STUDIES ON ACTIONS OF TUMOR PROMOTERS BY ANALYSIS OF GENE EXPRESSION IN CELL TRANSFORMATION

本研究旨在通过在肿瘤促进剂改变其表达的基因中寻找调节肿瘤促进的基因来阐明肿瘤促进的分子过程。BALB/3T3细胞的两阶段转化实验作为两阶段致癌实验的模型。12-O-十四酰佛波醇-13-乙酸酯(TPA)、冈田酸和原钒酸对细胞转化有较强的促进作用。在与转化实验促进阶段相同的条件下，提取细胞的总RNA，并用荧光mRNA差异显示法对其进行显示。分离到G09和C05两个基因片段。G09是三种肿瘤促进剂共同作用的基因片段，其序列与小鼠核蛋白Np95的2198-2708序列完全一致。C05是通常被…降低的mRNA297bp片段。在GenBank+EMBL+DDBJ+PDB数据库中没有发现C05基因的同源序列，用特异性引物RT-PCR和Northern blotting证实了这两个基因的表达变化。据报道，Np95蛋白在正常小鼠胸腺细胞中出现在细胞周期的S期，并在所有小鼠淋巴瘤细胞中不规则表达。在BALB/3T3成纤维细胞中，以BrdU摄取测定的DNA合成和Np95的表达在血清的增殖刺激下均有增加，并且在不同时间，随着Np95表达的增加，DNA合成也增加。这些结果表明，Np95的表达与成纤维细胞的细胞增殖有关。这两个基因的表达通常被肿瘤促进剂改变，这一事实表明这两个化学物质在促进肿瘤中可能存在一个共同的机制，尽管还需要进一步研究这两个基因在促肿瘤中的作用。较少

This study was intended to clarify the molecular process of tumor promotion by finding genes that regulate tumor promotion among genes whose expressions are altered by tumor promoters. A two-stage transformation assay in BALB/3T3 cells was used as a model of two-stage carcinogenicity tests. 12-O-tradecanoylphorbol-13-acetate (TPA), okadaic acid and orthovanadate were found to be potent promoters of cell transformation. The total RNA was extracted from the cells being treated with one of these three chemicals under the same conditions as in the promotion phase of the transformation assay, and the mRNA was visualized by a fluorescent mRNA differential display method. Two cDNA fragments, G09 and C05, were isolated. G09 was the cDNA fragment from the mRNA that was commonly increased by three tumor promoters, and the sequence of G09 was entirely identical to 2198-2708 of cDNA (3488 bp) of Np95, a murine nuclear protein. C05 was the 297 bp fragment from the mRNA that was commonly decreased b … More y the tumor promoters, and any homologous sequence of C05 has not been found in data base presented by GenBank + EMBL + DDBJ + PDB.The altered expressions of these two genes were confirmed by RT-PCR using specific primers and northern blotting. It is reported that Np95 protein appears in the S phase of the cell cycle in normal mouse thymocytes and is irregularly expressed in all the murine lymphoma cells. In BALB/3T3 fibroblasts the DNA synthesis measured by BrdU uptake and the expression of Np95 were elevated by proliferative stimuli of serum, and the DNA synthesis was increased in parallel with the elevation of Np95 expression at various times under the treatment by tumor promoters. These results suggest that the expression of Np95 also associates with cell proliferation in the fibroblasts. The fact that the expression of two genes were commonly altered by tumor promoters suggests that there may exist a common mechanism in tumor promotion by these chemicals, though further investigation is required to find the roles of two genes in tumor promotion. Less