Novobiocin- and phorbol-12-myristate-13-acetate-induced differentiation of human leukemia cells associated with a reduction in topoisomerase II activity

Studies were conducted to determine the possible involvement of DNA topoisomerase II (Topo II) in the induction of differentiation in two human promyelocytic HL-60 leukemia cell variants that are either susceptible or resistant to differentiation induced by phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator. The acquisition of maturation markers and changes in the activity, level, and phosphorylation of Topo II were determined after treatment with either novobiocin, a Topo II inhibitor, or PMA. Novobiocin at 50-150 microM induced differentiation in the HL-205 cells but not in the HL-525 cells, although both cell types were equally susceptible to novobiocin-evoked cytotoxicity. In both cell types, novobiocin induced similar reductions in topoisomerase I activity but different reductions in Topo II activity. Treatment with novobiocin at 150 microM for 6 h or at 2 mM for 30 min resulted in a 4-fold or higher reduction in Topo II activity in the differentiation-susceptible HL-205 cells but not in the differentiation-resistant HL-525 cells. A differential response in Topo II activity was also observed after treatment with PMA. The novobiocin-evoked decrease in Topo II activity seems to be due to an enhanced enzyme proteolysis, whereas the PMA-elicited decrease in Topo II activity is associated with an increase in Topo II phosphorylation. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine, which is an inhibitor of protein kinases, including protein kinase C, diminished the novobiocin-elicited proteolysis of Topo II and the PMA-induced Topo II phosphorylation, as well as the decrease in Topo II activity and the acquisition of differentiation markers induced by either novobiocin or PMA. These results suggest that induction of differentiation in HL-60 cells by novobiocin or PMA is associated with a reduction in Topo II activity, mediated directly or indirectly by a protein kinase(s), perhaps protein kinase C.     

A new derivative of camptothecin, irinotecan hydrochloride (cpt-11) induces programmed cell-death in leukemia-lymphoma cell-lines

Irinotecan Hydrochloride (CPT-11) and 7-ethyl-10-hydroxycamptothecin (SN-38), which are both topoisomerase I inhibitors with potent antitumor effects in vivo and in vitro, were tested for the induction of programmed cell death (PCD) in leukemia/lymphoma cell lines. When the human T-cell leukemia cell line HUT-102 and the human promyelocytic leukemia cell line HL-60 cells were exposed to CPT-11, PCD characterized by a DNA fragmentation ladder of 180-200 bp in agarose gel electrophoresis and loss of cell viability was induced. The PCD inducing activity of SN-38, an active metabolite of CPT-11, was much more powerful than that of CPT-11. Besides inducing PCD in HUT-102 and HL-60 cells, SN-38 also induced PCD in the human erythroblast leukemia cell line K-562, which was resistant to CPT-11. Induction of PCD by SN-38 and CPT-11 was dose- and time-dependent. PCD in HUT-102 cells induced by SN-38 was prevented neither by aurintricarboxylic acid (ATA), an endonuclease inhibitor, as determine by DNA electrophoretic profiles and the number of viable cells, nor by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). The present data suggest that the topoisomerase I inhibitors, SN-38 and CPT-11 exert antitumor activity through induction of PCD in involved cells, at least in part. The PCD-inducing activity of the topoisomerase II inhibitor VP-16 was also tested in the above three cell lines and compared with CPT-11 and SN-38.     

Phorbol ester effects on topoisomerase II activity and gene expression in HL-60 human leukemia cells with different proclivities toward monocytoid differentiation

We examined the effects of phorbol ester treatment on topoisomerase II-mediated events in two human leukemia cell lines with different proclivities toward phorbol ester-induced monocytoid differentiation. HL-60 is the parent line that will terminally differentiate; 1E3 is a derived line that will not terminally differentiate. Within 24 h of phorbol ester treatment, etoposide-induced, topoisomerase II-mediated DNA cleavage declined 10-fold, whereas 4'-(9-acridinylamino)-methanesulfon-m-anisidide- induced DNA cleavage declined 3-fold in HL-60. In phorbol-treated 1E3, etoposide-induced DNA cleavage declined only 2-fold, whereas 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced cleavage was barely affected. There was a 2- to 3-fold decline in topoisomerase II activity within the nuclear extracts from phorbol-treated HL-60 cells but not from phorbol-treated 1E3 cells. Immunoblotting experiments with anti-topoisomerase II antibodies indicated that phorbol treatment produced a structural change in the immunoreactive topiosomerase II in HL-60 nuclear extracts but produced no change in 1E3 topoisomerase II. Phorbol ester treatment also produced a decline in the level of topoisomerase II gene expression in HL-60 but not in 1E3 cells. By contrast, the cytotoxicity of etoposide in both lines was decreased following phorbol treatment. Thus, phorbols may uncouple the mechanisms linking drug-induced, topoisomerase II-DNA cleavable complex stabilization with drug-induced cytotoxicity, particularly in 1E3.     

