We have shown previously that overexpression of the ε isoform of protein kinase C (PKCε) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal ...transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/ε cells (overexpressing PKCε), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCε-transformed D/ε cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCε-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic effect of PKCε. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCε cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCε, and that overexpression of PKCε circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCε exerts its oncogenic activity in rat colonic cells by affecting the ras signaling cascade at the level of Raf-1 activation.
We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKC delta , PKC epsilon and PKC\g?\, in an in vitro model of colon carcinogenesis consisting of the ...nontumorigenic rat colonic epithelial cell line D/WT, and a derivative src-transformed line D/src. While PKC\g?\ and PKC epsilon showed similar protein levels, PKC delta was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKC delta was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKC delta . Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKC delta was involved in growth control and transformation in this model, we overexpressed PKC delta in D/src cells. Transfected cells underwent marked growth and morphological modifications toward the D/WT phenotype. In a late stage in culture, transfected cells ceased to proliferate, rounded up and degenerated into multinucleated, giant-like cells. We conclude that PKC delta can reverse the transformed phenotype and act as a suppressor of cell growth in D/src cells. Moreover, our data show that downregulation of this isoenzyme of PKC may cooperate in the neoplastic transformation induced by the src oncogene in D/WT cells.
We have analysed the expression of five protein kinase C PKC isoforms in an in vitro model using nontumorigenic rat colonic epithelial cells FRC/TEX CL D D/WT and in the related tumorigenic ...Ha-ras-transformed FRC/TEX CL D/H-ras line D/ras. The PKC subspecies alpha, delta, epsilon and xi were expressed at the protein level in both D/WT and D/ras cells, while beta PKC was undetectable in both lines. The levels of expression of the delta and xi isoforms were similar in D/WT and D/ras cells. Alpha PKC expression was decreased and epsilon PKC was increased in D/ras cells compared to the D/WT line. To assess whether overexpression of epsilon PKC was linked to the transformed phenotype, we have generated from D/WT cells two clones (D/epsilon-5 and D/epsilon-9) which stably overexpress epsilon PKC about fivefold. Overexpression of epsilon PKC caused marked morphological changes in both transfected clones, which were accompanied by increased saturation densities and anchorage-independent colony formation in semisolid agar. These growth effects were attenuated or reversed by chronic incubation with phorbol 12-myristate 13-acetate. Furthermore, D/epsilon-5 and D/epsilon-9 cells formed tumors in athymic nude mice with 100% incidence while the parental D/WT or vector alone (D/MV12) controls produced no tumors. We conclude that epsilon PKC can act as an oncoprotein when modestly overproduced in nontumorigenic D/WT colonic cells, and that this isoform of PKC may be linked to ras-modulated signal transduction leading to neoplastic transformation in colonic epithelium.
PEMT and CT activities were reciprocally regulated during the perinatal period. Consistently, PEMT2 expression was undetectable before birth when CT was highly expressed. Surprisingly, PEMT2 was ...relatively highly expressed at birth when the cell division and CT expression were still high. During development liver cell growth was associated with enhanced levels in the activity of β, zeta and, particularly, α PKC. The activity of δPKC was lower in foetal, higher in the newborn and again slightly lower than adult liver 10 days after birth. These data show that CT expression and α, β and zeta PKC activities are positively, whereas PEMT2 expression and δ PKC activity are negatively associated with the liver cell division during development.
Protein kinase C isozyme (α, β, δ and zeta) activities have been studied in compensatory cell proliferation at 20 and 24 hours after partial hepatectomy. Chromatographic analysis of PKC isozymes in ...male rats showed an activation of δ at 20 h after partial hepatectomy and of α, β and zeta at 24 in correspondence with S phase peak. A peculiar sexual dimorphism, which matched sexual differences in DNA synthesis, has been observed. The administration of an excess of choline was able to modulate the protein kinase C isozyme pattern in females in relation to DNA synthesis and c-myc expression. Our data suggest that α, β and zeta PKC positively regulated, while δ negatively controlled the liver compensatory growth after partial hepatectomy.
The profiles of the calcium-dependent protein kinase C (PKC) isozymes alpha, beta, and gamma were examined in subcellular fractions from Fischer 344 rat liver during the early stages (48 h, 96 h, 7 ...d, and 60 d) of diethylnitrosamine (DEN)-induced carcinogenesis, using the Solt-Farber "resistant hepatocyte" model (DEN-2-acetylaminofluorene-partial hepatectomy; DEN-AAF-PH), and then related to the presence of focal or nodular gamma-glutamyl transpeptidase (GGT)-positive morphologic changes in the liver. After DEAE and hydroxyapatite column chromatography, two peaks, immunologically identified as PKC-alpha and -beta isoforms, were detected in the liver of normal (alpha/beta ratio = 4.0) and treated rats. In DEN-AAF-PH hepatocarcinogenesis an increase in PKC-alpha expression was found after PH (+43 +/- 19% at 48 h, alpha/beta ratio = 5.1; +125 +/- 25% at 96 h, alpha/beta ratio = 4.8), whereas the PKC-beta isoform appeared less significantly modified (+11 +/- 3% at 48 h and +89 +/- 17% at 96 h). Seven and 60 days after PH, a marked increase in the PKC-alpha (+96 +/- 20% and +150 +/- 48%, respectively) and PKC-beta isoforms (+158 +/- 41%, alpha/beta ratio = 3.1 and +130 +/- 26%, alpha/beta ratio = 4.4, respectively), occurred along with the appearance of GGT-positive altered hepatic foci and nodules in the liver sections. Sham hepatectomy caused PKC-alpha and -beta isoform activities similar to those of normal controls. In contrast, saline-AAF-PH-treated rats had downregulation of PKC-alpha after PH (alpha/beta ratio = 1.8 at 96 h), possibly due to the mitoinhibitory effect of the carcinogen AAF on normal uninitiated hepatocytes. Immunohistochemical analysis with monoclonal antibodies to PKC-alpha and -beta revealed diffuse positive cytoplasmic signals in GGT-positive foci and nodules in rat liver. Taken together, these preliminary results, using the Solt-Farber model of liver carcinogenesis, suggest a role for PKC in tumor promotion. They also suggest that the PKC-alpha isoform may play a specific role in clonal expansion of DEN-initiated hepatocytes after PH.
Due to the growing number of recently cloned isoenzymes, purification and assay of protein kinase C (PKC) have become increasingly cumbersome. This paper reports the development of a shortened ...protocol for partial purification and assay of alpha, beta, delta and zeta PKC from rat liver, allowing the determination of a PKC subspecies activity pattern on a single tissue preparation. Calcium-dependent alpha and beta PKC subspecies were resolved by application of a DEAE eluate to a hydroxylapatite column, delta PKC was separated with SP-Sepharose and phenyl-Sepharose chromatography, whereas three column passages were necessary to isolate zeta PKC: DEAE-Sepharose, phenyl-Sepharose and heparin-Sepharose. This procedure allows reproducible separation and assay as well as constant recovery of the four liver PKC isoenzymes.
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