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 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.
Hiwasa
T
T
Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba, Japan.
Sawada
T
T
Sakiyama
S
S
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002273:Carcinogens / Q000494:pharmacology*; D002448:Cell Adhesion; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D015891:Cystatins / Q000096:biosynthesis / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004357:Drug Synergism; D015870:Gene Expression / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism
D002273:Carcinogens; D015891:Cystatins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D003907:Dexamethasone; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate