NIH3T3 transfectant containing human K-ras oncogene shows enhanced metastatic activity after in vivo tumor growth or co-culture with fibroblasts

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Abstract
A clone of NIH3T3 transformant (H-3), obtained by transfecting genomic DNA of a human colon carcinoma cell line, contains human K-ras oncogene and yields metastatic pulmonary nodules after intravenous injection of the cells into nude mice. This metastatic ability was enhanced remarkably after in vivo tumor growth (subcutaneous tumor formation in nude mice) accompanied by increased mRNA expression and gene amplification of the human-derived K-ras oncogene, while it declined gradually as the passage number increased in vitro, with corresponding decreases of gene amplification and mRNA expression. Six subclones were randomly selected from H-3 cells which had been subcultured to passage 22. All of the clones in culture showed almost the same low level of metastatic ability and exhibited little K-ras oncogene amplification with correspondingly low mRNA expression. However, after they formed tumors in nude mice, every clone acquired high metastatic ability and the gene amplification increased, with elevated mRNA expression. These experimental facts indicated that acquisition of metastatic ability coupled with the function of K-ras oncogene was conditional in nature, being strongly affected by in vivo tumor circumstances. The low metastatic and G-418-resistant H-3 cells were co-cultured with BALB/c3T3 fibroblasts for 2–4 weeks. After removal of fibroblasts by exposure to G-418, the tumor cells exhibited increased metastatic ability and human K-ras oncogene mRNA, suggesting an intimate interaction between H-3 cells and fibroblasts influencing the function of transfected human K-ras oncogene. Fibroblasts of the host animal may thus have an important role in generating enhanced metastatic activity of H-3 cells.