Wang Q, Zambetti G P, Suttle D P
Department of Pharmacology, College of Medicine, University of Tennessee, Memphis 38163, USA.
Mol Cell Biol. 1997 Jan;17(1):389-97. doi: 10.1128/MCB.17.1.389.
DNA topoisomerase II (topo II) is an essential nuclear enzyme involved in major cellular functions such as DNA replication, transcription, recombination, and mitosis. While an elevated level of topo II alpha is associated with cell proliferation, wild-type (wt) p53 inhibits the expression of various growth-stimulatory genes. To determine if p53 downregulates topo II alpha gene expression, a murine cell line, (10)1val, that expresses a temperature-sensitive p53 was utilized. The (10)1val cells had significantly lower levels of topo II alpha mRNA and protein following incubation for 24 h at 32 degrees C (p53 with wt conformation) than at 39 degrees C (p53 with mutant conformation). The effect of p53 on the human topo II alpha gene promoter was determined by using luciferase reporter plasmids containing varying lengths of the topo II alpha promoter transiently cotransfected into p53-deficient (10)1 cells together with wt or mutant p53 expression plasmids. Transcription from the full-length (bp -557 to +90) topo II alpha promoter was decreased 15-fold by wt p53 in a concentration-dependent manner, whereas mutant p53 exerted much weaker inhibition. Consecutive deletion of the five inverted CCAAT elements (ICEs) from the topo II alpha promoter reduced both the basal promoter activity and wt p53-induced suppression. Transcription of the minimal promoter (-32 to +90), which contains no ICE, was slightly stimulated by wt or mutant p53 expression. When point mutations were introduced into the most proximal ICE (-68), the inhibitory effect of wt p53 was alleviated and stimulation of topo II alpha expression resulted. Our study suggests that wt p53 functions as a transcriptional repressor of topo II alpha gene expression, possibly through a functional interaction with specific ICEs. Inactivation of wt p53 may reduce normal regulatory suppression of topo II alpha and contribute to abortive cell cycle checkpoints, accelerated cell proliferation, and alterations in genomic stability associated with neoplasia.
DNA拓扑异构酶II（拓扑异构酶II）是一种重要的核酶，参与DNA复制、转录、重组和有丝分裂等主要细胞功能。虽然拓扑异构酶IIα水平升高与细胞增殖相关，但野生型（wt）p53抑制多种生长刺激基因的表达。为了确定p53是否下调拓扑异构酶IIα基因表达，使用了一种表达温度敏感型p53的小鼠细胞系(10)1val。(10)1val细胞在32℃（具有野生型构象的p53）孵育24小时后，拓扑异构酶IIα mRNA和蛋白水平显著低于在39℃（具有突变型构象的p53）时。通过使用含有不同长度拓扑异构酶IIα启动子的荧光素酶报告质粒，将其与野生型或突变型p53表达质粒一起瞬时共转染到p53缺陷的(10)1细胞中，来确定p53对人拓扑异构酶IIα基因启动子的影响。野生型p53以浓度依赖的方式使全长（-557至+90碱基对）拓扑异构酶IIα启动子的转录降低15倍，而突变型p53的抑制作用则弱得多。从拓扑异构酶IIα启动子连续缺失五个反向CCAAT元件（ICEs）会降低基础启动子活性和野生型p53诱导的抑制作用。不含ICE的最小启动子（-32至+90）的转录受到野生型或突变型p53表达的轻微刺激。当在最靠近的ICE（-68）中引入点突变时，野生型p53的抑制作用减轻，导致拓扑异构酶IIα表达受到刺激。我们的研究表明，野生型p53可能通过与特定ICEs的功能相互作用，作为拓扑异构酶IIα基因表达的转录抑制因子发挥作用。野生型p53的失活可能会降低对拓扑异构酶IIα的正常调节抑制，并导致细胞周期检查点失败、细胞增殖加速以及与肿瘤形成相关的基因组稳定性改变。