Decreased expression of type II protein kinase C in HL-60 variant cells resistant to induction of cell differentiation by phorbol diester

To evaluate the molecular basis for susceptibility of the cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), we examined biochemical activities and expression of subspecies of protein kinase C from HL-60 cells that are susceptible to differentiation induced by TPA and HL-60R cells, HL-60 variant cells that are resistant to such induction. Analysis of the subcellular distribution of protein kinase C revealed that the activity of this kinase in the cytosol from HL-60R cells was 30% of that from parental HL-60 cells but that the enzyme activities in the membrane showed similar values in these cells. Treatment of HL-60 cells with 100 nM TPA for 30 min resulted in a 75% decrease in protein kinase C activity in the cytosol and a 300% increase in this activity in the membrane. A minor subcellular redistribution of the enzyme activity was found in HL-60R cells after TPA treatment. Based on analysis of protein kinase C isozymes by hydroxyapatite column chromatography, the relative activities of types I, II, and III in the cytosol of HL-60 cells were 11, 80, and 9%, whereas those in HL-60R cells were 27, 36, and 37%, respectively. Type II isozyme was a major protein kinase C in the cytosol of HL-60 cells, but type II was less in the HL-60R cells. Among the three isozymes, type II enzyme was most sensitive to TPA with histone H1 as the substrate, although all three isozymes were activated Ca2+-dependently in the presence of phosphatidylserine. We suggest that the acquired resistance of HL-60R cells toward induction of cell differentiation by TPA may be associated with a decrease in the expression of the type II isozyme of protein kinase C.     

Camptothecin hyper-resistant P388 cells: drug-dependent reduction in topoisomerase I content.

A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.     

Changes in thymidine kinase activity during differentiation of HL-60 leukemic cells

Induction of granulocyte maturation in HL-60 leukemic cells by DMSO (1.2%) or RA (1 microM) is accompanied by a 50-60% decrease in cellular thymidine kinase activity. Similarly, the differentiation of HL-60 cells into monocyte-macrophage phenotype by the addition of PMA is paralleled by a 60-80% suppression of thymidine kinase specific activity. Measurement of thymidine kinase kinetic parameters shows that the Vmax decreases from 0.7 pmol/min in control cells to 0.43 pmol/min in PMA-treated cells and to 0.38 pmol/min in RA-treated cells. The Km of the enzyme is not affected by either inducing agent and remains at 2.1 microM. Studies with PMA analogs suggest that thymidine kinase modulation is coupled to HL-60 differentiation.     

The effect of staurosporine on drug-induced,topoisomerase II-mediated DNA cleavage in human leukemia cells

Summary Phorbol-12-myristate 13-acetate (PMA), a stimulator of protein kinase C, dramatically decreased topoisomerase II-reactive drug-induced DNA cleavage in HL-60 human cells. The effect of staurosporine, an inhibitor of protein kinase C, on drug-induced, topoisomerase II-mediated DNA cleavage was quantified in the same cells. Staurosporine decreased the magnitude of 4-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)- and etoposide-induced DNA cleavage in a dose-and time-dependent fashion. Measurement of several parameters of cell proliferation revealed no clear and uniform correlation between staurosporine's inhibition of these parameters and its effects on drug-induced DNA cleavage. A direct comparison with PMA's effects on drug-induced DNA cleavage showed that whereas PMA's inhibition of etoposide-induced cleavage was much greater than its inhibition ofm-AMSA-induced cleavage, the magnitude of staurosporine's effect on the cleavage produced by the two topoisomerase II-reactive drugs was similar. Thus, although PMA stimulates protein kinase C and staurosporine inhibits this enzyme, it is unlikely that the actions of either on topoisomerase II-reactive, drug-induced DNA cleavage are mediated directly via protein kinase C. Furthermore, it is likely that the mechanisms by which PMA and staurosporine inhibit topoisomerase II-reactive drug-induced cleavage are different.Abbreviations m-AMSA 4-(9-Acridinylamino)methanesulfon-m-anisidide; etoposide - VP-16 4-demethylepipodiphyllotoxin 4-(4,6-O-ethylidene--d-glucopyranoside) - STSN staurosporine - PMA phorbol-12-myristate 13-acetate - DMSO dimethylsulfoxide - AD adherent cells - SN supernatant cells     

