Positive regulation of ERK activation and MKP-1 expression by peroxovanadium complex bpV (phen)

Lower micromolar concentrations of peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV (phen)] stimulate RINm5F cell metabolic activity. 1 and 3 mol/L bpV (phen) induces strong and sustained activation of extracellular signal-regulated kinase (ERK). However, it seems that bpV (phen) does not effect c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, bpV (phen) induces mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. We found that ERK activation could be completely abolished if RINm5F cells were incubated with both bpV (phen) and PD 98059, a specific inhibitor of upstream ERK kinase MEK1. On the other hand, this combined treatment up-regulated activation of stress kinases, JNK and p38 MAPK, significantly suppressed MKP-1 expression and induced cell death. Thus, our results suggest that the mechanism underlying bpV (phen) survival-enhancing effect could be associated with induced ERK activation and MKP-1 expression.     

Activation of MAPK by potassium bisperoxo(1,10-phenanthroline)oxovanadate (V).

Potassium bisperoxo(1,10-phenantroline)oxovanadate (V) [bpV(phen)] is a potent protein tyrocine phosphatase inhibitor which mediates a variety of biological effects. The aim of these studies was to examine the role(s) of mitogen activated protein kinase (MAPK) pathways in PC12 cell proliferation and toxicity by bpV(phen). BpV(phen) exerts a bimodal effect in PC12 cells: proliferation at low and cell death at higher micromolar concentrations. Activation of MAPK by bpV(phen) depends on time and concentration. The phosphorylation pattern of extracellular regulated kinases (ERK 1/2), c-jun N-terminal activated kinases (JNK) and p38 in PC12 cells is strikingly different. Activation of JNK is sustained in PC12 cells. In contrast, ERK 1/2 activation is transient and treatment with PD98059 indicates that ERK activation by bpV(phen) is partly independent from the ras-MEK pathway. Stability studies of bpV(phen) in DMEM and PBS showed linear relationship with T1/2 about 6 h and 10 days in DMEM and PBS, respectively. Comparison between the time courses of MAPK activation and kinetics of bpV(phen) decomposition as assessed by 51V-NMR analysis show that the initial and maximal phosphorylation signals are produced in the presence of the complex bpV(phen) and not caused by the decomposition products of bpV(phen).     

Involvement of p38 MAP kinase during iron chelator-mediated apoptotic cell death

Iron is an essential element for the neoplastic cell growth, and iron chelators have been tested for their potential anti-proliferative and cytotoxic effects. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during apoptosis induced by iron chelators. We report that the chelator deferoxamine (DFO) strongly activates both p38 MAP kinase and extracellular signal-regulated kinase (ERK) at an early stage of incubation, but slightly activates c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at a late stage of incubation. Among three MAP kinase blockers used, however, the selective p38 MAP kinase inhibitor SB203580 could only protect HL-60 cells from chelator-induced cell death, indicating that p38 MAP kinase serves as a major mediator of apoptosis induced by iron chelator. DFO also caused release of cytochrome c from mitochondria and induced activation of caspase 3 and caspase 8. Interestingly, treatment of HL-60 cells with SB203580 greatly abolished cytochrome c release, and activation of caspase 3 and caspase 8. Collectively, the current study reveals that p38 MAP kinase plays an important role in iron chelator-mediated cell death of HL-60 cells by activating downstream apoptotic cascade that executes cell death pathway.     

Cobalt chloride-induced apoptosis and extracellular signal-regulated protein kinase 1/2 activation in rat C6 glioma cells

Brain ischemia brings about hypoxic insults. Hypoxia is one of the major pathological factors inducing neuronal injury and central nervous system infection. We studied the involvement of mitogen-activated protein (MAP) kinase in hypoxia-induced apoptosis using cobalt chloride in C6 glioma cells. In vitro cytotoxicity of cobalt chloride was tested by MTT assay. Its IC(50) value was 400 microM. The DNA fragment became evident after incubation of the cells with 300 microM cobalt chloride for 24 h. We also evidenced nuclear cleavage with morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signal pathway of cobalt chloride-induced apoptosis in C6 cells. The activation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) started to increase at 1 h and was activated further at 6 h after treatment of 400 M cobalt chloride. In addition, pretreatment of PD98059 inhibited cobalt chloride-induced apoptotic cell morphology in Electron Microscopy. These results suggest that cobalt chloride is able to induce the apoptotic activity in C6 glioma cells, and its apoptotic mechanism may be associated with signal transduction via MAP kinase (ERK 1/2).     

