SYNERGISTIC INDUCTION OF ANCHORAGE-INDEPENDENT GROWTH OF NIH3T3 MOUSEFIBROBLASTS BY CYSTEINE PROTEINASE-INHIBITORS AND A TUMOR PROMOTOR

We have previously reported that Ras protein is a potent cysteine prot einase 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 alpha is a cystein e proteinase-specific inhibitor and has some amino acid sequence homol ogy with Ras. Ras has a CAAX motif (C, cysteine; A, aliphatic amino ac id; X, any amino acid) at the carboxyl terminus, which is indispensabl e for the biological activity. Thus, cystatin alpha carrying a CAAX mo tif (cystatin alpha-CVLS) was examined. A v-Ha-Ras deletion mutant, Ra s Delta 42-49, has undetectable GTP binding activity, yet it retains a similar protease inhibitory activity to that of wild-type v-Ras. Thes e genes were inserted into a eukaryotic inducible expression vector an d transfected into NIH3T3 cells, The expression was effectively induce d by treatment with a glucocorticoid hormone, dexamethasone. The expre ssion of cystatin alpha-CVLS or Ras Delta 42-49 alone induced neither transformation nor morphological changes. However, when their expressi on was induced in the presence of a tumor-promoting phorbol ester, a r emarkable increase in the anchorage-independent growth was observed in cystatin alpha-CVLS- and Ras Delta 42-49-transfected clones. These re sults suggest that cysteine proteinase inhibitors and a tumor promoter synergistically transformed NIH3T3 cells. It is thus possible that th e cysteine proteinase-inhibitory activity of Ras might play a key role in the early stage of carcinogenesis.