A novel antitumor compound,NC-190, induces topoisomerase II-dependent DNA cleavage and DNA fragmentation

 A novel benzophenazine derivative, NC-190, is a potent antitumor compound. NC-190 has been shown to inhibit the DNA strand-passing activity of DNA topoisomerase II. We investigated further the mode of action of NC-190 against DNA topoisomerase II and DNA fragmentation. NC-190 inhibited the decatenation activity of purified topoisomerase II, but had only a weak inhibitory effect against topoisomerase I. A topoisomerase II-dependent DNA cleavage assay showed that NC-190 inhibited the enzyme activity by stabilizing a topoisomerase II–DNA cleavable complex. NC-190 induced growth inhibition, protein-linked DNA breaks, and DNA fragmentation in cultured HL-60 cells in a dose-dependent manner. These activities of NC-190 in HL-60 cells were comparable to those of etoposide (VP-16). These results demonstrate a good correlation among growth inhibition, topoisomerase II-dependent DNA cleavage, and DNA fragmentation induced by NC-190. A DNA unwinding assay showed that NC-190 had intercalating activity, but its activity appeared to be weaker than those of ethidium bromide and adriamycin. These results indicate that the mechanism by which NC-190 exhibits antitumor activity may be the inhibition of topoisomerase II. Received: 23 September 1994/Accepted: 2 August 1995     

Protein kinase C activities with different characteristics, including substrate specificity, from two human HL-60 leukemia cell variants

To evaluate the role of protein kinase C in the cellular maturation processes induced by phorbol diesters, we examined the biochemical activity of protein kinase C from HL-205, a cell variant from the human promyelocytic HL-60 leukemia that is susceptible to differentiation induced by phorbol 12-myristate 13-acetate, and from HL-525, an HL-60 variant that is resistant to such an induction. The activities of protein kinase C from the two cell types differed in their requirements for the cofactors Ca2+ and lipids. These enzyme activities also differed in their abilities to phosphorylate protamine and a series of four oligopeptides. We suggest that the differences in vitro in the activities of protein kinase C between HL-205 and HL-525 cells, especially in their substrate specificity, are closely related to the different phosphorylation patterns induced in vivo by phorbol 12-myristate 13-acetate in these cells. We also suggest that these differences may be responsible for the different susceptibilities of the two cell types to maturation induced by phorbol diesters.     

The cyclin-dependent kinase inhibitor (CDKI) flavopiridol disrupts phorbol 12-myristate 13-acetate-induced differentiation and CDKI expression while enhancing apoptosis in human myeloid leukemia cells

Interactions between the cyclin-dependent kinase inhibitor (CDKI) flavopiridol (FP) and phorbol 12-myristate 13-acetate (PMA) were examined in U937 human leukemia cells in relation to differentiation and apoptosis. Simultaneous, but not sequential, exposure of U937 cells to 100 nM FP and 10 nM PMA significantly increased apoptosis manifested by characteristic morphological features, mitochondrial dysfunction, caspase activation, and poly(ADP-ribose) polymerase cleavage while markedly inhibiting cellular differentiation, as reflected by diminished plastic adherence and CD11b expression. Enhanced apoptosis in U937 cells was associated with an early caspase-independent increase in cytochrome c release and accompanied by a substantial decline in leukemic cell clonogenicity. Moreover, PMA/FP cotreatment significantly increased apoptosis in HL-60 promyelocytic leukemia cells and in U937 cells ectopically expressing the Bcl-2 protein. In U937 cells, coadministration of FP blocked PMA-induced expression and reporter activity of the CDKI p21WAF/CIP1 and triggered caspase-mediated cleavage of the CDKI p27KIP1. Coexposure to FP also resulted in a more pronounced and sustained activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase cascade after PMA treatment, although disruption of this pathway by the mitogen-activated protein kinase kinase 1 inhibitor U0126 did not prevent potentiation of apoptosis. FP accelerated PMA-mediated dephosphorylation of the retinoblastoma protein (pRb), an event followed by pRb cleavage culminating in the complete loss of underphosphorylated pRb (approximately Mr 110,000) by 24 h. Finally, gel shift analysis revealed that coadministration of FP with PMA for 8 h led to diminished E2F/pRb binding compared to the effects of PMA alone. Collectively, these findings indicate that FP modulates the expression/activity of multiple signaling and cell cycle regulatory proteins in PMA-treated leukemia cells and that such alterations are associated with mitochondrial damage and apoptosis rather than maturation. These observations also raise the possibility that combining CDKIs and differentiation-inducing agents may represent a novel antileukemic strategy.     

