PATHOLOGY OF CHEMICAL CARCINOGENISIS

本提案将探讨一些相互关联的共性。 由化学致癌物在肝细胞中产生，包括 核质RNA转运改变，核增大， 和核膜核苷三磷酸酶的改变 (NTPase)，一种被认为调节RNA运输的酶。 致癌物选择性地产生永久性的活性升高 46kD的NTPase。最近的测序数据表明 46-kD的NTPase代表来自N-的3个α-螺旋结构域 基于差分的板层A/C端部区域 光亲和标记和磷酸化，我们假设 NTPase(也显示出与一些 核癌基因蛋白和大肠杆菌ATPase)可能代表 使完整的核纤层磷酸化的激酶活性； 层粘连蛋白的磷酸化与细胞(核)密切相关 生长和有丝分裂。老鼠将被喂食致癌的食物和 在不同的时间间隔后被牺牲。增生性病灶(与 背景：肝)和肝细胞癌 检查过了。为鉴定提供生物学背景 与致癌有关的变化，我们还将检查肝脏 在经历急性期反应后，肝脏从 化学和外科侮辱，以及一次暴露后的肝脏 致癌物质。将对这些准备工作进行分析 改变：1)RNA处理和分隔，使用 多种cdna探针和体外转运试验 保持对核RNA序列的适当限制；2) 核大小/倍性和核孔数量/密度，使用Flow 细胞学和冷冻断裂技术；3)层蛋白A/C 磷酸化；以及4)NTPase活性，使用酶分析 和cDNA探针。将从大鼠身上获得NTPase cDNA探针 利用合成的多核苷酸构建的cDNA文库 氨基酸测序数据，或通过杂交大鼠的cDNA 带有人层粘连蛋白A/C探针的文库，并将被克隆和 已排序。这些探头也将被用来检查 致癌物会导致NTPase基因发生变化。我们的基础 假说是致癌物改变了细胞核的表达 板层NTPase，导致核结构改变(VIA Lamin磷酸化)和RNA运输：这些变化将 产生一连串的表型变化，导致(在 适当的促进影响)到最终的恶性 转换，从而提供了启发式解释 表型表达改变与癌变有关。

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.