INCREASED PROTEIN KINASE-C-ALPHA EXPRESSION IN HUMAN COLONIC CACO-2 CELLS AFTER INSERTION OF HUMAN HA-RAS OR POLYOMA-VIRUS MIDDLE T-ONCOGENES

The proteins encoded by ras and src protooncogenes are frequently acti vated in a constitutive state in human colorectal cancers. To investig ate 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 cel ls) or Py-MT oncogene, a constitutive activator of pp60c-src tyrosine kinase activity (Caco-2-MT cells), were analyzed for tumorigenicity, p rotein kinase C (PKC) isoform expression, and PKC activity. As compare d with control vector Caco-2-H cells, Caco-2-T and Caco-2-MT cells dis played: (a) an enhanced tumorigenicity in nude mice; (b) a 4-fold incr ease in the level of PKC-alpha 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-alpha immunoreactive protein content (3-fold), total PKC catalytic activity (3.5-fold), an d total cell number of [H-3]phorbol-12,13-dibutyrate binding sites (4- fold); and (d) a 1.7-fold higher membrane-bound/total PKC activity rat io together with 1.8- and 1.5-fold increases in [H-3]arachidonate- and [H-3]myristate-labeled diacylglycerol levels. tn conclusion, the tumo rigenic progression induced by oncogenic p21ras or the Py-MT/pp60c-src complex in Caco-2 cells is associated with increased PKC-alpha gene t ranscription and PKC-alpha expression as welt as with constitutive PKC activation. These results provide the first evidence that the PKC-alp ha gene is a target for the signaling pathways of oncogenically activa ted p21ras and pp60c-src in human colonic cells. They raise the possib ility that PKC-alpha is an effector of these oncoproteins for activati on of Caco-2 cell tumorigenic potential.