Up regulation of the phorbol ester receptor-protein kinase C in HL-60 variant cells

The human promyelocytic leukemia cell line HL-60 can be induced to differentiate into macrophage-like cells by nanomolar concentrations of phorbol esters. A phorbol ester-resistant variant R1B6 obtained by culturing HL-60 cells with increasing concentrations of 12-O-tetradecanoylphorbol-13-acetate, is reversibly resistant. These cells have been growing continuously in the presence of phorbol esters for more than 1 yr, but when the phorbol ester is removed, the cells gradually regain their sensitivity and express characteristics of macrophage-like cells upon readdition of phorbol ester. The concentration of phorbol ester receptors in R1B6 is about one-third that in the parental HL-60 cells. The reversion of the variants to sensitivity to phorbol esters is associated with the up regulation of the cytosol and membrane phorbol ester receptors. When partially purified, these receptor populations contain protein kinase C activity, in support of the identity of protein kinase C and the receptor. This study demonstrates that a phenotypic change in a clonal cell population correlates with the up regulation of the phorbol ester receptor-calcium-activated phospholipid-dependent protein kinase. This variant cell line is a useful model for analyzing the relationship between phorbol ester binding and protein kinase C during differentiation of HL-60 cells.     

The pro-apoptotic drug camptothecin stimulates phospholipase D activity and diacylglycerol production in the nucleus of HL-60 human promyelocytic leukemia cells.

It has recently been reported (T. Shimizu et al., J. Biol. Chem., 273: 8669-8674, 1998) that the pro-apoptotic drug, camptothecin, an inhibitor of topoisomerase I, induces a protein kinase C-alpha-mediated phosphorylation of lamin B in HL-60 cells, which precedes both degradation of lamin B and fragmentation of DNA. In this paper, we report that, in HL-60 cells exposed to camptothecin, there is a rapid and sustained increase of nuclear protein kinase C-alpha activity that is due to an increase in the amount of protein kinase C-alpha present in the nucleus. The enhancement of nuclear kinase C activity is preceded by an increase in the mass of nuclear diacylglycerol. As demonstrated by its sensitivity to propranolol, the nuclear diacylglycerol mass increase is due to the activation of a phospholipase D. Indeed, inhibitors of neither phosphatidylcholine-specific phospholipase C nor phosphoinositide-specific phospholipase C blocked the rise in nuclear diacylglycerol. In vitro assays also demonstrated the activation of a nuclear phospholipase D, but not of a phosphoinositide-specific phospholipase C, after treatment with camptothecin. Propranolol was also able to block the rise in nuclear protein kinase C-alpha activity, thus suggesting that the increase in diacylglycerol mass is important for the activation of the kinase at the nuclear level. Moreover, propranolol was capable of drastically reducing the number of HL-60 cells that underwent apoptosis after treatment with camptothecin. Our results show the activation during apoptosis of a phospholipase D-mediated signaling pathway operating at the nuclear level. This pathway may represent an attractive therapeutic target for the modulation of apoptotic events in human disease.     

Identification of a point mutation in the topoisomerase II gene from a human leukemia cell line containing an amsacrine-resistant form of topoisomerase II.

