ISOLATION OF GENES REQUIRED FOR HPV-MEDIATED ORAL CANCER

口腔鳞癌是世界上最常见的十大癌症之一。口腔恶性肿瘤患者的总体五年预后约为50%，低于可比的宫颈鳞癌。当与吸烟和饮酒这两个突出的危险因素相关时，宫颈癌和口腔癌的发病率都会增加。此外，宫颈癌与HPV感染密切相关。90%的宫颈癌患者存在HPV基因组的高危血清型。另一方面，关于口腔癌和HPV感染之间的因果关系的数据一直是多种多样的。尽管HPV在口腔和生殖器粘膜癌变过程中的作用相似，但在细胞特性和环境特性上也存在显著差异。这可能会导致癌症发展途径的不同。为了研究细胞转化为肿瘤时基因的差异表达，我们将利用Park等人开发的体外模型系统。在该模型中，正常口腔角质形成细胞感染HPV成为永生化细胞。然后，这些细胞暴露在一种与烟草相关的致癌物质中，这种致癌物质会将细胞转化为肿瘤。利用该模型系统，随着细胞向恶性表型的发展，我们将通过基因差异显示(MRNADD)分析来分离和鉴定差异表达基因S。然后，我们将通过Northern分析来验证mRNA DD分析，以观察在各种已建立的细胞系和原代培养中的表达模式和表达的稳定性。此外，RT-PCR和原位杂交将与患者活检组织一起进行，以确定分离的mRNA DD产物的细胞类型和阶段特异性。从mRNA DD分析中获得的上调或下调的cDNA克隆将用于表达或直接克隆，以研究其在HPV介导的致癌中的作用。该项目可能最终确定在肿瘤转化的某些阶段持续存在的特定生物标记物。然后，可以开发一种快速诊断方法，通过敏感、经济有效的程序对患者进行常规筛查。在正常的办公室探视期间可以提供早期检测，以增加口腔癌的存活率。

Oral squamous carcinoma is among the top ten most frequently occurring cancers in the world. The overall five year prognosis for patients with oral malignancies is about 50 percent, which is lower than the comparable squamous carcinoma of the cervix. The incidence of both cervical and oral carcinomas increase when associated with two prominent risk factors, cigarette smoking and alcohol consumption. In addition, cervical cancer is strongly correlated with HPV infection. Ninety percent of cervical cancer harbor "high-risk" serotype of HPV genome. On the other hand, data on the causal relationship between oral cancer and HPV infection have been variable. In spite of the similarities in the role of HPV in the development of cancer in the oral and genital mucosa, there are also significant differences in the cellular and the environmental properties of the two types of mucosa. This will likely result in differences in the pathways of cancer development. To investigate the differential expression of the genes as cells transforms to tumor, we will utilize an in vitro model system developed by Park et al. In this model, normal oral keratinocytes are infected with HPV to become immortalized cells. Then the cells are exposed to a tobacco-related carcinogen, which converts the cells to a tumor. Using this model system, we will isolate and characterize differentially expressed gene(s) by the mRNA differential display (mRNA DD) analysis, as the cells progress to the malignant phenotype. We will then confirm the mRNA DD analysis by the Northern analysis to observe the expression pattern and the stability of expression in various established cell lines and primary cultures. In addition, RT-PCR and in situ hybridization will be performed with patient biopsies to determine the cell type and the stage specificity of the isolated mRNA DD products. The up-or down-regulated cDNA clones obtained from the mRNA DD analysis will be used for either expression or direct cloning to study its function in conferring HPV-mediated carcinogenesis. This project may eventually identify specific biomarkers that are consistently present in certain stages of neoplastic conversion. A quick diagnostic method can then be developed to screen patients routinely by a sensitive, cost-effective procedure. Early detection can be provided during normal office visits to increase the chance of survival from oral cancer.