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.     

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     

Rapid increase in the activity of DNA topoisomerase I, but not topoisomerase II, in HL-60 promyelocytic leukemia cells treated with a phorbol diester

There is significant evidence to suggest that protein kinase C and DNA topoisomerases are functionally linked in signal transduction pathways. Much of this is based on the observation that phosphorylation of topoisomerase II by protein kinase C may lead to its activation in vitro and that inhibitors of topoisomerase II block phorbol diester-induced differentiation in HL-60 cells. In the present study, the activities of the DNA topoisomerases I and II have been quantitated to examine their regulation in phorbol diester-treated HL-60 cells undergoing differentiation. The activity of topoisomerase I increased rapidly after treatment with phorbol myristate acetate (PMA); it increased maximally (150% of control activity) at 3 hr post-treatment and remained elevated for at least 24 hr. Conversely, from the onset of exposure to PMA through 12 hr, there was no measurable alteration in topoisomerase II activity in PMA-treated cells. Moreover, there was a measurable decrease in topoisomerase II activity at the later time points, a result that occurred concomitantly with the loss of proliferative potential in differentiating HL-60 cells. Similar results were obtained when the activities of both enzymes were measured in nuclear extracts. The apparent increase in topoisomerase I activity was not due to an increase in the mass of the enzyme after PMA treatment, as measured by both western blotting and by the formation of camptothecin-dependent, topoisomerase I-DNA complexes. Taken together, these data suggest that the activities of the topoisomerases I and II may have been regulated independently in PMA-treated HL-60 cells, that the activity of topoisomerase II was not increased under conditions in which protein kinase C was activated in vivo, and that an increase in the activity of topoisomerase I may have had a role in the mechanism through which HL-60 cells underwent monocytic maturation in response to phorbol diesters.     

Relative activity of structural analogues of amsacrine against human leukemia cell lines containing amsacrine-sensitive or -resistant forms of topoisomerase II: use of computer simulations in new drug development.

Anilino analogues of amsacrine showed increased activity against amsacrine (AMSA)-resistant cell lines when compared with the parent compound, but the mechanisms of amsacrine resistance in these lines were unknown (Finlay, G. J., Baguley, B. C., Snow, K., and Judd, W., J. Natl. Cancer Inst., 82: 662-667, 1990). We tested the cytotoxic and DNA-cleaving activities of two amsacrine analogues which were derivatives of 9-anilinoacridine (1'-methylcarbamate and 1'-benzenesulfonamide) against an amsacrine-resistant human leukemia cell line (HL-60/AMSA) whose resistance is due to an amsacrine-resistant topoisomerase II. Neither agent could overcome the amsacrine resistance of HL-60/AMSA. Neither agent could induce HL-60/AMSA topoisomerase II-mediated cleavage of DNA in an isolated biochemical system, although at high concentrations the two analogues could inhibit HL-60/AMSA topoisomerase II-mediated DNA strand passage. Both analogues were at least as active, if not more active, than amsacrine against amsacrine-sensitive HL-60 and its topoisomerase II. Comparison of the cellular and biochemical results with those from computer simulation of the energy-minimized structures of amsacrine, its inactive isomer o-AMSA, and the two new active analogues suggests the following possibilities: (a) the positioning of the potential topoisomerase II-binding site (1'-anilino group) of the two new drugs resembles the positioning of this site in amsacrine; (b) the HL-60 topoisomerase II has a binding site which interacts with amsacrine and the two anilino analogues but not with o-AMSA, an analogue with altered positioning of the methoxy group; (c) the HL-60/AMSA topoisomerase II interacts with reduced affinity with amsacrine and the two anilino analogues, although HL-60/AMSA topoisomerase II still interacts with the structurally distinct topoisomerase II-reactive nonintercalator, etoposide; (d) because of their higher DNA binding affinity or the greater possible positions of their side groups in comparison to amsacrine, the two analogues can, at high concentrations, inhibit the strand-passing activity of HL-60/AMSA topoisomerase II.     

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.     

