Racial differences in CYP1B1 polymorphisms and risks for prostate cancer

 描述(申请人提供)：在美国，非裔美国人(AFA)男性前列腺癌的发病率和死亡率高于高加索男性(CAU)。这种差异的原因尚不清楚，但研究表明，激活致癌物的细胞色素P450 1B1基因的单核苷酸多态(SNPs)参与了前列腺癌的发病。这个项目的目标是确定CYP1B1 SNP是否是种族相关前列腺癌的危险因素，我们假设这些变异：1)在AFA和CAU之间不同，2)导致种族相关前列腺癌及其进展，(3)与前列腺组织水平有关，以及(4)导致酶水平和活性的差异。为实现该项目的目标，提出了两个具体目标。目的#1将调查细胞色素P1B1的SNPs是否是与种族相关的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症(BPH)的AFA和CAU患者以及不同阶段和级别的前列腺癌患者身上采集；以及来自年龄匹配的健康男性志愿者的血液DNA。将进行以下实验：(A)通过序列特异性聚合酶链式反应和基因组直接测序来确定CYP1B1启动子和编码区的SNPs。将确定CYP1B1 SNPs和单倍型与种族、前列腺癌易感性和进展的相关性。(B)将通过免疫组织化学方法监测细胞色素P1B1在前列腺增生症和癌组织中的定位。通过交叉参考SNPs和单倍型，将确定多态对组织CYP1B1水平的影响。在目标#2中，将研究在目标#1中确定的CYP1B1单核苷酸多态的机制效应。将进行以下实验：(A)通过克隆构建CYP1B1野生型质粒，并通过定点突变产生多态变体；(B)将启动子构建导入RACE特异性前列腺细胞系，并通过荧光素酶报告实验确定其对基因表达的影响；(C)将SNP构建体导入大肠杆菌表达改变的蛋白，并利用CYP1B1检测其酶活性。这项拟议研究的成功完成将阐明(A)作为种族相关前列腺癌易感性和进展的危险因素和生物标记物的CYP1B1多态的作用，以及(B)CYP1B1变异对酶水平和活性的影响。了解CYP1B1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.