Protein kinase Czeta mediated Raf-1/extracellular-regulated kinase activation by daunorubicin.

In light of the emerging concept of a protective function of the mitogen-activated protein kinase (MAPK) pathway under stress conditions, we investigated the influence of the anthracycline daunorubicin (DNR) on MAPK signaling and its possible contribution to DNR-induced cytotoxicity. We show that DNR increased phosphorylation of extracellular-regulated kinases (ERKs) and stimulated activities of both Raf-1 and extracellular-regulated kinase 1 (ERK1) within 10 to 30 minutes in U937 cells. ERK1 stimulation was completely blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor PD98059 or the Raf-1 inhibitor 8-bromo-cAMP (cyclic adenosine monophosphate). However, only partial inhibition of Raf-1 and ERK1 stimulation was observed with the antioxidant N-acetylcysteine (N-Ac). Moreover, the xanthogenate compound D609 that inhibits DNR-induced phosphatidylcholine (PC) hydrolysis and subsequent diacylglycerol (DAG) production, as well as wortmannin that blocks phosphoinositide-3 kinase (PI3K) stimulation, only partially inhibited Raf-1 and ERK1 stimulation. We also observed that DNR stimulated protein kinase C zeta (PKCzeta), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNR-triggered PKCzeta activation. Finally, we found that the expression of PKCzeta kinase-defective mutant resulted in the abrogation of DNR-induced ERK phosphorylation. Altogether, these results demonstrate that DNR activates the classical Raf-1/MEK/ERK pathway and that Raf-1 activation is mediated through complex signaling pathways that involve at least 2 contributors: PC-derived DAG and PI3K products that converge toward PKCzeta. Moreover, we show that both Raf-1 and MEK inhibitors, as well as PKCzeta inhibition, sensitized cells to DNR-induced cytotoxicity.

Overexpression of the atypical protein kinase C zeta reduces topoisomerase II catalytic activity, cleavable complexes formation, and drug-induced cytotoxicity in monocytic U937 leukemia cells.

In this study, we evaluated the influence of protein kinase C zeta (PKC zeta) on topoisomerase II inhibitor-induced cytotoxicity in monocytic U937 cells. In U937-zeta J and U937-zeta B cells, enforced PKC zeta expression, conferred by stable transfection of PKC zeta cDNA, resulted in total inhibition of VP-16- and mitoxantrone-induced apoptosis and decreased drug-induced cytotoxicity, compared with U937-neo control cells. In PKC zeta-overexpressing cells, drug resistance correlated with decreased VP-16-induced DNA strand breaks and DNA protein cross-links measured by alkaline elution. Kinetoplast decatenation assay revealed that PKC zeta overexpression resulted in reduced global topoisomerase II activity. Moreover, in PKC zeta-overexpressing cells, we found that PKC zeta interacted with both alpha and beta isoforms of topoisomerase II, and these two enzymes were constitutively phosphorylated. However, when human recombinant PKC zeta (rH-PKC zeta) was incubated with purified topoisomerase II isoforms, rH-PKC zeta interacted with topoisomerase II beta but not with topoisomerase II alpha. PKC zeta/topoisomerase II beta interaction resulted in phosphorylation of this enzyme and in decrease of its catalytic activity. Finally, this report shows for the first time that topoisomerase II beta is a substrate for PKC zeta, and that PKC zeta may significantly influence topoisomerase II inhibitor-induced cytotoxicity by altering topoisomerase II beta activity through its kinase function.

Implication of protein kinase C in the regulation of DNA mismatch repair protein expression and function.

The DNA mismatch repair (MMR) proteins are essential for the maintenance of genomic stability of human cells. Compared with hereditary or even sporadic carcinomas, MMR gene mutations are very uncommon in leukemia. However, genetic instability, attested by either loss of heterozygosity or microsatellite instability, has been extensively documented in chronic or acute malignant myeloid disorders. This observation suggests that in leukemia some internal or external signals may interfere with MMR protein expression and/or function. We investigated the effects of protein kinase C (PKC) stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA) on MMR protein expression and activity in human myeloid leukemia cell lines. First, we show here that unstimulated U937 cells displayed low level of PKC activity as well as MMR protein expression and activity compared with a panel of myeloid cell lines. Second, treatment of U937 cells with TPA significantly increased (3-5-fold) hMSH2 expression and, to a lesser extent, hMSH6 and hPMS2 expression, correlated to a restoration of MMR function. In addition, diacylglycerol, a physiological PKC agonist, induced a significant increase in hMSH2 expression, whereas chelerythrine or calphostin C, two PKC inhibitors, significantly decreased TPA-induced hMSH2 expression. Reciprocally, treatment of HEL and KG1a cells that exhibited a high level of PKC expression, with chelerythrine significantly decreased hMSH2 and hMSH6 expression. Moreover, the alteration of MMR protein expression paralleled the difference in microsatellite instability and cell sensitivity to 6-thioguanine. Our results suggest that PKC could play a role in regulating MMR protein expression and function in some myeloid leukemia cells.