Park Y G, Park S, Lim S O, Lee M S, Ryu C K, Kim I, Cho-Chung Y S
Department of Biochemistry, Korea University College of Medicine, Seoul, 136-701, Korea.
Biochem Biophys Res Commun. 2001 Mar;281(5):1213-9. doi: 10.1006/bbrc.2001.4521.
We have previously demonstrated that the activation of p53 signaling may contribute to tumor growth inhibition by the CRE-decoy oligonucleotide containing CRE sequence (5'-TGACGTCA-3') (Lee et al., Biochemistry 39, 4863-4868, 2000). However, growth inhibition by CRE-decoy treatment was also observed in tumor cells containing a mutant p53 (Park et al., J. Biol. Chem. 274, 1573-1580, 1999). To understand additional mechanisms of the decoy oligonucleotide, we investigated the effect on cyclin D1 expression and a cyclin D1/Cdk4/retinoblastoma protein (pRB) signaling pathway. Here we show that in MCF7 breast cancer cells the CRE-decoy competed with cyclin D1-CRE (5'-TAACGTCA-3') for binding transcription factors and reduced cyclin D1 gene expression (in reporter gene assay, Northern blotting and Western blotting) to modulate cyclin D1/Cdk4/pRB signaling and G1-S progression in a steady state and/or under estrogen stimulation. Decrease of cyclin D1 protein level by CRE-decoy treatment was also observed in p53-mutated cancer cells. Cyclin D1 expression was also diminished in MCF7 cells stably expressing dominant negative mutant CREB indicating that the nonspecific effect of oligonucleotide or its degradation products could be excluded. These data suggest that inhibition of cyclin D1 expression contributes to the growth inhibition induced by the decoy oligonucleotide in MCF7 cells through a cyclin D1/Cdk4/pRB signaling pathway. Downregulation of cyclin D1 expression also provides a mechanism of CRE-decoy-induced growth inhibition in tumor cells having p53 mutation.
我们之前已经证明，含CRE序列（5'-TGACGTCA-3'）的CRE诱饵寡核苷酸激活p53信号通路可能有助于抑制肿瘤生长（Lee等人，《生物化学》39卷，4863 - 4868页，2000年）。然而，在含有突变型p53的肿瘤细胞中也观察到了CRE诱饵处理对生长的抑制作用（Park等人，《生物化学杂志》274卷，1573 - 1580页，1999年）。为了了解诱饵寡核苷酸的其他作用机制，我们研究了其对细胞周期蛋白D1表达以及细胞周期蛋白D1/Cdk4/视网膜母细胞瘤蛋白（pRB）信号通路的影响。在此我们表明，在MCF7乳腺癌细胞中，CRE诱饵与细胞周期蛋白D1 - CRE（5'-TAACGTCA-3'）竞争结合转录因子，并降低细胞周期蛋白D1基因表达（在报告基因检测、Northern印迹和Western印迹中），以在稳态和/或雌激素刺激下调节细胞周期蛋白D1/Cdk4/pRB信号通路和G1 - S期进程。在p53突变的癌细胞中也观察到CRE诱饵处理使细胞周期蛋白D1蛋白水平降低。在稳定表达显性负性突变型CREB的MCF7细胞中，细胞周期蛋白D1的表达也减少，这表明可以排除寡核苷酸或其降解产物的非特异性作用。这些数据表明，抑制细胞周期蛋白D1表达通过细胞周期蛋白D1/Cdk4/pRB信号通路导致MCF7细胞中诱饵寡核苷酸诱导的生长抑制。细胞周期蛋白D1表达的下调也为p53突变的肿瘤细胞中CRE诱饵诱导的生长抑制提供了一种机制。
