De Siervi Adriana, Marinissen Maria, Diggs Jessica, Wang Xiao-Fan, Pages Gilles, Senderowicz Adrian
Molecular Therapeutics Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland 20892, USA.
Cancer Res. 2004 Jan 15;64(2):743-50. doi: 10.1158/0008-5472.can-03-2505.
Alkylphospholipids (ALKs) are a novel class of antitumor agents with an unknown mechanism of action. The first ALK tested in the clinic, miltefosine, has been approved recently in Europe for the local treatment of patients with cutaneous metastasis. Perifosine, the only available oral ALK, is being studied currently in human cancer clinical trials. We have shown previously that perifosine induces p21(waf1/cip1) in a p53-independent fashion and that induction of p21(waf1/cip1) is required for the perifosine-induced cell cycle arrest because cell lines lacking p21(waf1/cip1) are refractory to perifosine. In this report, we investigated the mechanism by which perifosine induces p21(waf1/cip1) protein expression. We observed that perifosine induces the accumulation of p21(waf1/cip1) mRNA without affecting p21(waf1/cip1) mRNA stability. Using several p21(waf1/cip1) promoter-driven luciferase reporter plasmids, we observed that perifosine activates the 2.4-kb full-length p21(waf1/cip1) promoter as well as a p21 promoter construct lacking p53-binding sites, suggesting that perifosine activates the p21(waf1/cip1) promoter independent of p53. The minimal p21 promoter region required for perifosine-induced p21 promoter activation contains four consensus Sp1-binding sites. Mutations in each particular Sp1 site block perifosine-induced p21(waf1/cip1) expression. Moreover, we showed that perifosine activates the mitogen-activated protein/extracellular signal-regulated kinase pathway, and this activation promotes the phosphorylation of Sp1 in known mitogen-activated protein kinase residues (threonine 453 and 739), thereby leading to increased Sp1 binding and enhanced p21(waf1/cip1) transcription. These results represent a novel mechanism by which alkylphospholipids modulate transcription, and may contribute to the discovery of new signal transduction pathways crucial for normal and neoplastic cell cycle control.
烷基磷脂（ALKs）是一类作用机制不明的新型抗肿瘤药物。首个进入临床测试的ALK——米替福新，最近在欧洲已获批用于局部治疗皮肤转移患者。目前，唯一可用的口服ALK——哌立福新正在进行人类癌症临床试验研究。我们之前已经表明，哌立福新以不依赖p53的方式诱导p21（waf1/cip1）表达，并且哌立福新诱导的细胞周期停滞需要p21（waf1/cip1）的诱导，因为缺乏p21（waf1/cip1）的细胞系对哌立福新不敏感。在本报告中，我们研究了哌立福新诱导p21（waf1/cip1）蛋白表达的机制。我们观察到哌立福新诱导p21（waf1/cip1）mRNA的积累，但不影响p21（waf1/cip1）mRNA的稳定性。使用几种p21（waf1/cip1）启动子驱动的荧光素酶报告质粒，我们观察到哌立福新激活2.4 kb全长p21（waf1/cip1）启动子以及缺乏p53结合位点的p21启动子构建体，这表明哌立福新独立于p53激活p21（waf1/cip1）启动子。哌立福新诱导p21启动子激活所需的最小p21启动子区域包含四个共有Sp1结合位点。每个特定Sp1位点的突变均会阻断哌立福新诱导的p21（waf1/cip1）表达。此外，我们表明哌立福新激活丝裂原活化蛋白/细胞外信号调节激酶途径，并且这种激活促进Sp1在已知的丝裂原活化蛋白激酶残基（苏氨酸453和739）处的磷酸化，从而导致Sp1结合增加和p21（waf1/cip1）转录增强。这些结果代表了一种烷基磷脂调节转录的新机制，可能有助于发现对正常和肿瘤细胞周期控制至关重要的新信号转导途径。
