The proteins encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancers. To investigate the mechanism(s) whereby oncogenic p21ras and pp60c-src contribute to malignant transformation of intestine, human colonic Caco-2 cells transfected with an activated (Val 12) human Ha-ras gene (Caco-2-T cells) or Py-MT oncogene, a constitutive activator of pp60c-arc tyrosine kinase activity (Caco-2-MT cells), were analyzed for tumorigenicity, protein kinase C (PKC) isoform expression, and PKC activity. As compared with control vector Caco-2-H cells, Caco-2-T and Caco-2-MT cells displayed: (a) an enhanced tumorigenicity in nude mice; (b) a 4-fold increase in the level of PKC-α mRNA which was not due to enhanced mRNA stability and was mediated through a PKC-independent pathway since it persisted after PKC depletion; (c) increased PKC-α immunoreactive protein content (3-fold), total PKC catalytic activity (3.5-fold), and total cell number of [3H]phorbol-12,13-dibutyrate binding sites (4-fold); and (d) a 1.7-fold higher membrane-bound/total PKC activity ratio together with 1.8- and 1.5-fold increases in [3H]arachidonate- and [3H]myristate-labeled diacylglycerol levels. In conclusion, the tumorigenic progression induced by oncogenic p21ras or the Py-MT/pp60c-src complex in Caco-2 cells is associated with increased PKC-α gene transcription and PKC-α expression as well as with constitutive PKC activation. These results provide the first evidence that the PKC-α gene is a target for the signaling pathways of oncogenically activated p21ras and pp60c-src in human colonic cells. They raise the possibility that PKC-α is an effector of these oncoproteins for activation of Caco-2 cell tumorigenic potential.
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, INSERM-U.181 and INSERM-U.55 (France), the Association pour la Recherche contre le Cancer (A. R. C., Villejuif, France) and the Fondation pour la Recherche Médicale (F. R. M., Paris, France).