GENETIC POLYMORPHISM IN PROSTATE CANCER

我们描述了NKX3.1的遗传多态，它是一种同源框基因，具有 器官特异性表达仅限于成人的前列腺。这个 多态通过用T代替C来改变密码子154，从而 导致错义密码子52，用半胱氨酸取代精氨酸 (R52C)。在对DNA样本的调查中，我们发现NKX3.1 R52C是 在4%的正常捐赠者中存在，但在12%的前列腺中存在 癌症患者，这表明NKX3.1 R52C可能增加3倍的风险 前列腺癌。此外，编码的氨基酸变化 多态增加了NKX3.1蛋白与DNA结合的亲和力。 因此，这种多态代表了一种功能获得突变 似乎会影响患前列腺癌的风险。该提案的重点是DNA NKX3.1野生型和NKX3.1的结合和转录激活 多态蛋白质。我们将确定真正的DNA结合位点 NKX3.1。我们将构建与NKX3.1结合的报告质粒 站点，以研究突变和多态之间的差异 并对NKX3.1进行缺失分析。我们将建造 嵌合NKX3.1/GAL4融合蛋白对NKX3.1作用的研究 同源结构域两侧的序列对DNA结合活性的影响 激活已知的转录因子。以增强体外培养的 表型影响氨基酸52突变，我们产生其他氨基酸 酸取代，并在报告基因分析中对它们进行表征。 最后，通过差异显示技术，我们将识别基因 由NKX3.1调节以开始理解信令 NKX3.1激活的通路及其对NKX3.1反应的研究 启动子是蛋白质在体内的靶标。

We have described a genetic polymorphism of NKX3.1, a homeobox gene with organ-specific expression confined to the prostate in the adult. The polymorphism changes codon 154 by substituting a T for a C, thereby resulting in a missense codon 52 that replaces arginine with cysteine (R52C). In a survey of DNA samples we found that NKX3.1 R52C was present in 4 percent of normal donors, but in 12 percent of prostate cancer patients, suggesting that NKX3.1 R52C may confer 3-fold risk for prostate cancer. Moreover, the amino acid alteration coded by the polymorphism increases the avidity of DNA binding by the NKX3.1 protein. Therefore, this polymorphism represents a gain-of-function mutation that appears to affect prostate cancer risk. The proposal focuses on the DNA binding and transcriptional activation by NKX3.1 wild type and polymorphic proteins. We will identify the true DNA binding site for NKX3.1. We will construct reporter plasmids with the NKX3.1 binding site in order to study differences between mutant and polymorphic proteins and to perform deletion analysis of NKX3.1. We will construct chimaeric NKX3.1/GAL4 fusion proteins to study the effects of NKX3.1 sequences flanking the homeodomain on the activity of DNA binding and activation of a known transcription factor. To enhance the in vitro phenotypic impact of the amino acid 52 mutation, we generate other amino acid substitutions and characterize them in reporter gene assays. Lastly, through differential display technology, we will identify genes that are regulated by NKX3.1 to begin to understand the signaling pathways that are activated by NKX3.1 and to study NKX3.1-responsive promoters that are in vivo targets for the protein.