HPV, Genetic Instability & Oral Cancer

描述：（申请人提供）人类频繁感染 口腔中的乳头瘤病毒 (HPV) 已被发现与 HIV 免疫功能低下者有关 儿童和成人。 HIV感染者更容易被感染 具有多种 HPV 亚型，包括 16 型和 18 型。这些“高风险”HPV 与恶性口腔癌的发生密切相关的是： 病毒 DNA 常见于口腔癌细胞和组织中。而且， 用克隆的“高 风险”HPV基因组使这些细胞永生化，这些细胞可以完全转化为 当接触化学致癌物时，细胞会发生转化。由于 (1) 相同 化学致癌剂不能转化 NHOK 细胞以及 (2) 基因组的丢失 完整性是肿瘤细胞的标志，“高危”HPV必须发挥作用 通过破坏 NHOK 细胞的恶性转化过程中发挥关键作用 细胞维持基因组完整性的能力。保持基因组完整性 通过不断修复DNA损伤；因此，DNA 修复的干扰会导致 突变，最终导致细胞恶性转化。的 该项目的中心假设是 NHOK 细胞感染“高 风险”HPV致癌基因破坏DNA修复；以及抑制 HPV 癌基因 表达允许肿瘤前人类口腔上皮细胞高表达 风险”HPV致癌基因恢复其DNA修复活性和基因组 诚信。为了检验这一假设，申请人提出以下建议 具体目的：（1）确定基础DNA和基因毒剂诱导的DNA NHOK细胞的修复活性，转染HPV-16基因组的HOK细胞， 和肿瘤前口腔上皮细胞（来自病变活检） 表达“高危”HPV； (2)探讨HPV-16癌基因的影响 对 NHOK 细胞基础和基因毒剂诱导的 DNA 修复活性的影响；和 (3)研究“高危”HPV核酶对DNA修复活性的影响 次黄嘌呤磷酸核糖基转移酶的突变频率（和率） (hprt) 人口腔上皮细胞癌前和癌变基因 （表示“高风险”HPV）源自病变活检。申请人 希望回答以下问题：“高危”HPV 是否会扰乱 修复NHOK细胞的DNA损伤？如果是的话，哪个 DNA 修复过程是 受损？病毒致癌基因是否造成了这种破坏？如果是的话，是 HPV 癌基因导致 p53 或 pRB 失活，这完全是导致 破坏？肿瘤前期或肿瘤细胞（表达“高危”HPV） 来自人类口腔病变活检的DNA修复具有相同的范围 NHOK细胞转染“高危”HPV基因组是否存在缺陷？是否 病毒癌基因转录物的破坏可恢复 DNA 修复活性， HPV 永生化 HOK 细胞（肿瘤前期和肿瘤）的基因组完整性 人类口腔上皮细胞（来自病变活检）表达“高风险”HPV 癌基因？

DESCRIPTION: (Provided by Applicant) Frequent infection with human papillomavirus (HPV) in the oral cavity has been noted in HIV immunocompromised children and adults. HIV-infected individuals are more susceptible to infection with multiple HPV subtypes, including types 16 and 18. These "high risk" HPVs that are closely associated with development of malignant oral cancer: the viral DNA is frequently found in oral cancer cells and tissue. Moreover, transfection of normal human oral keratinocyte (NHOK) cells with cloned "high risk" HPV genome immortalizes these cells, which can convert to fully transformed cells when exposed to chemical carcinogens. Since (1) the same chemical carcinogens cannot transform NHOK cells and (2) the loss of genomic integrity is the hallmark of neoplastic cells, "high risk" HPV must play a critical role in the malignant transformation of NHOK cells by disrupting cells' ability to maintain genomic integrity. Genomic integrity is maintained by constant repair of DNA damage; thus, disturbance of DNA repair results in mutations, which ultimately induces malignant transformation of cells. The central hypothesis of the project is that infection of NHOK cells with "high risk" HPV oncogenes disrupts DNA repair; and that inhibition of HPV oncogene expression allows pre-neoplastic human oral epithelial cells expressing high risk" HPV oncogenes to regain their DNA repair activities and genomic integrity. To test this hypothesis, the applicants propose the following specific aims: (1) to determine the basal and genotoxic agent-induced DNA repair activities of NHOK cells, HOK cells transfected with the HPV-16 genome, and pre-neoplastic oral epithelial cells (derived from lesion biopsies) expressing "high risk" HPV; (2) to investigate the effects of HPV-16 oncogenes on basal and genotoxic agent-induced DNA repair activities of NHOK cells; and (3) to study the effect of "high risk" HPV ribozymes on DNA repair activities and mutation frequency (and rate) of hypoxanthine phosphoribosyl transferase (hprt) gene of pre-neoplastic and neoplastic human oral epithelial cells (expressing "high risk" HPV) derived from lesion biopsies. The applicants expect to answer the following questions: Does "high risk" HPV disrupt the repair of DNA damage in NHOK cells? If so, which DNA repair process is impaired? Are viral oncogenes responsible for such disruption? If so, is the inactivation of p53 or pRB by HPV oncogenes solely responsible for the disruption? Do pre-neoplastic or neoplastic cells (expressing "high risk" HPV) derived from human oral lesion biopsies have the same spectrum of DNA repair defects as NHOK cells transfected with "high risk" HPV genome? Does the disruption of viral oncogene transcripts restore the DNA repair activites and genomic integrity of HPV-immortalized HOK cells, pre-neoplastic and neoplastic human oral epithelial cells (from lesion biopsies) expressing "high risk" HPV oncogenes?