Lee Te-Hsiu, Chang Hui-Chiu, Chuang Lea-Yea, Hung Wen-Chun
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC.
Biochem Pharmacol. 2003 Aug 1;66(3):371-7. doi: 10.1016/s0006-2952(03)00258-2.
We have previously shown that tamoxifen (Tam) inhibits proliferation of estrogen receptor-negative human non-small cell lung cancer cells and this inhibition is associated with induction of p27(Kip1). In this study, we investigated the mechanism by which Tam increases p27(Kip1) expression. Because intracellular p27(Kip1) protein level is mainly controlled via posttranslational regulation, we first tested whether Tam might affect protein stability of p27(Kip1). Metabolic labeling and pulse chase assays showed that Tam did not affect the half-life of this protein. We next examined whether Tam enhanced p27(Kip1) expression through transcriptional activation. Our results demonstrated that Tam directly stimulated the p27(Kip1) promoter in lung cancer cells. Deletion and mutation analysis revealed that two Sp1 consensus sites located between -545 and -532bp from the transcription start site were crucial for the induction of p27(Kip1) by Tam. Conversely, mutation in a CTF site (-525/-520) nearby these two Sp1 sites had little effect. Electromobility shift assays showed that Sp1 transcription factor bound to these consensus sites and the DNA binding activity of Sp1 was enhanced by Tam. Our data also demonstrated that induction of p27(Kip1) by Tam was inhibited by protein kinase A inhibitor H89, but not by protein kinase C inhibitor calphostin C and mitogen-activated kinase kinase inhibitor PD98059. Taken together, our results suggest that Tam transcriptionally activates p27(Kip1) expression via the Sp1 consensus sites in the p27(Kip1) promoter and PKA is involved in this process.
我们之前已经表明，他莫昔芬（Tam）可抑制雌激素受体阴性的人非小细胞肺癌细胞的增殖，且这种抑制作用与p27（Kip1）的诱导有关。在本研究中，我们调查了Tam增加p27（Kip1）表达的机制。由于细胞内p27（Kip1）蛋白水平主要通过翻译后调控来控制，我们首先测试了Tam是否可能影响p27（Kip1）的蛋白稳定性。代谢标记和脉冲追踪实验表明，Tam不影响该蛋白的半衰期。接下来，我们检测了Tam是否通过转录激活来增强p27（Kip1）的表达。我们的结果表明，Tam可直接刺激肺癌细胞中的p27（Kip1）启动子。缺失和突变分析显示，位于转录起始位点-545至-532bp之间的两个Sp1共有序列位点对于Tam诱导p27（Kip1）至关重要。相反，这两个Sp1位点附近的CTF位点（-525 / -520）发生突变影响很小。电泳迁移率变动分析表明，Sp1转录因子与这些共有序列位点结合，并且Tam可增强Sp1的DNA结合活性。我们的数据还表明，蛋白激酶A抑制剂H89可抑制Tam对p27（Kip1）的诱导，但蛋白激酶C抑制剂钙泊三醇C和丝裂原活化激酶激酶抑制剂PD98059则无此作用。综上所述，我们的结果表明，Tam通过p27（Kip1）启动子中的Sp1共有序列位点转录激活p27（Kip1）表达，并且PKA参与了这一过程。