Differential regulation of JNK activation and MKP-1 expression by peroxovanadium complexes

Bisperoxovanadium complexes have been identified as insulinomimetic agents and protein tyrosine phosphatase inhibitors. The aim of the present study was to examine the effects of the most potent bisperoxovanadium complex, potassium bisperoxo (1,10-phenanthroline) oxovanadate (V) [bpV(phen)], on expression and activation of c-jun N-terminal protein kinases (JNK) and on expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) in different cell lines. We compared the effects of bpV(phen) with the effects of tumor necrosis factor-alpha (TNF-alpha), a known regulator of JNK phosphorylation and inducer of MKP-1. Treatment with bpV(phen) causes significant and sustained down-regulation of MKP-1 expression both in PC12 and HeLa cells. In contrast, TNF-alpha induces MKP-1 expression in PC12 cells and does not alter MKP-1 expression in HeLa cells. Both bpV(phen) and TNF-alpha induce MKP-1 expression in OVCAR-3 cell line but with different dynamics: TNF-alpha causes transient and bpV(phen) sustained induction of MKP-1 expression. Temporal pattern of level of MKP-1 expression correlates with the regulation of JNK phosphorylation by bpV(phen) and TNF-alpha in PC12 cells. However, no detectable phospho-JNK signal is observed in either OVCAR-3 or HeLa cells treated with bpV(phen). In contrast, TNF-alpha causes strong and sustained JNK phosphorylation in OVCAR-3 cell line, and strong but transient JNK activation in HeLa cells. BpV(phen) and TNF-alpha does not alter JNK expression in any of the cell lines studied. We demonstrate that the effect of two stressors, bpV(phen) and TNF-alpha, on MKP-1 expression and JNK phosphorylation are strikingly different, depending on the cell type. These results suggest the possible role of MKP-1 in regulation of JNK phosphorylation in both PC12 and OVCAR-3 cell lines treated with bpV(phen).     

Modulation of nitric oxide-induced apoptotic death of HL-60 cells by protein kinase C and protein kinase A through mitogen-activated protein kinases and CPP32-like protease pathways.

To define the signaling pathways during NO-induced apoptotic events and their possible modulation by two protein kinase systems, we explored the involvement of three structurally related mitogen-activated protein kinase subfamilies. Exposure of HL-60 cells to sodium nitroprusside (SNP) strongly activated p38 kinase, but did not activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In addition, SNP-induced apoptosis was markedly blocked by the selective p38 kinase inhibitor (SB203580) but not by MEK1 kinase inhibitor (PD098059), indicating that p38 kinase serves as a mediator of NO-induced apoptosis. In contrast, treatment of cells with phorbol 12-myristate 13-acetate (PMA) strongly activated not only JNK but also ERK, while not affecting p38 kinase. However, although SNP by itself weakly activated CPP32-like protease, SNP in combination with PMA markedly increased the extent of CPP32-like protease activation. Interestingly, N6,O2-dibutylyl cAMP (DB-cAMP) significantly blocked SNP- or SNP plus PMA-induced activation of CPP32-like protease and the resulting induction of apoptosis. DB-cAMP also blocked PMA-induced JNK activation. Collectively, these findings demonstrate the presence of specific up- or down-modulatory mechanisms of cell death pathway by NO in which (1) p38 kinase serves as a mediator of NO-induced apoptosis, (2) PKC acts at the point and/or upstream of JNK and provides signals to potentiate NO-induced CPP32-like protease activation, and (3) PKA lies upstream of either JNK or CPP32-like protease to protect NO- or NO plus PMA-induced apoptotic cell death in HL-60 cells.     

Cell cycle regulation and induction of apoptosis by beta-carotene in U937 and HL-60 leukemia cells

In this communication, we report the efficacy of beta-carotene towards differentiation and apoptosis of leukemia cells. Dose (20 microM) and time dependence (12 h) tests of beta- carotene showed a higher magnitude of decrease (significance p < 0.05) in cell numbers and cell viability in HL-60 cells than U937 cells but not normal cell like Peripheral blood mononuclear cell (PBMC). Microscopical observation of beta-carotene treated cells showed a distinct pattern of morphological abnormalities with inclusion of apoptotic bodies in both leukemia cell lines. When cells were treated with 20 microM of beta-carotene, total genomic DNA showed a fragmentation pattern and this pattern was clear in HL-60 than U937 cells. Both the cell lines, on treatment with beta- carotene, showed a clear shift in G(1) phase of the cell cycle. In addition the study also revealed anti-oxidant properties of beta-carotene since there was reduction in relative fluorescent when treated than the control at lower concentration. Collectively this study shows the dual phenomenon of apoptosis and differentiation of leukemia cells on treatment with beta-carotene.     

