Synergistic Induction of Anchorage-independent Growth of NIH3T3 Mouse Fibroblasts by Cysteine Proteinase Inhibitors and a Tumor Promoter (∗)

We have previously reported that Ras protein is a potent cysteine proteinase inhibitor. In order to examine whether the cysteine proteinase-inhibitory activity of Ras is involved in carcinogenesis, the effects of the following probes were investigated. Cystatin α is a cysteine proteinase-specific inhibitor and has some amino acid sequence homology with Ras. Ras has a CAAXmotif (C, cysteine; A, aliphatic amino acid; X, any amino acid) at the carboxyl terminus, which is indispensable for the biological activity. Thus, cystatin α carrying a CAAXmotif (cystatin α-CVLS) was examined. A v-Ha-Ras deletion mutant, RasΔ42-49, has undetectable GTP binding activity, yet it retains a similar protease inhibitory activity to that of wild-type v-Ras. These genes were inserted into a eukaryotic inducible expression vector and transfected into NIH3T3 cells. The expression was effectively induced by treatment with a glucocorticoid hormone, dexamethasone. The expression of cystatin α-CVLS or RasΔ42-49 alone induced neither transformation nor morphological changes. However, when their expression was induced in the presence of a tumor-promoting phorbol ester, a remarkable increase in the anchorage-independent growth was observed in cystatin α-CVLS- and RasΔ42-49-transfected clones. These results suggest that cysteine proteinase inhibitors and a tumor promoter synergistically transformed NIH3T3 cells. It is thus possible that the cysteine proteinase-inhibitory activity of Ras might play a key role in the early stage of carcinogenesis.