REGULATION OF HUMAN PAPILLOMAVIRUSES IN ORAL MUCOSA

人类乳头瘤病毒(HPV)与人类口腔和宫颈有关 癌症。其中大多数存在特定类型的HPV 肿瘤，这些病毒的E6和E7基因可以将细胞转化为 恶性表型。然而，细胞中存在致癌的HPV 不足以使细胞恶性过度表达 转化基因必须受到其他因素的刺激。这些因素有 在人乳头瘤病毒相关的口腔癌中未见研究。因此，该项目将 从两个方面阐明控制转化基因的因素 HPV-16和-18；HPV的长控制区(LCR)的结构 病毒，以及与LCR相互作用的细胞蛋白的作用。至 研究HPV-16或-18的LCR，将从 用聚合酶链式反应方法对口腔癌细胞和组织的基因组DNA进行了检测。 可能控制病毒表达的调节元件 基因将通过对序列的检查来识别。它们的功能 将通过构建荧光素酶表达载体和 几种细胞类型的转染法。基本序列，以及那些 那些在癌症中过度活跃或被消除的电话将被定位 利用正常和转化的口腔上皮细胞进行诱变实验 细胞。与LCR相互作用的细胞因子将在 使用体外足迹技术的细胞提取物及其在细胞提取物中的检测 使用体外足迹和电泳迁移率变化分析 紫外光交联和免疫印迹的辅助。他们在促进发展方面的作用 与LCR相邻的基因的表达将通过 定量体外转录分析和体内荧光素酶 瞬时转基因..在颁奖期结束时，这部作品 将提供关于乳头瘤病毒是如何在 正常口腔上皮细胞及其在口腔中的表达 癌细胞。已识别的细胞因子可能是靶点，而 特定的HPV LCR可以被修饰为基因治疗的启动子 未来。

Human papillomaviruses (HPV) are associated with human oral and cervical cancers. Specific types of HPV are present in the majority of these tumors, and the E6 and E7 genes of these viruses can transform cells to a malignant phenotype. However, the presence of an oncogenic HPV in a cell is not sufficient to make a cell malignant - over expression of the transforming gene must be stimulated by other factors. These factors have not been studied in HPV-related oral cancer. This project will therefore clarify two aspects of the factors that control the transforming genes from HPV-16 and -18; the structure of the long control region (LCR) of the virus, and the role of cellular proteins that interact with the LCR. to study the LCR of HPV-16 or -18, it will be isolated and sequenced from genomic DNA of oral cancer cells and tissues by a PCR method.The cis- regulating elements which probably control the expression of the viral genes will be recognized by examination of the sequence. Their function will be confirmed by construction of a luciferase expression vector and transfection of several cell types. The essential sequences, and those which are over-active or eliminated in cancer calls will be located by mutagenesis experiments, using normal and transformed oral epithelial cells. Cellular factors that interact with the LCR will be detected in cell extracts using in vitro footprinting and detected in cell extracts using in vitro footprinting and electrophoretic mobility shift assays with the aid of UV-crosslinking and immunoblotting. Their role in promoting expression of genes that are adjacent to the LCR will be examined by a quantitative in vitro transcription assay and an in vivo luciferase transient transfection.. At the conclusion of the award period, this work will have provided a description of how papillomaviruses are regulated in normal oral epithelium, and how their expression in increased in oral cancer cells. The identified cell factors could be targets and the specified HPV LCR could be modified as a promoter for gene therapy in the future.