Marreiros Alexandra, Dudgeon Kip, Dao Vinh, Grimm Marc-Oliver, Czolij Robert, Crossley Merlin, Jackson Paul
Oncology Research Centre, Prince of Wales Hospital, Randwick, NSW, Australia.
Oncogene. 2005 Jan 20;24(4):637-49. doi: 10.1038/sj.onc.1208216.
A molecular mechanism to explain reduced KAI1 expression in invasive and metastatic tumour cells remains elusive. In this report, we extend an earlier study in bladder cells to confirm that a 76 bp region of the KAI1 promoter (residues -922 to -847), with binding motifs for p53, AP1 and AP2, is required for high level activity of a KAI1 reporter in prostate cancer cell lines. Gel shift and supershift experiments supported binding of p53, junB and heterodimers of AP2alpha/AP2gamma or AP2beta/AP2gamma to this sequence. Introduction of mutations into specific motifs demonstrated an essential requirement for p53 and junB to reporter activity, and that functional synergy between these two factors enhanced activity. A further elevation of reporter activity required AP2. Roles of individual p53, junB and AP2 proteins, as well as functional synergy between p53 and junB, were confirmed in transfection experiments. Western blotting analysis showed that an absence of wild-type p53, and/or a loss of junB and AP2 protein expression, correlated with downregulation of KAI1 mRNA levels in a series of prostate cancer cell lines. A loss of p53 function and/or expression of junB, combined with reduced expression of specific AP2 proteins may underly downregulated KAI1 expression in tumour cells.
目前仍不清楚一种能够解释侵袭性和转移性肿瘤细胞中KAI1表达降低的分子机制。在本报告中，我们扩展了早期对膀胱细胞的研究，以证实KAI1启动子的一个76bp区域（-922至-847位残基）对前列腺癌细胞系中KAI1报告基因的高水平活性是必需的，该区域具有p53、AP1和AP2的结合基序。凝胶迁移和超迁移实验支持p53、junB以及AP2α/AP2γ或AP2β/AP2γ异二聚体与该序列的结合。对特定基序引入突变表明，p53和junB对报告基因活性至关重要，并且这两个因子之间的功能协同作用增强了活性。报告基因活性的进一步提高需要AP2。在转染实验中证实了单个p53、junB和AP2蛋白的作用，以及p53和junB之间的功能协同作用。蛋白质印迹分析表明，在一系列前列腺癌细胞系中，野生型p53的缺失和/或junB和AP2蛋白表达的丧失与KAI1 mRNA水平的下调相关。p53功能和/或junB表达的丧失，以及特定AP2蛋白表达的降低，可能是肿瘤细胞中KAI1表达下调的基础。
