrescine, spermidine, and spermine) play important roles in breast cancer cell growth and differentiation. We examined the effects of E2 and spermine on the phosphorylation and DNA binding of activating transcription factor-2 (ATF-2) in MCF-7 breast cancer cells. ATF-2 is a transcription factor involved in estrogenic regulation of cyclin D1 gene, and thereby cell cycle progression. DNA affinity immunoblot assays showed a six- to eightfold increase in the binding of ATF-2 to a 74-mer ATF/CRE oligonucleotide (ODN1) from cyclin D1 promoter in the presence of 4 nM E2 and 0.5 mM spermine, compared to untreated control. Individual treatments with E2 or spermine caused a twofold or lower increase in ATF-2 binding to ODN1. Immunoblotting with phospho-ATF-2 antibody showed that increased DNA binding of ATF-2 was associated with its phosphorylation. A p38 MAP kinase inhibitor, PD169316, inhibited ATF-2 phosphorylation. In contrast, the MEK-ERK1/2 inhibitor, PD98059, or the JNK inhibitor, SP600125, had no significant effect on DNA binding of ATF-2. Cyclin D1 promoter (-1745CD1) activity increased by approximately 12-fold (above control) in the presence of E2 and spermine, compared to a sixfold increase in the presence of E2 alone and a twofold increase with spermine. Cells transfected with a dominant negative mutant of ATF-2 showed decreased transactivation of cyclin D1 promoter in response to E2 and spermine. These results indicate that spermine can enhance E2-induced cell signaling and cyclin D1 transcription by activation of the p38 MAP kinase and phosphorylation of ATF-2, contributing to breast cancer cell proliferation.
雌二醇（E2）和天然存在的多胺（腐胺、亚精胺和精胺）在乳腺癌细胞的生长和分化中发挥着重要作用。我们研究了E2和精胺对MCF-7乳腺癌细胞中激活转录因子-2（ATF-2）磷酸化和DNA结合的影响。ATF-2是一种参与细胞周期蛋白D1基因雌激素调节从而影响细胞周期进程的转录因子。DNA亲和免疫印迹分析显示，与未处理的对照相比，在存在4 nM E2和0.5 mM精胺的情况下，ATF-2与细胞周期蛋白D1启动子的74聚体ATF/CRE寡核苷酸（ODN1）的结合增加了6至8倍。单独用E2或精胺处理导致ATF-2与ODN1的结合增加两倍或更低。用磷酸化ATF-2抗体进行免疫印迹显示，ATF-2 DNA结合增加与其磷酸化有关。p38丝裂原活化蛋白激酶抑制剂PD169316抑制ATF-2磷酸化。相反，MEK-ERK1/2抑制剂PD98059或JNK抑制剂SP600125对ATF-2的DNA结合没有显著影响。与单独存在E2时增加6倍和精胺时增加2倍相比，在存在E2和精胺的情况下，细胞周期蛋白D1启动子（-1745CD1）活性增加了约12倍（高于对照）。用ATF-2显性负突变体转染的细胞显示，对E2和精胺的反应中细胞周期蛋白D1启动子的反式激活降低。这些结果表明，精胺可通过激活p38丝裂原活化蛋白激酶和ATF-2磷酸化来增强E2诱导的细胞信号传导和细胞周期蛋白D1转录，从而促进乳腺癌细胞增殖。
