Racial differences in CYP1B1 polymorphisms and risks for prostate cancer

 描述（由申请人提供）：在美国，与高加索人（Cau）相比，非洲裔美国人（AfA）男性前列腺癌的发病率和死亡率更高。这种差异的原因尚不清楚，但研究表明，细胞色素P450 1B 1（CYP 1B 1）的单核苷酸多态性（SNP），一种激活致癌物质的基因，参与前列腺癌的发病机制。该项目的目标是确定CYP 1B 1 SNP是否是种族相关前列腺癌的风险因素，我们假设这些变体：1）AfA和Cau之间存在差异，2）有助于种族相关前列腺癌及其进展，（3）与前列腺组织水平相关，（4）导致酶水平和活性差异。为实现该项目的目标，提出了两个具体目标。目的#1将研究CYP 1B 1的SNPs是否是种族相关前列腺癌的危险因素。血液DNA和前列腺组织将从患有良性前列腺增生（BPH）和各种阶段和等级的前列腺癌的AfA和Cau患者中收集;以及从年龄匹配的健康男性志愿者中收集血液DNA。将进行以下实验：（a）通过序列特异性PCR和直接基因组测序确定CYP 1B 1启动子和编码区中的SNP。将确定CYP 1B 1 SNP和单倍型与种族和前列腺癌易感性和进展的相关性。(b)将通过免疫组织化学监测BPH和癌组织中CYP 1B 1的定位。通过交叉引用SNP和单倍型，将确定多态性对组织CYP 1B 1水平的影响。在目标#2中，将研究目标#1中确定的CYP 1B 1 SNP的机制效应。将进行以下实验：（a）通过克隆构建CYP 1B 1野生型质粒，并使用定点诱变产生多态性变体，（B）将启动子构建体转染到种族特异性前列腺细胞系中，并通过荧光素酶报告基因测定法测定其对基因表达的影响，（c）将SNP构建体转染到大肠杆菌中以表达改变的蛋白质，并使用CYP 1B 1测定法测定其酶活性。成功完成拟议研究将阐明（a）CYP 1B 1多态性作为种族相关前列腺癌易感性和进展的风险因素和生物标志物的作用，以及（B）CYP 1B 1变体对酶水平和活性的影响。了解CYP 1B 1基因变异在种族特异性前列腺癌中的功能作用将为这种疾病的管理提供改进的策略。

 DESCRIPTION (provided by applicant): In the USA, African-American (AfA) men have higher incidence and death rates due to prostate cancer when compared to Caucasians (Cau). The reasons for this discrepancy are unknown but studies have shown that single nucleotide polymorphisms (SNPs) of cytochrome P450 1B1 (CYP1B1), a gene that activates carcinogens, to be involved in prostate cancer pathogenesis. The goal of this project is to determine if CYP1B1 SNPs are risk factors for race-related prostate cancer and we hypothesize that these variants: 1) differ between AfA and Cau, 2) contribute to race-related prostate cancer and its progression, (3) are associated with prostate tissue levels, and (4) cause differences in enzyme levels and activity. Two specific aims are proposed to achieve the goals of the project. Aim #1 will investigate whether SNPs of CYP1B1 are risk factors for race- related prostate cancer. Blood DNA and prostate tissue will be collected from AfA and Cau patients with benign prostatic hyperplasia (BPH) and various stages and grades of prostate cancer; as well as blood DNA from age- matched, healthy male volunteers. The following experiments will be performed: (a) SNPs in the CYP1B1 promoter and coding region will be determined by sequence-specific PCR and direct genomic sequencing. The correlation of CYP1B1 SNPs and haplotypes with race and prostate cancer susceptibility and progression will be determined. (b) Localization of CYP1B1 in BPH and cancer tissues will be monitored by immunohistochemistry. By cross-referencing SNPs and haplotypes, the effects of polymorphisms on tissue CYP1B1 levels will be determined. In Aim #2, the mechanistic effects of CYP1B1 SNPs identified in Aim #1 will be investigated. The following experiments will be performed: (a) Construct CYP1B1 wildtype plasmids by cloning and generate polymorphic variants using site-directed mutagenesis, (b) Transfect promoter constructs into race-specific prostate cell lines and determine their effects on gene expression by luciferase reporter assay, (c) Transfect SNP constructs into E coli to express altered proteins and determine their enzymatic activity by utilizing a CYP1B1 assay. Successful completion of the proposed research will elucidate the (a) role of CYP1B1 polymorphisms as a risk factor and biomarker for race-related prostate cancer susceptibility and progression, and (b) effect of CYP1B1 variants on enzymatic levels and activities. Understanding the functional role of the CYP1B1 gene variants in race-specific prostate cancer will provide improved strategies for the management of this disease.