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

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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