TUMOR SUPPRESSOR P53 GENE IN VIRAL HEPATOCARCINOMA

病毒性肝癌中的肿瘤抑制 P53 基因

• 批准号: 3199143
• 负责人: MEHMET OZTURK
• 金额: $ 18.22万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-25 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3199143
• 关键词: athymic mouse; gene deletion mutation; gene expression; hepatitis B virus group; human subject; human tissue; immunocytochemistry; molecular cloning; neoplasm /cancer genetics; polymerase chain reaction; transfection; tumor suppressor genes; viral carcinogenesis; western blottings

The p53 gene, which encodes a growth regulatory protein, is frequently inactivated in human cancer and is considered a tumor suppressor gene. In exploring potential molecular mechanisms of hepatocellular carcinogenesis, we discovered p53 gene inactivations in primary human hepatocellular carcinoma (PHC). The frequency of such mutations in these tumors remains to be determined, but in our preliminary studies, we found p53 abnormalities in 8 out of 9 PHC-derived cell lines. Our results strongly suggest that the inactivation of p53 gene is a significant genetic lesion in hepatocellular carcinoma (HCC). We are beginning studies to determine the relationship between p53 gene inactivation and other suspected co- factors in the process of hepatocellular carcinogenesis, particularly Hepatitis B Virus (HBV). We hypothesize that HBV could play a role in HCC by directly altering the structure or expression of p53 gene. Alternatively, inactivation of p53 function and chronic infection with HBV may have cumulative effects in the multi-step process of hepatocellular transformation. We will begin our studies by determining the frequency of p53 gene inactivation in PHCs. Tumors, as well as uninvolved liver samples from the same patients, will be analyzed at the protein level using immunochemical methods; studies of the p53 gene will include restriction enzyme analysis of genomic DNA and sequencing of conserved regions after polymerase chain reaction amplification. PHCs with demonstrated p53 gene abnormalities will be used for preparation of genomic libraries, from which p53 genes as well as HBV integration sites will be cloned and sequenced. To further investigate the relationship between p53, HBV, and hepatocellular carcinogenesis, we will conduct transfection studies on three PHC-derived cell lines: 'minimally deviant' Hep G2 cells, which appear to express 'wide-type' p53, Hep 3B cells, which have no endogenous p53, and Huh 7 cells, which express a mutant p53 protein. These cell lines will be transfected with plasmids expressing 'wild-type' or mutant p53 and/or the HBV genome. Transfected cells will be assessed for consequent phenotypic changes, such as altered growth capability and tumorigenicity. Through our demonstration that the p53 gene is inactivated in HCC, we have developed a new perspective in our studies of hepatocellular carcinogenesis. We aim to determine whether HBV interacts directly with the tumor suppressor function of p53 or contributes independently to the transformation process. We believe that these studies will be instrumental in clarifying the pathogenesis of HCC.

p53基因编码一种生长调节蛋白， 在人类癌症中失活，被认为是肿瘤抑制基因。 在 探索肝细胞癌发生的潜在分子机制， 我们在原发性人肝细胞中发现p53基因失活， 肝癌（PHC）。 这些肿瘤中此类突变的频率仍然存在 但在我们的初步研究中，我们发现p53 在9个PHC衍生的细胞系中的8个中存在异常。 我们的研究结果强烈 提示p53基因失活是一种重要的遗传损伤 肝细胞癌（HCC）。 我们正在着手研究 p53基因失活与其他疑似共- 肝细胞癌发生过程中的因素，特别是 B型肝炎病毒（HBV）。 我们推测HBV可能在HCC中发挥作用， 直接改变p53基因的结构或表达。 或者，p53功能失活和慢性HBV感染 在肝细胞的多步骤过程中可能具有累积效应， 转型 我们将开始研究， PHCs中p53基因失活。 肿瘤以及未受累的肝脏样本 将在蛋白质水平上使用 免疫化学方法; p53基因的研究将包括限制性内切酶酶切 基因组DNA的酶分析和保守区域的测序， 聚合酶链反应扩增 携带p53基因的PHCs 异常将用于制备基因组文库， p53基因以及HBV整合位点将被克隆和测序。 为进一步研究p53与HBV、HBV DNA之间的关系， 肝细胞癌发生，我们将进行转染研究， 三种PHC衍生的细胞系：“最小偏差”Hep G2细胞， 似乎表达“野生型”p53，Hep 3B细胞，其没有内源性 p53和Huh 7细胞，其表达突变型p53蛋白。 这些细胞系 将用表达“野生型”或突变型p53的质粒转染 和/或HBV基因组。 将评估转染细胞的后续结果。 表型变化，如改变的生长能力和致瘤性。 通过我们证明p53基因在HCC中失活，我们有 为肝细胞癌的研究开辟了新的视角， 致癌作用 我们的目标是确定HBV是否直接与 p53的肿瘤抑制功能或独立地促进肿瘤的发生， 转化过程 我们相信这些研究将有助于 阐明HCC的发病机制。