title: Regulation of transformed state by calpastatin via PKCepsilon in NIH3T3 mouse fibroblasts.
abstract: Ca(2+)-activated neutral protease calpain is ubiquitously expressed and may have pleiotropic biological functions. We have previously reported that repeated treatment of NIH3T3 mouse fibroblasts with the calpain inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) resulted in the induction of transformed foci [T. Hiwasa, T. Sawada, and S. Sakiyama (1990) Carcinogenesis 11, 75-80]. To elucidate further the effects of calpain in malignant transformation of NIH3T3 cells, calpastatin, an endogenous specific inhibitor of calpain, was expressed in NIH3T3 cells by transfection with cDNA. G418-selected calpastatin-expressing clones showed a significant increase in the anchorage-independent growth ability. A similar increase in cloning efficiency in soft agar medium was also observed in calpain small-subunit-transfected clones. On the other hand, reduced expression of calpastatin achieved by transfection with calpastatin antisense cDNA in Ha-ras-transformed NIH3T3 (ras-NIH) cells caused morphological reversion as well as a decrease in anchorage-independent growth. When NIH3T3 cells were treated with ALLN for 3 days, cell growth was stimulated by approximately 10%. This growth stimulation by ALLN was not observed in ras-NIH cells, but recovered by expression of a dominant negative form of protein kinase C (PKC)epsilon but not by that of PKCalpha. Western blotting analysis showed that an increase in PKCepsilon was much more prominent than that of PKCalpha in NIH3T3 cells after treatment with ALLN. These results are concordant with the notion that calpain suppresses malignant transformation by predominant degradation of PKCepsilon.
authors.affiliation: Department of Biochemistry, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan. hiwasa@med.m.chiba-u.ac.jp
authors.forename: Takaki
authors.initials: T
authors.lastname: Hiwasa
authors.forename: Mitsuyuki
authors.initials: M
authors.lastname: Nakata
authors.forename: Miho
authors.initials: M
authors.lastname: Nakata
authors.forename: Shigeo
authors.initials: S
authors.lastname: Ohno
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Maki
authors.forename: Koichi
authors.initials: K
authors.lastname: Suzuki
authors.forename: Masaki
authors.initials: M
authors.lastname: Takiguchi
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002455:Cell Division; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D010641:Phenotype; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013997:Time Factors; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D007527:Isoenzymes; D007976:Leupeptins; C063261:acetylleucyl-leucyl-norleucinal; C033668:calpastatin; C497256:Prkce protein, mouse; C496628:PRKCA protein, human; C497255:PRKCE protein, human; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D018631:ras Proteins; D002118:Calcium