Immunochemical evidence that three protein kinase C isozymes increase in abundance during HL-60 differentiation induced by dimethyl sulfoxide and retinoic acid

Activity of the Ca2+/phospholipid-dependent protein kinase C has been shown to increase during differentiation of the human promyelocytic leukemia cell line HL-60 by dimethyl sulfoxide and retinoic acid (Zylber-Katz, E., and Glazer, R. I. (1985) Cancer Res. 45, 5159-5164). Antipeptide antibodies were prepared that specifically recognize the alpha, beta, and gamma isozymes of protein kinase C in rat brain cytosol and HL-60 cell extracts. The three isozymes do not share a common tissue distribution pattern. The gamma enzyme is abundant in brain but a relatively minor component in HL-60 cells; the opposite is true for the alpha enzyme. All three isozymes increase at least 2-fold in abundance in HL-60 cells exposed to 1.2% dimethyl sulfoxide for 48 h. The increase in abundance of the alpha and beta isoforms reaches 7- and 5-fold, respectively, by 96 h without further increase in the abundance of the gamma isozyme. Similarly, all three isozymes increase at least 1.5-fold in abundance after 48 h and 3-fold after 96 h with 1 microM retinoic acid. No further increase in the abundance of any of the isozymes is seen between 96 and 144 h of incubation with retinoic acid. The increase in protein kinase C activity is not limited to the cytosolic forms of the enzyme; a parallel increase in membrane-associated protein kinase C is also observed during differentiation. Approximately 10% of total protein kinase C activity is membrane-associated in both control and differentiating cells. These studies provide the first immunochemical evidence that all three protein kinase C isozymes increase during HL-60 cell differentiation, and they suggest that the increase in the isozyme levels may be coordinately regulated.     

BpV (phen) induces apoptosis of RINm5F cells by modulation of MAPKs and MKP-1

We investigated the mechanism of toxicity of peroxovanadium complex bpV (phen) in RINm5F cells. Treatment with bpV (phen) provoked cell death, predominantly by apoptosis. This compound induced strong and sustained JNK and p38 MAPK activation. However, ERK phosphorylation was not affected. The level of expression of MAPK phosphatase MKP-1 was suppressed after bpV (phen) treatment. In addition, this compound did not stimulate proteolytic processing of procaspase-3, suggesting that caspase-3 is not activated during the course of bpV (phen)-induced apoptosis. A correlative inhibition of JNK activation by immunosuppressive drug FK 506 induced ERK activation and MKP-1 expression, and suppressed RINm5F cell death. A specific p38 inhibitor SB 203580 also stimulated ERK activation and cell survival. Furthermore, simultaneous pretreatment with both FK 506 and SB 203580 almost completely abolished cell death. Thus, our results suggest that stress kinases and MKP-1 have a role in bpV (phen)-induced apoptosis of RINm5F cells.     

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.     

Curcumin abolishes apoptosis resistance of calcitriol-differentiated HL-60 cells

Exposure of HL-60 cells to 1,25-dihydroxyvitamin D(3) (calcitriol) induces their differentiation into monocytes. This terminal differentiation is associated with acquired resistance to many proapoptotic stimuli. Here we show that differentiated HL-60 cells undergo apoptosis upon curcumin treatment although they retain resistance to apoptosis induced by a topoisomerase poison - etoposide. Curcumin induced changes of nuclear morphology, DNA fragmentation, release of cytochrome c as well as caspase activation in both differentiated and undifferentiated cells. Experiments performed in other laboratories suggested that curcumin initiates apoptosis by DNA damage that results from topoisomerase II poisoning. We measured gammaH2AX expression, a marker of DNA double strand breaks, in both undifferentiated and differentiated HL-60 cells treated with curcumin or etoposide. In etoposide-treated undifferentiated cells early gammaH2AX expression correlated with initiation phase of apoptosis. In contrast, in curcumin-treated cells gammaH2AX expression correlated with apoptotic DNA fragmentation, which is characteristic for the execution phase of apoptosis. Our experiments show that curcumin overcomes the resistance of calcitriol-differentiated HL-60 cells to DNA-damage-induced apoptosis by activating other cell signaling pathways leading to cell death of HL-60.     

