TWO-DIMENSIONAL GEL ANALYSIS OF CARCINOGENESIS

致癌作用的二维凝胶分析

• 批准号: 3774790
• 负责人: M J MILLER
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3774790
• 关键词: biphenyl compounds; carcinogenesis; chemical carcinogen; chemical carcinogenesis; epithelium; gel electrophoresis; gene induction /repression; genetic promoter element; genetic strain; haptoglobins; hemolysis; laboratory rat; molecular oncology; neoplastic transformation; phosphorylation; plasmids; protein biosynthesis; proteins; protooncogene; transfection; western blottings; zinc

The objective of this project is to study the mechanism of carcinogenesis using quantitative two-dimensional gel electrophoresis (2D-gel). This technique lets us examine both qualitative and quantitative changes in the synthesis of thousands of cellular polypeptides as the cell undergoes neoplastic transformation. Research has focused on two areas: a study of the changes induced in dog plasma by the known urinary bladder carcinogen 4-aminobiphenyl (4-ABP), and an examination of the effects of an inducible c-raf proto-oncogene on the 2D-gel patterns of rat liver epithelial (RLE) cells. The first study, involving the effects of 4-ABP on dog plasma, was done to determine if there were deletions in plasma proteins specific to 4-ABP, and to identify such proteins. Two sets of proteins, which were greatly reduced during 4-ABP treatment, were observed. One was identified as the beta-chain of haptoglobin. We hypothesize that 4-ABP binds to hemoglobin, causing hemolysis. This ABP-hemoglobin complex then binds to haptoglobin and is purged from the blood stream. A second set of three spots (MW = 65 kD, pI = 6.5-6.6) disappears much more rapidly than the haptoglobin and recovers more quickly after dosing stops. Despite intense efforts, we have not been able to identify this protein. The second study has employed RLE cells transfected with a plasmid containing the c-raf-1 proto-oncogene under the control of an inducible metallo-thionein promoter. We anticipated that the raf gene would be induced in the trans- fectants by appropriate concentrations of zinc and by examining proteins that were phosphorylated in response to increased raf synthesis we might identify potential effector proteins for raf. Twenty subclones were isolated and at least two were shown to be inducible by high concentrations of zinc. However, no consistent changes were observed in the 2D-gel pattern of induced versus uninduced cells. Western blot analysis with three different anti-raf antibodies failed to demonstrate the presence of induced raf protein. It appears, then, that the plasmid raf mRNA is induced, but not translated in vivo.

本项目的目的是研究癌症发生的机制 使用定量二维凝胶电泳（2D-gel）。 这 这项技术让我们可以检查在过去的一年中， 合成数以千计的细胞多肽， 肿瘤性转化 研究集中在两个领域： 已知膀胱致癌物在犬血浆中诱导的变化 4-氨基联苯（4-ABP），并检查诱导的 c-raf原癌基因在大鼠肝上皮细胞（RLE）二维凝胶电泳中的表达 细胞 第一项研究涉及4-ABP对犬血浆的影响， 以确定是否存在特异于 4-ABP，并鉴定这些蛋白。 两组蛋白质， 在4-ABP处理期间，观察到显著降低。 一个被确认为 结合珠蛋白的β链 我们假设4-ABP与 血红蛋白，导致溶血。 然后，这种ABP-血红蛋白复合物结合到 结合珠蛋白并从血流中清除。 第二组三 斑点（MW = 65 kD，pI = 6.5-6.6）的消失速度比 在停止给药后，结合珠蛋白和恢复得更快。 尽管激烈 但是，我们还没有找到这种蛋白质。 第二项研究 使用了用含有c-raf-1的质粒转染的RLE细胞 受诱导型金属硫蛋白控制的原癌基因 启动子 我们预期raf基因会在反式- 通过适当浓度的锌和检测蛋白质 在对raf合成增加的反应中被磷酸化， 鉴定RAF潜在效应蛋白。 20个亚克隆 分离，至少有两个被证明是诱导高 锌的浓度。 然而，没有观察到一致的变化， 诱导细胞与未诱导细胞的2D凝胶模式。 Western blot 用三种不同的抗RAF抗体进行的分析未能证实 诱导的raf蛋白的存在。 看来，质粒 raf mRNA在体内被诱导，但不翻译。