TWO-DIMENSIONAL GEL ANALYSIS OF CARCINOGENESIS

本项目的目的是研究致癌机制。 采用定量双向凝胶电泳法(2D-Gel)。这 技术可以让我们检查质量和数量上的变化 细胞经历过程中数千种细胞多肽的合成 肿瘤性转化。研究集中在三个领域：研究 V-ras癌基因对黑色素瘤多肽合成的影响 二倍体人成纤维细胞系(LG1)的诱变研究 犬血浆中已知的膀胱癌物质4-氨基联苯(4-氨基联苯) ABP)；以及对可诱导的c-raf原-的影响的检查- 癌基因在大鼠肝上皮(RLE)细胞的2D-凝胶图谱上。这个 LG1细胞的2D-凝胶图谱已经与非致瘤细胞、 永生化生长变异体VO和转化的细胞株 V-H-ras或v-N-ras。LG1、VO和ras转化细胞 产生了明显的2D-凝胶图案，很容易通过 聚类分析技术。细微但始终如一的差异是 在RAS转化的细胞之间也检测到。第二项研究， 包括4-ABP对狗血浆的影响 4-ABP特异的血浆蛋白有缺失， 鉴定这些蛋白质。两组蛋白质，它们被大大减少 在4-ABP治疗过程中观察。其中一个被确认为测试版- 结合珠蛋白链。第二组三个斑点(Mw=65kD，等电点=6.5- 6.6)，比结合珠蛋白消失得快得多，恢复得更快 停止服药后迅速服用。第三项研究使用了RLE细胞 用含c-raf-1原癌基因的重组质粒 可诱导金属硫蛋白启动子的控制。预计 通过适当的诱导，可以在转染体中诱导出raf基因。 锌的浓度。我们打算研究的蛋白质是 磷酸化是对RAF合成增加的反应，希望发现 RAF的潜在效应蛋白。已分离出20个亚克隆 目前正在努力确定工业生产的适当条件。 RAF在这些转染体中的表达。

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 three areas: study of the effects of v-ras oncogenes on the synthesis of polypeptides in a diploid human fibroblast line (LG1); 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 in rat liver epithelial (RLE) cells. The 2D-gel patterns of LG1 cells have been compared with a non-tumorigenic, immortalized growth variant, VO, and with cell strains transformed with either v-H-ras or v-N-ras. LG1, VO and the ras transformed cells resulted in distinct 2D-gel patterns which were easily distinguished by cluster analysis techniques. Subtle, but consistent differences were also detected between the ras transformed cells. The second 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 are greatly reduced during 4-ABP treatment, were observed. One was identified as the beta- chain of haptoglobin. 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. The third study has employed RLE cells transfected with plasmids containing the c-raf-1 proto-oncogene under the control of an inducible metallothione promoter. It is anticipated that the raf gene can be induced in the transfectants by appropriate concentrations of zinc. We intend to examine proteins that are phosphorylated in response to increased raf synthesis in hopes of finding potential effector proteins for raf. Twenty subclones have been isolated and work is underway to determine the appropriate conditions for induc- tion of raf in these transfectants.