Regulation of glycogen synthase in rat hepatocytes. Evidence for multiple signaling pathways.

We examined the signaling pathways regulating glycogen synthase (GS) in primary cultures of rat hepatocytes. The activation of GS by insulin and glucose was completely reversed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Wortmannin also inhibited insulin-induced phosphorylation and activation of protein kinase B/Akt (PKB/Akt) as well as insulin-induced inactivation of GS kinase-3 (GSK-3), consistent with a role for the phosphatidylinositol 3-kinase/PKB-Akt/GSK-3 axis in insulin-induced GS activation. Although wortmannin completely inhibited the significantly greater level of GS activation produced by the insulin-mimetic bisperoxovanadium 1,10-phenanthroline (bpV(phen)), there was only minimal accompanying inhibition of bpV(phen)-induced phosphorylation and activation of PKB/Akt, and inactivation of GSK-3. Thus, PKB/Akt activation and GSK-3 inactivation may be necessary but are not sufficient to induce GS activation in rat hepatocytes. Rapamycin partially inhibited the GS activation induced by bpV(phen) but not that effected by insulin. Both insulin- and bpV(phen)-induced activation of the atypical protein kinase C (zeta/lambda) (PKC (zeta/lambda)) was reversed by wortmannin. Inhibition of PKC (zeta/lambda) with a pseudosubstrate peptide had no effect on GS activation by insulin, but substantially reversed GS activation by bpV(phen). The combination of this inhibitor with rapamycin produced an additive inhibitory effect on bpV(phen)-mediated GS activation. Taken together, our results indicate that the signaling components mammalian target of rapamycin and PKC (zeta/lambda) as well as other yet to be defined effector(s) contribute to the modulation of GS in rat hepatocytes.     

Inhibition of N-linked glycosylation induces early apoptosis in human promyelocytic HL-60 cells

Inhibition of protein N-glycosylation by tunicamycin induced morphological changes characteristic of apoptosis in human promyelocytic HL-60 cells. Internu-cleosomal DMA fragmentation could be detected after short-time incubation (between 6 and 9 h) of HL-60 cells with low doses of tunicamycin (0.05 μg/ml). Under these conditions the synthesis of glycoproteins was reduced to 17% of control values, while no significant changes in the rates of total protein synthesis could be observed. Tunicamycin ability to induce DNA fragmentation was in good correlation with its potency as glycosylation inhibitor in several myeloid cell lines. Tunicamycin-induced apoptosis was potentiated by activation of protein kinease C (PKC) by phorbol esters and partially prevented by the PKC inhibitor staurosporine. Inhibitors of RNA and protein synthesis displayed a protective effect. Treatment of HL-60 cells with tunicamycin did not elicit the expression of cell surface differentiation antigens or their ability to generate superoxide anion. In contrast, tunicamycin significantly inhibited these processes during dimethyl sulfoxide (DMSO)-induced myeloid differentiation. These observations indicate that the main effect of tunicamycin in HL-60 cells is the induction of apoptosis. © 1995 Wiley-Liss, Inc.     

Bisperoxovanadium complex promotes dopamine exocytosis in PC12 cells.

The effects of the peroxovanadium complex potassium bisperoxo(1,10-phenanthroline)-oxovanadate (bpV[phen]) have been studied on dopamine (DA) exocytosis in PC12 cells. Bisperoxo(1,10-phenanthroline)-oxovanadate does not elicit dopamine secretion in PC12 cells. However, treatment of PC12 cells with 30 microM bpV[phen] for 20 min significantly enhances the secretion induced by the Ca(2+)-ionophore A23187. The effects appear to be irreversible, and strikingly different from the transient and suppressing effects of orthovanadate, which, like bpV[phen], is also a protein tyrosine phosphatase inhibitor. Contrastingly, the short-lived peroxovanadates, formed in situ by the addition of hydrogen peroxide and orthovanadate, are relatively ineffective. The Ca(2+) chelating agent EGTA abolishes bpV[phen]-enhanced dopamine release. The extracellular-regulated protein kinases (ERK) and synaptophysin, proteins implicated in exocytosis, are both tyrosine-phosphorylated by bpV[phen] in a dose- and time-dependent manner, with a maximal effect at 30 microM. Pre-treatment of cells with PD98059 significantly reduced dopamine release (P<0.05). These results suggest that this peroxovanadium complex enhances dopamine exocytosis, at least in part, by ERK-mediated signaling pathway and synaptophysin-associated phosphatase(s).     

