CELL CYCLE, P53, AND DNA REPAIR IN ORAL CARCINOGENESIS

有令人信服的证据表明致癌是一个多步骤的过程。 而多种基因损伤是人类发生癌症所必需的。 沿着遗传损伤，P53蛋白的改变(由于突变 P53基因感染或“高危”人乳头瘤病毒感染， 例如，16型或18型HPV)是 包括口腔癌在内的人类癌症。一大堆证据 支持P53突变是口腔癌发生的早期事件。我们的 研究还表明，人类口腔角质形成细胞(HOK)含有 可以忽略不计的野生型(Wt)P53蛋白(由于HPV DNA 整合)和HOK表达突变型(Mt)p53蛋白是不朽的，但 不会致癌的。当接触到这些细胞时，这些细胞会转化为肿瘤细胞 烟草--致癌物质。而具有正常P53补体的细胞则不是， 这表明P53功能障碍似乎是口腔疾病的早期事件 致癌。因此，P53蛋白的功能障碍似乎是一种 至少在口腔癌发生的早期事件，也是必要的 其他基因的后续遗传性疾病将正常细胞转化为 人体口腔中的肿瘤细胞。 由于wt-p53蛋白在细胞的调节中起主要作用。 周期停滞，我们假设正常人口腔角质形成细胞 含有wt p53蛋白的DNA修复比口服更有效 P53功能缺陷的角质形成细胞。正如许多研究表明的那样 包括我们的初步数据，表达wt p53蛋白的细胞具有 在细胞周期进程中建立暂时性延迟的能力 当暴露在遗传毒性物质中时，但具有P53功能缺陷的细胞会 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是必需的进展 受损的DNA模板和受损的染色体分离，细胞 P53功能缺陷可能会失败或修复能力有限 当接触到遗传毒性物质时，DNA会受到损伤。在建议的研究中，我们 将通过确定主要烟草的影响来检验上述假设- 致癌物对(1)细胞周期的进展，对主要 生长停滞和DNA损伤诱导基因(例如，P53、WAF1/CIP1和 GADD45)和细胞周期蛋白依赖性蛋白激酶(CDKs)的活性； 宿主染色体DNA(DNA加合物和单链DNA)的遗传毒性 (3)受损DNA的修复；(4)突变频率 正常HOK表达Wt P53，HOK表达HPV-16或 HPV-18E6蛋白、HOK表达突变型P53蛋白和HPV永生化 口腔角质形成细胞。这些拟议的研究应该会帮助我们获得更多 烟草相关口腔癌发生的分子机制研究。

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.