PATHOLOGY OF CHEMICAL CARCINOGENISIS

本提案将探讨若干相互关联的共同点 由化学致癌物在肝细胞中产生，包括 改变核质RNA转运，核增大， 核纤层核苷三磷酸酶的改变 （NTR），一种被认为调节RNA运输的酶。 致癌物选择性地产生活性的永久性升高 46 kD NTR的分子量。 最近的测序数据表明， 46-kD NTR代表来自N-末端的3个α-螺旋结构域， 核纤层蛋白A/C末端区。 基于差分 光亲和标记和磷酸化，我们假设， NTR（它也显示出与许多 核癌基因蛋白和E. coli ATP酶）可能代表 磷酸化完整核纤层蛋白的激酶活性; 核纤层蛋白的磷酸化与细胞（核） 生长和有丝分裂。 老鼠将被喂食致癌食物， 在不同的时间间隔后处死。 增生灶（vs. 背景肝）和坦率的肝细胞癌将被 考察 提供生物学背景以识别 与致癌作用有关的变化，我们还将检查肝脏 经历急性期反应，肝脏从 化学和手术损伤，以及单次暴露后的肝脏 致癌物的反应 将分析这些制剂， 改变：1）RNA加工和区室化，使用 多种cDNA探针和体外转运试验， 维持核RNA序列的适当限制; 2） 核大小/倍性和核孔数/密度，使用流式细胞仪 细胞计数和冷冻断裂技术; 3）核纤层蛋白A/C 磷酸化;和4）NTR活性，使用酶测定 和cDNA探针。 NTR cDNA探针将从大鼠中获得 使用由以下设计的合成多核苷酸的cDNA文库： 氨基酸测序数据，或通过交叉杂交大鼠cDNA 用人核纤层蛋白A/C探针构建文库，并将其克隆， 测序 这些探测器还将用于检查 致癌物质会导致NTR基因发生变化。 我们的基本 假设致癌物改变了核表达， 核纤层，导致核结构的改变（通过 核纤层蛋白磷酸化）和RNA转运：这些变化将 产生一系列的表型改变，导致（根据 适当的促进影响）到最终的恶性 转换，从而提供了一个启发式的解释， 与癌变相关的表型表达改变。

This proposal will explore a number of interrelated commonalities produced in hepatocytes by chemical carcinogens, including altered nucleocytoplasmic RNA transport, nuclear enlargement, and alterations in nuclear lamina nucleosidetriphosphatase (NTPase), an enzyme thought to regulate RNA transport. Carcinogens selectively produce permanent elevations in activity of the 46-kD NTPase. Recent sequencing data demonstrate that the 46-kD NTPase represents 3 alpha-helical domains from the N- terminal region of lamins A/C. On the basis of differential photoaffinity labeling and phosphorylation, we hypothesize that the NTPase (which also shows weak homology to a number of nuclear oncogene proteins and an E. coli ATPase) may represent the kinase activity which phosphorylates intact nuclear lamins; phosphorylation of lamins is integrally related to cellular (nuclear) growth and mitosis. Rats will be fed a carcinogenic diet and sacrificed after various intervals. Hyperplastic foci (vs. background liver) and frank hepatocellular carcinomas will be examined. To provide a biological context for identification of changes pertinent to carcinogenesis, we will also examine livers undergoing the acute phase response, livers regenerating from chemical and surgical insult, and livers following a single exposure to hepatocarcinogens. These preparations will be analyzed for alterations in: 1) RNA processing and compartamentation, using a variety of cDNA probes and an in vitro transport assay which maintains appropriate restriction of nuclear RNA sequences; 2) nuclear size/ploidy and nuclear pore number/density, using flow cytometric and freeze-fracture techniques; 3) lamin A/C phosphorylation; and 4) NTPase activity, using enzymatic assays and cDNA probes. NTPase cDNA probes will be obtained from rat cDNA libraries using synthetic polynucleotides designed from amino acid sequencing data, or by cross-hybridizing rat cDNA libraries with human lamin A/C probes, and will be cloned and sequenced. These probes will also be used to examine whether carcinogens produce changes in the NTPase gene. Our basic hypothesis is that carcinogens modify expression of the nuclear lamina NTPase, leading to alterations in nuclear structure (via lamin phosphorylation) and RNA transport: These changes would produce a cascade of phenotypic alterations, leading (under suitable promotional influences) to eventual malignant transformation, thus providing a heuristic explanation for the altered phenotyic expression associated with carcinogenesis.