Inhibition of extracellular signal-regulated protein kinase or c-Jun N-terminal protein kinase cascade, differentially activated by cisplatin, sensitizes human ovarian cancer cell line.

We have studied the roles of c-Jun N-terminal protein kinase (JNK) and extracellular signal-regulated protein kinase (ERK) cascade in both the cisplatin-resistant Caov-3 and the cisplatin-sensitive A2780 human ovarian cancer cell lines. Treatment of both cells with cisplatin but not transplatin isomer activates JNK and ERK. Activation of JNK by cisplatin occurred at 30 min, reached a plateau at 3 h, and declined thereafter, whereas activation of ERK by cisplatin showed a biphasic pattern, indicating the different time frame. Activation of JNK by cisplatin was maximal at 1000 microM, whereas activation of ERK was maximal at 100 microM and was less at higher concentrations, indicating the different dose dependence. Cisplatin-induced JNK activation was neither extracellular and intracellular Ca(2+)- nor protein kinase C-dependent, whereas cisplatin-induced ERK activation was extracellular and intracellular Ca(2+)- dependent and protein kinase C-dependent. A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, PD98059, had no effect on the cisplatin-induced JNK activity, suggesting an absence of cross-talk between the ERK and JNK cascades. We further examined the effect of each cascade on the viability following cisplatin treatment. Either exogenous expression of dominant negative c-Jun or the treatment by PD98059 induced sensitivity to cisplatin in both cells. Our findings suggest that cisplatin-induced DNA damage differentially activates JNK and ERK cascades and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin.     

Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60

Focal adhesion kinase (FAK) has an anti-apoptotic role in anchorage-dependent cells via an unknown mechanism. To elucidate the role of FAK in anti-apoptosis, we have established several FAK cDNA-transfected HL-60 cell lines and examined whether FAK-transfected cells have resistance to apoptotic stimuli. FAK-transfected HL-60 (HL-60/FAK) cells were highly resistant to apoptosis induced with hydrogen peroxide (1 mm) and etoposide (50 microg/ml) compared with the parental HL-60 cells or the vector-transfected cells, when determined using viability assay, DNA fragmentation, and flow cytometry analysis. Because no proteolytic cleavage of pro-caspase 3 to mature caspase 3 fragment was observed in HL-60/FAK cells, FAK was presumed to inhibit an upstream signal pathway leading to the activation of caspase 3. HL-60/FAK activated the phosphatidylinositide 3'-OH-kinase-Akt survival pathway and exhibited significant activation of NF-kappaB with marked induction of inhibitor-of-apoptosis proteins (IAPs: cIAP-1, cIAP-2, XIAP), regardless of the hydrogen peroxide-treated or untreated conditions, whereas no significant IAPs were detected in the parental or vector-transfected HL-60 cells. Apoptotic agents induced higher NF-kappaB activation in HL-60/FAK cells than in HL-60/Vect cells, and it appeared that sustained NF-kappaB activation is critical to the anti-apoptotic states in HL-60/FAK cells. Mutagenesis of FAK cDNA revealed that Y397 and Y925, which are involved in the tyrosine-phosphorylation sites, were prerequisite for the anti-apoptotic activity as well as induction of IAPs, and that K454, which is involved in the kinase activity, was also required for the full anti-apoptotic activity of FAK. Taken together, we have demonstrated definitively that FAK-transfected HL-60 cells, otherwise sensitive to apoptosis, become resistant to the apoptotic stimuli. We conclude that FAK activates the phosphatidylinositide 3'-OH-kinase-Akt survival pathway with the concomitant activation of NF-kB and induction of IAPs, which ultimately inhibit apoptosis by inhibiting caspase-3 cascade.     

Phorbol diester-induced alterations in the expression of protein kinase C isozymes and their mRNAs. Analysis in wild-type and phorbol diester-resistant HL-60 cell clones.

In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild-type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA-induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA.