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

本研究的目的是通过在肿瘤促进剂改变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。采用BALB/3 T3细胞中的两阶段转化试验作为两阶段致癌性试验的模型。发现12-O-十四烷酰基佛波醇-13-乙酸酯（TPA）、冈田酸和原钒酸盐是细胞转化的有效促进剂。在与转化测定的促进阶段相同的条件下，从用这三种化学品之一处理的细胞中提取总RNA，并通过荧光mRNA差异显示法使mRNA可视化。分离到两个cDNA片段，G 09和C 05。G 09是三种肿瘤促进剂共同增加的mRNA的cDNA片段，并且G 09的序列与小鼠核蛋白Np 95的cDNA（3488 bp）的2198-2708完全相同。C 05是来自mRNA的297 bp片段，其通常在B ...更多信息 在GenBank + EMBL + DDBJ + PDB数据库中未发现C 05基因的同源序列。用特异性引物进行RT-PCR和北方杂交证实了这两个基因的表达改变。据报道，Np 95蛋白在正常小鼠胸腺细胞中出现于细胞周期的S期，在所有小鼠淋巴瘤细胞中不规则表达。在BALB/3 T3成纤维细胞的DNA合成测定BrdU摄取和Np 95的表达升高的血清的增殖刺激，和DNA合成的增加与Np 95表达的升高在不同的时间下的肿瘤促进剂的治疗平行。这些结果表明，Np 95的表达也与成纤维细胞中的细胞增殖有关。这两个基因的表达通常被肿瘤促进剂改变的事实表明，这些化学物质在肿瘤促进中可能存在共同的机制，但需要进一步的研究来发现这两个基因在肿瘤促进中的作用。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