Gian Paolo Perletti

Protein kinase Cε is oncogenic in colon epithelial cells by interaction with the ras signal transduction pathway

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.

PKCδ acts as a growth and tumor suppressor in rat colonic epithelial cells

We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKCδ, PKCε and PKCζ, 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 PKCζ and PKCε showed similar protein levels, PKCδ was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKCδ was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKCδ. Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKCδ was involved in growth control and transformation in this model, we overexpressed PKCδ 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 PKCδ 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.

Cardiotoxicity induced by doxorubicin in vivo: Protective activity of the spin trap alpha-phenyl-tert-butyl nitrone

The role of free radical generation in the development of the acute cardiotoxicity induced by doxorubicin (DXR) in the rat and the protective activity of anti-radical drugs were investigated in in vivo experiments by evaluating the body weight curve, ECG, contractile performance and coronary flow up to 10 days after DXR. A lipophilic spin trap (alpha-phenyl-tert-butyl nitrone, PBN) was continuously administered at a dose of 0.65 mg/kg every hour for 2 weeks by an intraperitoneal osmotic pump. DXR was administered i.v. at a dose of 9 mg/kg 3 days after beginning the PBN infusion. DXR impaired ECG and body weight gain after 3 days (partly reversible at later times), while contractility and coronary flow were significantly impaired throughout the experimental time. PBN was shown to prevent the DXR-induced alterations of contractility and coronary flow, while ECG was non-significantly improved. The body weight curve was not affected. Since the dose of PBN used does not produce pharmacological effects, the protective activity in rats receiving DXR indicates that free radicals may play a causal role in the acute cardiotoxicity in vivo. The use of suitable spin traps and administration schedules seems to be an interesting approach for the prevention of radical-dependent pathologies.

PARTIAL PURIFICATION OF PROTEIN KINASE C ISOENZYMES FROM RAT LIVER

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.