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 alpha is a
cysteine proteinase-specific inhibitor and has some amino acid sequence homology with Ras. Ras has a CAAX motif (C,
cysteine; A, aliphatic
amino acid; X, any
amino acid) at the carboxyl terminus, which is indispensable for the
biological activity. Thus,
cystatin alpha carrying a CAAX motif (
cystatin alpha-CVLS) was examined. A v-Ha-Ras deletion mutant, Ras delta 42-49, 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 alpha-CVLS or Ras delta 42-49 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 alpha-CVLS- and Ras delta 42-49-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.