Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Cancer Cell Int. 2010 Feb 19;10:3. doi: 10.1186/1475-2867-10-3.
p27(Kip1) is a cyclin-dependent kinase inhibitor that inhibits G1-to-S phase transition of the cell cycle. It is known that a relatively large number of nutritional and chemopreventive anti-cancer agents specifically up-regulate expression of p27 without directly affecting the expression of other G1-to-S phase cell cycle regulatory proteins including p21(Cip1Waf1). However, the upstream molecular signaling pathways of how these agents up-regulate the expression of p27 have not been well characterized. The objective of this study was to identify such pathways in human breast cancer cells in vitro using 4-hydroxytamoxifen, dexamethasone, and various retinoic acids as examples of such anti-cancer agents.
Experimental evidence presented in the first half of this report was obtained by transfecting human breast cancer cells in vitro with proximal upstream region of p27 gene-luciferase reporter plasmids. 1) The evidence indicated that 4-hydroxytamoxifen, dexamethasone, and various retinoic acids up-regulated expression of p27 in both estrogen receptor-positive and negative human breast cancer cells in vitro. 2) The degree of up-regulation of p27 expression by these anti-cancer agents in human breast cancer cells in vitro linearly correlated with the degree of inhibition of methylnitrosourea (MNU)-induced rat mammary adenocarcinoma in vivo. 3) Lastly, up-regulation of the expression of p27 was likely due to the activation of translation initiation rather than transcription of p27 gene. The experimental evidence presented in the second half of this report was obtained by a combination of Western immunoblot analysis and transfection analysis. It indicated that 4-hydroxytamoxifen and dexamethasone up-regulated expression of p27 by down-regulating phosphorylation of eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) at Ser65 and this phosphorylation was likely to be mediated by upstream receptor tyrosine kinases/phosphoinositide-3-kinase/Akt/5'-AMP-activated protein kinase/mammalian target of rapamycin (RTKs/PI3K/Akt/AMPK/mTOR) protein kinase signaling pathways. Retinoic acids up-regulated expression of p27 without using either 4E-BP1 or RTKs/PI3K/Akt/AMPK/mTOR protein kinase signaling pathways.
4-Hydroxytamoxifen and dexamethasone up-regulated translation initiation of p27 by down-regulating 4E-BP1 phosphorylated at Ser65 and this down-regulation seemed to be mediated by upstream RTKs/PI3K/Akt/AMPK/mTOR protein kinase signaling pathways. Retinoic acids also up-regulated translation initiation of p27, but without using any of these pathways.
p27(Kip1)是一种细胞周期蛋白依赖性激酶抑制剂，可抑制细胞周期的 G1 期向 S 期的转变。已知，大量营养和化学预防抗癌剂特异性地上调 p27 的表达，而不直接影响其他 G1 期至 S 期细胞周期调节蛋白的表达，包括 p21(Cip1/Waf1)。然而，这些试剂如何上调 p27 表达的上游分子信号通路尚未得到很好的描述。本研究的目的是使用 4-羟基他莫昔芬、地塞米松和各种维甲酸作为抗癌剂的例子，在体外鉴定人乳腺癌细胞中的这些途径。
本报告前半部分的实验证据是通过将 p27 基因启动子区域的报告质粒转染入体外培养的人乳腺癌细胞获得的。1) 这些证据表明，4-羟基他莫昔芬、地塞米松和各种维甲酸在体外均能上调雌激素受体阳性和阴性的人乳腺癌细胞中 p27 的表达。2) 这些抗癌剂在体外对人乳腺癌细胞中 p27 表达的上调程度与甲基亚硝脲（MNU）诱导的大鼠乳腺癌在体内的抑制程度呈线性相关。3) 最后，p27 表达的上调可能是由于翻译起始的激活而不是 p27 基因的转录。本报告后半部分的实验证据是通过 Western 免疫印迹分析和转染分析相结合获得的。结果表明，4-羟基他莫昔芬和地塞米松通过下调真核翻译起始因子 4E（eIF4E）结合蛋白 1（4E-BP1）在丝氨酸 65 位的磷酸化而上调 p27 的表达，这种磷酸化可能是由上游受体酪氨酸激酶/磷脂酰肌醇 3-激酶/蛋白激酶 B（Akt）/5'-AMP 激活蛋白激酶/哺乳动物雷帕霉素靶蛋白（RTKs/PI3K/Akt/AMPK/mTOR）蛋白激酶信号通路介导的。维甲酸上调 p27 的表达而不使用 4E-BP1 或 RTKs/PI3K/Akt/AMPK/mTOR 蛋白激酶信号通路。
4-羟基他莫昔芬和地塞米松通过下调丝氨酸 65 位磷酸化的 4E-BP1 而上调 p27 的翻译起始，这种下调似乎是由上游 RTKs/PI3K/Akt/AMPK/mTOR 蛋白激酶信号通路介导的。维甲酸也上调了 p27 的翻译起始，但不使用这些途径。
