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.

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-bromocAMP (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 (PKC), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNRtriggered PKC activation. Finally, we found that the expression of PKC ki-

In this study, we evaluated the influence of protein kinase C (PKC ) on topoisomerase II inhibitor-induced cytotoxicity in monocytic U937 cells. In U937-J and U937-B cells, enforced PKC expression, conferred by stable transfection of PKC 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 -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 overexpression resulted in reduced global topoisomerase II activity. Moreover, in PKC -overexpressing cells, we found that PKC interacted with both ␣ and ␤ isoforms of topoisomerase II, and these two enzymes were constitutively phosphorylated. However, when human recombinant PKC (rH-PKC ) was incubated with purified topoisomerase II isoforms, rH-PKC interacted with topoisomerase II␤ but not with topoisomerase II␣. PKC /topoisomerase II␤ 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␤ is a substrate for PKC , and that PKC may significantly influence topoisomerase II inhibitor-induced cytotoxicity by altering topoisomerase II␤ activity through its kinase function.DNA topoisomerases II are nuclear enzymes that modify DNA topology by their ability to break and reseal both strands in concert. Topoisomerases II have important functions in DNA replication and can serve as a cancer chemotherapy target. Indeed, drugs such as etoposide (VP-16) or mitoxantrone, form drug-topoisomerase II-DNA ternary complexes referred to as "cleavable complex." The primary cytotoxic effect of these socalled "topoisomerase II inhibitors" is not by inhibition of topoisomerase II activity but rather by stabilizing topoisomerase II cleavable complexes. This interaction prevents the DNAresealing step normally catalyzed by topoisomerase II. The ternary complex constitutes a latent DNA-damaging state, which is ultimately converted to an irreversible DNA doublestrand break (DSB). 1 Although the mechanism by which complex formation mediates cell death is still poorly understood, it has been largely documented with few exceptions that the amount of cleavable complexes and the subsequent number of DNA breaks correlates with cytotoxicity (1). These observations suggest that abnormal intracellular distribution or a decrease in expression level, activity, and sensitivity of the inhibited topoisomerase may have major impacts on topoisomerase inhibitor clinical efficacy. This has been confirmed by the molecular characterization of the so-called atypical multidrug resistant phenotype (at-MDR) resulting from selection by topoisomerase II inhibitors. Indeed, at-MDR cells display crossresistance to other topoisomerase II inhibitors and have been associated with a number of functional and/or structural topoisomerase II alterations, including decrea...

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