Mutagenicity/Genotoxicity with Endogenous PGH Synthase-2

描述(申请人提供)：前列腺素H合成酶(PGHS)在 在炎症和癌变中起重要作用。PGHS表格2(PGHS-2) 有丝分裂原、生长因子和肿瘤促进剂诱导的突变和 遗传毒性。细菌检测不合适，因为反应性毒物 由PGHS-2产生的细菌寿命短，不容易与细菌 薄膜。 我们的目标是开发比色/化学发光和荧光 实时定量聚合酶链式反应(QPCR)定量检测DNA损伤 内源性人PGHS-2。在第一阶段，我们在人DNA中表达了人PGHS-2 修复缺陷的成纤维细胞系XPA用 苯并(A)芘-7，8-二氢二醇及16.2kb定量聚合酶链式反应检测DNA损伤 用比色法/化学发光法测定线粒体DNA。 在第二阶段，我们将改进比色法/化学发光法 内控DNA，建立荧光实时定量聚合酶链式反应方法。我们会 也可以用精通DNA损伤修复的人成纤维细胞进行QPCR检测 将含有PGHS-2基因的载体导入细胞后，建立细胞系。 表达XPA的人PGHS-1也将建立定量聚合酶链式反应。公用事业 将通过对细胞进行处理来探索其中的几种分析方法 环境毒物。 建议的商业应用： PGHS-2的高表达可能导致包括结肠癌在内的人类肿瘤的发展。对在真核细胞中表达的PGHS-2进行可靠的致突变性和遗传毒性检测是非常必要的。因此，利用人PGHS-2：在DNA修复缺陷和熟练成纤维细胞中表达的人PGHS-2，将建立简便的比色/ECL和荧光实时定量聚合酶链式反应(QPCR)检测方法。QPCR检测试剂盒将用于筛选依赖PGHS-2的前诱变剂和DNA损伤剂以及化学预防药物。

DESCRIPTION (provided by applicant): Prostaglandin H synthase (PGHS) plays a significant role in inflammation and carcinogenesis. PGHS form 2 (PGHS-2) induced by mitogens, growth factors and tumor promotors causes mutagenicity and genotoxicity. A bacterial test is not suitable because reactive toxicants produced by PGHS-2 are short-lived and cannot easily cross the bacterial membrane. Our objective is to develop colorimetric/chemiluminescent and fluorescent real-time quantitative PCR (QPCR) assays to quantitate DNA damage mediated by endogenous human PGHS-2. In Phase I, we expressed human PGHS-2 in human DNA repair deficient fibroblast cell line, XPA, treated the cells with benzo(a)pyrene-7,8-dihydrodiol and measured DNA damage by QPCR of 16.2 kb mitochondrial DNA using colorimetric/chemiluminescent assays. In Phase II, we will improve the colorimetric/chemiluminescent QPCR assay internal control DNAs and develop a fluorescent real-time QPCR assay. We will also develop QPCR assays with a DNA damage repair-proficient human fibroblast cell line after transfection of the cells with a PGHS-2 gene-containing vector. QPCR assays will also be developed with human PGHS-1 expressing XPA. Utilities of the assays will be explored by treatment of the cells with several environmental toxicants. PROPOSED COMMERCIAL APPLICATION: Increased expression of PGHS-2 may lead to development of human tumors including colon cancer. Reliable mutagenicity and genotoxicity assays with PGHS-2 expressed in eukaryotic cells is in great demand. Thus, facile colorimetric/ECL and fluorescent Real-Time quantitative PCR (QPCR) assays will be developed with human PGHS-2 :expressed in human DNA repair deficient and proficient fibroblasts. The QPCR assay kits will be marketed to screen PGHS-2-dependent promutagens and DNA damaging agents as well as chemopreventive agents.