HPV, Genetic Instability & Oral Cancer

描述：(申请人提供)经常感染人类 口腔中的乳头瘤病毒(HPV)已被发现与HIV免疫功能低下有关 无论是孩子还是成年人。感染艾滋病毒的人更容易感染 有多种HPV亚型，包括16型和18型。这些高危HPV 与恶性口腔癌的发展密切相关的： 病毒DNA经常在口腔癌细胞和组织中发现。此外， 克隆的HIGH基因转染人正常口腔角质形成细胞 HPV基因组使这些细胞永生化，这些细胞可以完全转化为 当接触到化学致癌物时，转化的细胞。由于(1)相同 化学致癌物不能转化NHOK细胞和(2)基因组的损失 诚信是肿瘤细胞的标志，“高危”HPV必须发挥作用 在NHOK细胞破坏恶性转化过程中的关键作用 细胞维持基因组完整性的能力。保持了基因组的完整性 通过不断修复DNA损伤；因此，DNA修复的干扰导致 突变，最终导致细胞恶性转化。这个 该项目的中心假设是，感染NHOK细胞的人 风险：HPV癌基因破坏DNA修复；抑制HPV癌基因 表达使癌前人类口腔上皮细胞高表达 HPV癌基因恢复DNA修复活性和基因组的风险 正直。为了验证这一假设，申请者提出了以下建议 具体目标：(1)确定基础DNA和遗传毒剂诱导的DNA NHOK细胞、转HPV-16基因组的HOK细胞的修复活性 和癌前口腔上皮细胞(来源于病变活检) 表达“高危”HPV；(2)研究HPV-16癌基因的作用 基础和遗传毒剂诱导的NHOK细胞的DNA修复活性；以及 (3)研究高危HPV核酶对DNA修复活性的影响 和次黄嘌呤磷酸核糖转移酶的突变频率(和突变率) 人口腔癌前病变及癌前病变口腔上皮细胞(HPRT)基因的研究 (表达“高危”HPV)来源于病变活检。申请者 预计将回答以下问题：“高风险”的HPV是否会扰乱 修复NHOK细胞中的DNA损伤？如果是这样的话，哪个DNA修复过程 受伤了吗？病毒致癌基因是造成这种干扰的原因吗？如果是这样，那么 人乳头瘤病毒癌基因导致的p53或pRb失活 颠覆？肿瘤前病变细胞或肿瘤细胞(表达“高危”HPV) 取自人类口腔病变活检组织的DNA修复谱相同 缺陷：NHOK细胞被高危的HPV基因组所感染？这是不是 破坏病毒癌基因转录本可以恢复DNA修复活性和 HPV永生化HOK细胞、癌前病变和肿瘤的基因组完整性 人类口腔上皮细胞(取自病变活检组织)表达“高危”HPV 致癌基因？

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?