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Abstract: Okadaic acid (OA) is a tumor promoter in two-stage carcinogenesis experiments. Nevertheless, the effects of OA on cell transformation, cell proliferation and apoptosis vary widely, and the molecular events underlying these effects of OA are not well understood. In the present study, we examined the promoting activity and the associated effects on cell growth and apoptosis mediated by OA in BALB/c 3T3 cells, and evaluated alterations of gene transcriptional expression by microarray analysis. The promoting activity of OA was estimated by a two-stage transformation assay, in which cells were treated first with a low dose of the initiator N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and then with OA for 14 days. It showed that OA, at concentrations of 7.8–31.3ng/ml, enhanced the transformation of MNNG-treated cells. In the promotion phase, cells exposed to OA (7.8ng/ml) grew slowly for the first 2 days and subsequently died. As determined by Hoechst 33342 fluorescent dye and Annexin-V/PI dual-colored flow cytometry, OA induced morphologically apoptotic cells and increased the percentage of early apoptotic cells. The gene expression profile induced by OA at five time points in the promotion phase was determined by use of a specific mouse toxicological microarray containing 1796 clones, and a total of 177 differentially expressed genes were identified. By gene ontology analysis, 31 of these were determined to be functionally involved with cell growth and/or maintenance. In this group, numerous genes associated with the cell proliferation and cell cycle progression were down-regulated at early and/or middle time points. Among these was a subset of genes associated with apoptosis, in which Bnip3, Cycs, Casp3 and Bag1 genes are involved in the mitochondrial pathway of apoptosis. Ier3, Mdm2 and Bnip3 genes may be p53 targets. Furthermore, real-time PCR confirmed the expression changes of five genes selected at random from the differentially expressed genes. We conclude that OA induces cell growth inhibition and apoptosis in the two-stage, MNNG-initiated transformation of BALB/c 3T3 cells. The results of gene expression profile analysis imply that multiple molecular pathways are involved in OA-induced proliferation inhibition and apoptosis. Mitochondrial and p53-associated apoptotic pathways also may contribute to OA-induced apoptosis. [Copyright &y& Elsevier]

Abstract: Identification of specific etiological carcinogens is one of the most important issues in environmental-toxicology studies. In this study, cDNA microarrays were used to analyze gene expression and discern chemical-associated profiles induced by a variety of tumor promoting agents in transformed cells. Two-stage transformation model of BALB/c 3T3 cells was established with MNNG as initiator, and 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid (OA), or cadmium chloride (CdCl2) as tumor promoters. Nine morphologically transformed foci were isolated and the anchorage-independent growth of transformed cells was verified. The gene expression alterations in foci were evaluated using cDNA microarray with 1796 mouse genes. Unsupervised hierarchical clustering analysis revealed that the nine foci were classified into three groups in concordance with the promoters used to induce them and characteristic clusters of genes were identified. In these clusters, genes associated with oxidative stress were specially upregulated following distinct promoter exposure. Moreover, common gene expression alterations were also observed in foci, including upregulated genes associated with cell proliferation and downregulated genes associated with extracellular matrix. Our results demonstrate the presence of unique gene expression profiles in transformed cells which reflect the etiological chemicals and indicate the importance of characteristic molecular alterations as potential biomarkers of exposure to tumor promoters. [Copyright &y& Elsevier]