HL-60/AMSA is a human leukemia cell line that is 50- to 100-fold more resistant to the cytotoxic actions of the topoisomerase II-reactive intercalator amsacrine than is its drug-sensitive HL-60 parent line. Previously, we have shown that the topoisomerase II from HL-60/AMSA is also resistant to inhibition by amsacrine and other intercalating agents. We therefore sought the molecular basis for the resistance of the topoisomerase II of HL-60/AMSA and, by inference, of the HL-60/AMSA line itself. We report the cloning and sequencing of the topoisomerase II genes from both the sensitive and resistant leukemia cell lines using polymerase chain reaction technology. We have identified a single base change associated with the drug-resistant form of topoisomerase II. This mutation is present in both cloned HL-60/AMSA complementary DNA and extracted HL-60/AMSA genomic DNA. A rapid assay for this mutation in clinical samples has been developed and applied to the DNA of cells from both normal volunteers and leukemia patients. Thus far, the HL-60/AMSA genotype has not been identified in the cells from any individual, suggesting that this genotype is indeed a mutation and not an allelic form of topoisomerase II. The novel assay developed will allow a rapid search for the prevalence of this mutation in clinical samples from patients with leukemia who have relapsed following intercalator therapy.     

Potentiation of topoisomerase inhibitor-induced DNA strand breakage and cytotoxicity by tumor necrosis factor: enhancement of topoisomerase activity as a mechanism of potentiation

A combination of tumor necrosis factor (TNF) and the topoisomerase I inhibitor, camptothecin, or the topoisomerase II inhibitors, teniposide and amsacrine, produced dose-dependent synergistic cytotoxicity against the murine L929 fibrosarcoma cells. Similar synergy was not observed with a combination of TNF and bleomycin. To define the role of TNF in the augmentation of tumor cell killing by topoisomerase I or II inhibitors, the effect of TNF on the production of enzyme-linked DNA strand breaks induced in cells by topoisomerase inhibitors was investigated. L929 cells incubated for 1 h with the topoisomerase inhibitors contained protein-linked strand breaks. In contrast, TNF alone did not induce DNA strand breakage. However, when cells were incubated simultaneously with TNF and camptothecin, amsacrine, Adriamycin, actinomycin D, teniposide, or etoposide, increased numbers of strand breaks were produced. Preincubation of the cells with TNF for 30 min or 3 h before the addition of camptothecin or etoposide resulted in no more strand breaks than that observed in cells incubated with the drugs alone. TNF treatment of L929 cells produced a rapid and transient increase in specific activity of extractable topoisomerases I and II. These increases were maximum at 2-5 min of TNF treatment and by 30 min the activities of extractable enzymes were equal to or less than those detected in extracts from untreated cell controls. The transient nature of the increase in extractable topoisomerase activity may explain the kinetics and significance of the order of addition of TNF and inhibitors for maximal synergistic activity. These data are consistent also with a role for topoisomerase-linked DNA lesions in the TNF-mediated potentiation of killing of L929 cells by topoisomerase inhibitors.     

Up-regulation of p21WAF1 expression in myeloid cells is activated by the protein kinase C pathway.

Phorbol-12-myristate-13-acetate (PMA) induces p21WAF-1 expression in human myeloid leukaemic HL-60 cells. We show that this induction is specifically mediated by protein kinase C (PKC). In addition, the PKC inhibitor Ro 31-8220 with predominant PKC-alpha isoform specificity almost completely inhibited PMA-induced up-regulation of p21WAF1 in HL-60 cells as well as in the myelomonocytic leukaemic U937 cells. Pretreatment of HL-60 cells with Ro 31-8220 also inhibited PMA-induced activation of c-raf-1, a known PKC alpha target. In the phorbol ester-tolerant HL-60 subline (PET) with PKC-beta isoform deficiency PMA or bryostatin-1 induced p21WAF1 expression, but to a lesser extent than in wild-type HL-60 cells. In PET cells, Ro 31-8220 also inhibited PMA and bryostatin-1-induced up-regulation of p21WAF1 expression. Our findings indicate that at least in HL-60 cells up-regulation of p21WAF-1 is specifically activated by PKC. We suggest that PKC isoforms other than beta, presumably the PKC-alpha isoform, are involved in this process.     

Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors

Most DNA intercalators and epipodophyllotoxins inhibit mammalian topoisomerase II by trapping the enzyme within DNA cleavage complexes that can be detected in cells as protein-associated DNA strand breaks. We have characterized previously a line of Chinese hamster cells (DC3F/9-OHE cells) the resistance of which to the cytotoxic effect of intercalators and etoposide is associated with a reduced formation of protein-associated DNA strand breaks. In the present study, topoisomerases of these cells were compared to those of the parental sensitive cells (DC3F). NaCl extracts (0.35 M) of isolated DC3F/9-OHE nuclei did not form 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA-protein linking, whereas DC3F nuclear extracts did. In addition, DC3F/9-OHE nuclear extract had an unusually high level of DNA linking activity in the absence of 4'-(9-acridinylamino)methanesulfon-m-anisidide. Topoisomerases II from DC3F/9-OHE and DC3F nuclei appeared similar qualitatively. DC3F/9-OHE nuclear extract had approximately twice less topoisomerase II molecules than did DC3F nuclear extract but similar topoisomerase II activity. Topoisomerase I activities appeared also similar in sensitive and resistant cells. However, part of DC3F/9-OHE topoisomerase I copurified with a DNA linking activity which was not present in DC3F nuclei. This unusual DNA linking activity was not sensitive to the stimulatory effect of 4'-(9-acridinylamino)methanesulfon-m-anisidide.     

Sequence effect of irinotecan (CPT-11) and topoisomerase II inhibitors in vivo

The DNA topoisomerases I and II are the target of several clinically important antineoplastic agents which produce DNA cleavage by stabilization of the covalent DNA-protein bond with resultant cell death after DNA synthesis is attempted. Depletion of the target topoisomerase and reciprocal changes in the other occur with drug treatment. Purpose and methods: To develop empiric treatment regimens of combinations and sequences of agents directed against topoisomerase I (irinotecan/CPT-11) and II (etoposide and doxorubicin), in vivo studies were performed in mice bearing the EMT-6 mammary tumor to assess efficacy, host tolerance and the resultant biochemical changes in topoisomerase mRNA and protein. Results: At 24 h after therapy, depletion of the target topoisomerase mRNA and protein with reciprocal increases in the alternate topoisomerase mRNA and, to a lesser extent, protein were noted. No therapeutic antagonism was found with any combination or sequence of agents, and therapeutic antagonism was noted with concurrent irinotecan/etoposide and sequential doxorubicin/irinotecan. Depletion of target topoisomerases by combined therapy beyond a threshold necessary for therapeutic efficacy produced no additional benefit. Conclusions: Antineoplastic therapy with combinations of topoisomerase I and II agents is feasible and may produce therapeutic synergy. The appropriate sequence may depend on the particular agents used. The rationale for such therapy, that topoisomerases I and II may have reciprocal and compensatory interactions, is supported by the biochemical data. Received: 8 September 1997 / Accepted: 15 January 1998     

The activation of PI 3-K and PKC zeta in PMA-induced differentiation of HL-60 cells

The human myelocytic leukemia cell line HL-60 is a useful model for the study of cellular differentiation. Phorbol 12-myristate 13-acetate (PMA) induces the monocyte/macrophage-like differentiation of HL-60 cells and results in growth arrest, increasing adherence. In PMA-induced differentiation of HL-60 cells, phosphoinositide 3-kinase (PI 3-K) activity was measured as phosphatidylinositol3P recovery from phosphatidylinositol by in vitro kinase assay. PI 3-K activity was increased in HL-60 cells that were stimulated by 20 nM PMA and the activity was inhibited by pretreatment with 20 microM LY294002, a specific inhibitor of PI 3-K. Members of the protein kinase C (PKC) family have been suggested to be one of the downstream targets of PI 3-K. PKC zeta is one of the atypical PKCs, non-diacylglycerol-responsive PKCs, and the activity was measured by the ability of phosphorylation onto myelin basic protein. PMA also induced the activation of PKC zeta during monocytic differentiation of HL-60 cells, and LY294002-pretreated cells failed to induce PKC zeta activation. The activity of PI 3-K is essential for PKC zeta activation, and LY294002 blocks both monocytic differentiation of HL-60 cells and activation of PKC zeta during PMA-induced cell differentiation. This implies that activated PI 3-K subsequently stimulates the PKC zeta in the process of PMA-induced monocytic differentiation.