ANALYSIS OF ALTERED GENE EXPRESSIONS BY TUMOR PROMOTERS DURING CELL TRANSFORMATION

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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