ANALYSIS OF ALTERED GENE EXPRESSIONS BY TUMOR PROMOTERS DURING CELL TRANSFORMATION

用BALB/3T3细胞进行的细胞转化实验能够检测到肿瘤促进剂作为转化促进剂。12-O-十四酰佛波醇-13-乙酸酯(TPA)、冈田酸(OA)、原钒酸(VA)和对-壬基酚(NP)能显著促进细胞转化。为了阐明转化促进剂的作用机制，并建立一种通过检测基因表达变化来鉴定肿瘤促进剂的短期检测方法，采用荧光mRNA差异显示分析方法检测了这些非遗传毒性转化促进剂对BALB/3T3细胞基因表达的影响，并用RT-PCR进行了验证。在表达发生改变的基因转录起始点上游1200bp的5‘侧翼区域搜索可能的转录因子结合序列。TPA可诱导As1、Ly6e和Nudt9基因表达增加，而OA、SSB和Sned1诱导则可诱导Plat和Lgals3BP基因表达增加。降低了快速…TPA和OA诱导的Thbs1和An EST(BY594155)，OA和NP诱导的Vim，TPA诱导的AI428795和Spare，OA诱导的Rbl3，NP诱导的ND1。这些基因中存在不同的转录因子结合位点。多数基因(12/13)存在E2A_CS、EKLF_CS、LYF/Ikaros_Site、YI-Consensus和Z_box(Zta)位点，而CNBP-SRE、Add1/SREBP_Site_(2)和Vacinia-Term-Site序列仅存在于表达增加的基因中。TPA和OA引起了几个基因表达的共同变化，表明TPA和OA在细胞上存在共同的作用。然而，这些变化在TPA和OA中的时间进程是不同的。四种非遗传毒性的转化启动子均未调控共同基因。虽然通过检测基因表达的增加和降低来开发一种简单的肿瘤促进剂的检测方法是困难的，但这些数据将有助于阐明非遗传毒性化学物质在细胞转化和致癌过程中的促进机制。较少

The cell transformation assay using BALB/3T3 cells is able to detect tumor promoters as transformation promoters. 12-O-Tetradecanoylphorbol 13-acetate(TPA), okadaic acid(OA), orthovanadate(VA) and p-nonylphenol(NP) significantly enhanced the cell transformation. To clarify the mechanism of transformation promotion and develop a short term assay identifying tumor promoters by the detection of altered gene expressions, alterations in the gene expressions of BALB/3T3 cells exposed to these non-genotoxic transformation promoters were examined using the fluorescent mRNA differential display analysis and confirmed by RT-PCR. Putative transcription factor binding sequences were computer-searched in the 5' flanking regions extending 1200 bp upstream of the points of the transcriptional initiation in the genes showing altered expressions. Elevated expressions of the following genes were induced : Ass1, Ly6e and Nudt9 by TPA and OA, Plat and Lgals3bp by OA, Ssb and Sned1 by NP. Decreased express … More ions of the following genes were induced : Thbs1 and an EST (BY594155) by TPA and OA, Vim by OA and NP, AI428795 and Spare by TPA, Rbl3 by OA, ND1 by NP. There were diverse transcription factor binding sites in these genes. The sites E2A_CS, EKLF_CS, LyF/Ikaros_site, Yi-consensus and Z_box(Zta) existed in most genes (12/13), and CNBP-SRE, ADD1/SREBP_site_(2) and vaccinia-term-sequence exclusively existed in the genes which were increased in the expressions. TPA and OA caused common changes in the expression of several genes suggesting the existence of common actions on the cells between TPA and OA. However, the time courses of these changes were different between TPA and OA. No common gene was regulated by the four non-genotoxic transformation promoters. Although it would be difficult to develop a simple assay method for tumor promoters utilizing the detection of increased and decreased gene expressions, these data will contribute to clarifying the mechanisms of promotion by non-genotoxic chemicals during cell transformation and carcinogenesis. Less