CELL CYCLE, P53, AND DNA REPAIR IN ORAL CARCINOGENESIS

有令人信服的证据表明，致癌作用是一个多步骤的过程 并且多种遗传损伤是人类发展癌症所必需的。 沿着遗传病变，p53蛋白的改变（由于突变 p53基因或“高危”人乳头瘤病毒[HPV]感染， 例如，在一个实施例中，16或18型HPV）是最常见的遗传性疾病， 人类癌症包括口腔癌。大量证据表明 支持p53突变是口腔癌发生的早期事件。我们 研究还表明，含有人口腔角质形成细胞（HOK）的人口腔角质形成细胞（HOK 可忽略量的野生型（wt）p53蛋白（由于HPV DNA 整合）和HOK表达突变体（mt）p53蛋白是永生的，但 而不是致瘤的。这些细胞在暴露于 烟草致癌物而具有正常p53补体的细胞则没有， 表明p53功能障碍似乎是口腔癌的早期事件， 致癌作用因此，p53蛋白的功能障碍可能是一个重要的因素。 至少在口腔癌发生的早期事件， 其他基因的遗传性疾病将正常细胞转化为 人类口腔中的肿瘤细胞。 由于野生型p53蛋白在调节细胞凋亡中起主要作用， 周期停滞，我们假设正常人口腔角质形成细胞 含有野生型p53蛋白修复受损的DNA比口服更有效 p53功能缺陷的角质形成细胞。许多研究表明， 包括我们的初步数据，表达野生型p53蛋白的细胞具有 在细胞周期进程中建立短暂延迟的能力 当暴露于遗传毒性剂时，但具有缺陷p53功能的细胞 不具备这样的能力。由于细胞周期的短暂停滞 在复制之前修复受损的DNA是必要的。 受损的DNA模板和受损的染色体分离，具有 有缺陷的p53功能可能失败或修复能力有限， 当暴露于遗传毒性物质时，DNA受损。在这项研究中，我们 将通过确定主要烟草的影响来测试上述假设- 致癌物对（1）细胞周期的进展，主要的表达 生长停滞和DNA损伤诱导基因（例如，p53、WAF 1/CIP 1和 gadd 45）和细胞周期蛋白依赖性激酶（cdks）的活性;（2） 宿主染色体DNA（DNA加合物和单链DNA）的遗传毒性 断裂）;（3）受损DNA的修复;（4）突变频率 和在表达wt p53的正常HOK、表达HPV-16的HOK或 HPV-18 E6蛋白、表达HOK的突变型p53蛋白和HPV永生化的 口腔角质形成细胞这些拟议的研究应该有助于我们获得更多 深入了解烟草相关口腔癌发生的分子机制。

There is compelling evidence that carcinogenesis is a multistep process and multiple genetic lesions are necessary to develop cancer in human. Along the genetic lesions, the alteration of p53 protein (due to mutation of p53 gene or by infection of "high risk" human papillomaviruses [HPV], e.g., type 16 or 18 HPV) is the most frequently found genetic disorder in human cancers including oral cancer. A significant body of evidence supports p53 mutation as an early event in oral carcinogenesis. Our studies have also shown that human oral keratinocytes (HOK) containing negligible amount of wild-type (wt) p53 protein (because of HPV DNA integration) and HOK expressing mutant (mt) p53 protein are immortal, but not tumorigenic. These cells convert to tumorigenic cells when exposed to tobacco-carcinogens. whereas cells with a normal complement of p53 do not, indicating that p53 dysfunction appears to be an early event in oral carcinogenesis. Therefore, the dysfunction of p53 protein appears be an early event at least in oral carcinogenesis and also be necessary for subsequent genetic disorders of other genes to convert normal cells to tumor cells in the human oral cavity. Inasmuch as wt p53 protein plays a major role in the regulation of cell cycle arrest, we hypothesize that normal human oral keratinocytes containing wt p53 protein repair damaged DNA more efficiently than oral keratinocytes with defective p53 function. As demonstrated by many studies including our preliminary data, cells expressing wt p53 protein have the ability to establish transient delays in the progression of cell cycle when exposed to genotoxic agents, but cells with defective p53 function do not possess such ability. Since the transient arrest of the cell cycle progression is necessary to repair damaged DNA prior to replication of damaged DNA template and segregation of damaged chromosome, cells with defective p53 function may fail or have limited ability to repair the damaged DNA when exposed to genotoxic agents. In the proposed study, we will test the above hypothesis by determining the effect of major tobacco- carcinogens on (1) the progression of cell cycle, the expression of major growth arrest and DNA damage inducible genes (e.g., p53, WAF1/CIP1, and gadd45), and the activity of cyclin-dependent kinases (cdks); (2) the genotoxicity of host chromosomal DNA (DNA adducts and single strand DNA breaks); (3) the repair of damaged DNA; and (4) the mutation frequencies and spectrum in normal HOK expressing wt p53, HOK expressing HPV-16 or HPV-18 E6 protein, HOK expressing mutant p53 protein, and HPV-immortalized oral keratinocytes. These proposed studies should help us gain more insight into molecular mechanisms of tobacco-related oral carcinogenesis.