ISOLATION OF HUMAN HEPATOCYTE ENHANCED GROWTH VARIANTS

人肝细胞增强生长变异体的分离

• 批准号: 2097712
• 负责人: David G. Kaufman
• 金额: $ 20.85万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-04 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2097712
• 关键词: SCID mouse; aflatoxins; athymic mouse; cell cycle; cell growth regulation; cell type; chemical carcinogen; chemical carcinogenesis; gene expression; hepatocellular carcinoma; human tissue; karyotype; liver cells; morphology; neoplasm /cancer genetics; neoplastic process; neoplastic transformation; preneoplastic state; tissue /cell culture; transfection

Hyperplastic foci that appear in livers of rats after treatments with hepatocarcinogens may represent progeny of clones of initiated hepatocytes. When we place liver cells from rats treated in this manner into cell culture soon after treatment, a number of hepatocytes proliferated under conditions in which normal hepatocytes failed to grow or senesced. We have suggested that hepatocytes with this enhanced- growth variant (EGV) phenotype (able to proliferate in cell culture) may be the initiated clonal progenitors for focus-forming hepatocytes in vivo. Since normal hepatocytes are programmed such that they do not to grow or grow for only a very limited duration in cell culture, presumably these EGV cells have undergone some alteration in their ability to negatively control cell proliferation. Negative control of cell proliferation is manifest at the level of initiation of DNA replication and mitosis. Whereas human cells normally delay entry into S and/or M phases when DNA is damaged, this capacity is lost in EGV's. We hypothesize that these two alterations of negative regulation of cell proliferation (EGV phenotype and checkpoint failure) many be manifestations of the same genetic defect. From this we predict that cells that develop the EGV phenotype also should have impaired ability to inhibit initiation of S and/or M phases (G1 and G2 checkpoint delays) and replicon initiation when their DNA is damaged. Hepatocyte EGV's have been generated after transfection of human fetal liver cells with selected oncogenes. To test our hypothesis we propose to induce several human hepatocyte EGV's by transfection of various growth promoting genes or by treatment with chemical oncogenes. To test our hypothesis we propose to induce several human hepatocyte EGV's by transfection of various growth promoting genes or by treatment with chemical carcinogens. Characteristics of cultured human hepatocytes will be evaluated including morphology, karyotype and expression of hepatocyte-specific genes. Hepatocytes will be transfected with various genes (ori-SV40, truncated human c-myc, HPV E6/E7, etc.) during the initial phase of active cell proliferation or treated with chemical carcinogens using a novel protocol involving conditional EGV's. EGV's will be isolated based on their abilities to grow under conditions in which normal cells fail to grow or senesce. We will determine if the hepatocyte EGV phenotype is consistently associated with a defect in negative regulation of cell cycle progression, regardless of how the EGV's are induced. The intactness of the G/D and G2/M checkpoints and the ability to inhibit replicon initiation will be evaluated. This should test whether alteration in control of cell proliferation and the EGV phenotype are characteristics of an early stage of neoplastic evolution.

大鼠肝脏经药物治疗后出现增生灶 肝癌物质可能代表起始的克隆的后代 肝细胞。 当我们将以这种方式处理的大鼠的肝细胞放入 治疗后不久进入细胞培养物，大量肝细胞 在正常肝细胞无法生长的条件下增殖 或衰老。 我们建议肝细胞具有这种增强- 生长变异（EGV）表型（能够在细胞培养物中增殖）可能 是形成病灶的肝细胞的起始克隆祖细胞 体内。 由于正常肝细胞的编程使其不会 大概在细胞培养物中生长或仅生长非常有限的时间 这些 EGV 细胞的能力发生了一些改变 负控制细胞增殖。 细胞阴性对照 增殖表现在 DNA 复制起始水平 和有丝分裂。 而人类细胞通常会延迟进入 S 和/或 M 当 DNA 受损时，EGV 就会丧失这种能力。 我们 假设细胞负调节的这两种改变 增殖（EGV 表型和检查点故障）很多是 相同基因缺陷的表现。 由此我们预测 形成 EGV 表型的细胞也应该具有受损的能力 抑制 S 和/或 M 期的启动（G1 和 G2 检查点延迟） 当 DNA 受损时，复制子就会启动。 肝细胞 EGV 有 转染人胎儿肝细胞后产生 选定的癌基因。 为了检验我们的假设，我们建议归纳几个 通过转染各种生长促进基因获得人肝细胞 EGV 或通过化学致癌基因治疗。 为了检验我们的假设，我们 提议通过转染诱导几种人肝细胞 EGV 各种生长促进基因或用化学致癌剂治疗。 将评估培养的人肝细胞的特征，包括 肝细胞特异性基因的形态、核型和表达。 肝细胞将转染各种基因（ori-SV40，截短的 人c-myc、HPV E6/E7等）在活跃细胞的初始阶段 增殖或使用新方案用化学致癌剂进行处理 涉及有条件的EGV。 EGV 将根据其情况进行隔离 在正常细胞无法生长的条件下生长的能力或 衰老。 我们将确定肝细胞 EGV 表型是否为 始终与细胞负调节缺陷相关 循环进程，无论 EGV 是如何诱导的。 这 G/D 和 G2/M 检查点的完整性以及抑制能力 将评估复制子的启动。 这应该测试是否 细胞增殖控制和 EGV 表型的改变 肿瘤演化早期阶段的特征。