JAK-STAT signaling involved in phorbol 12-myristate 13-acetate- and dimethyl sulfoxide-induced 2'-5' oligoadenylate synthetase expression in human HL-60 leukemia cells

The JAK-STAT signal transduction cascade participates in various cellular processes, including immune response, cell replication, differentiation and oncogenesis. Here, we report that this cascade is induced in two human myeloid HL-60 leukemia cell variants by the granulocyte differentiation inducer dimethyl sulfoxide (DMSO) and macrophage differentiation inducer phorbol 12-myristate 13-acetate (PMA). DMSO and PMA also induced the expression and catalytic activity of 2'-5' oligoadenylate synthetase (2-5A synthetase), a known interferon (IFN) inducible enzyme. The HL-60 cell variants included HL-205, which is susceptible to DMSO- and PMA-induced differentiation, and HL-525, which is susceptible to DMSO- but not to PMA-induced differentiation. Treatment of HL-205 and HL-525 cells with DMSO and HL-205 cells with PMA-induced JAK1 phosphorylation, JAK1/STAT1 association, formation of STAT1-STAT2 heterodimers, and the binding of the active IFN stimulating growth factor 3 (ISGF3) to the IFN-stimulated response element (ISRE) fragment isolated from the 2-5A synthetase promoter. These events were either reduced or absent in the resistant HL-525 cells treated with PMA. Taken together, our data implicate the above signaling cascade in DMSO- and PMA-induced 2-5A synthetase expression and catalytic activity in the HL-60 cell system.     

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.     

Intercalator-induced, topoisomerase II-mediated DNA cleavage and its modification by antineoplastic antimetabolites

Defining specific biochemical targets of active antineoplastic agents could aid in discovering better anticancer therapy and more thoroughly understanding the biochemical basis of malignancy. Through a series of cellular and biochemical studies, we and others have identified the nuclear enzyme topoisomerase II as the target of several active agents, including 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA). The interference with topoisomerase II produced by m-AMSA can be quantified in whole cells exposed to m-AMSA by using the alkaline elution technique to measure DNA cleavage. Antimetabolites such as ara-C, hydroxyurea, and 5-azacytidine can augment m-AMSA-induced, topoisomerase II-mediated DNA cleavage and, concurrently, m-AMSA-induced cell killing. Studies in proliferating and quiescent human cells and an m-AMSA-sensitive/resistant human leukemia cell pair further support the hypothesis that a connection exists between topoisomerase II-mediated DNA cleavage and the mechanism by which m-AMSA kills cells. Pharmacologic or hormonal modification of specific biochemical processes critical to drug-induced cytotoxicity may enhance the therapeutic index of clinically useful agents.     

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.     

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.     

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.     

DNA topoisomerase II as a target of antineoplastic drug therapy

Summary A major goal of cancer therapy research is identification of critical biochemical targets that mediate the ability of effective cancer chemotherapy to kill tumor cells while allowing the maintenance of normal cell function. A candidate for such a target is DNA topoisomerase II, a ubiquitous enzyme that alters three-dimensional conformation of supercoiled DNA. DNA intercalating agents and epipodophyllotoxins stabilize a DNA and topoisomerase II complex. The process of stabilization probably represents the poisoning of an intermediate state in the normal functioning of the enzyme. This stabilized intermediate state can be measured in whole cells using the filter elution method of Kohn to quantify protein-associated DNA cleavage produced when the cells are exposed to intercalators or epipodophyllotoxins. By altering cell populations in quantifiable ways, four factors appear to influence the magnitude of drug-induced, topoisomerase II-mediated DNA cleavage and cytotoxicity: (a) the proliferative state of the cell (proliferating cells are more sensitive than quiescent ones); (b) the cell cycle state (cells pharmacologically recruited into G₁-S are more sensitive than asynchronously growing cells); (c) the chromatin conformation (DNA methylation, polyamine depletion, and other chromosomal changes can alter the magnitude of topoisomerase II-mediated effects); (d) the cellular phenotype (in an as yet uncharacterized manner, malignant cells apparently are more sensitive to topoisomerase II-mediated events than normal cells). These data suggest that the biochemical basis of the therapeutic index of drugs such as the intercalating agents or epipodophyllotoxins may be the intrinsic hypersensitivity of the topoisomerase II in malignant cells to poisoning by these drugs.     

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.     

Comparison of DNA cleavage induced by etoposide and doxorubicin in two human small-cell lung cancer lines with different sensitivities to topoisomerase II inhibitors

In an attempt to clarify the role of drug-induced protein-associated DNA breaks (i.e., DNA topoisomerase II-mediated DNA cleavage) in the cytotoxic activity of doxorubicin and etoposide, their cellular effects were compared in 2 human small-cell lung cancer (SCLC) lines, characterized by differential sensitivity to DNA topoisomerase II inhibitors. These drugs were selected for comparative studies since they are among the most effective agents in the treatment of SCLC. H146 and N592 cell lines were obtained from pleural effusion and bone-marrow aspirate of pretreated patients, respectively. Both cell lines grew as floating aggregates with similar doubling times (30 and 33 hr for N592 and H146 cells, respectively). Although, immediately after 1 hr exposure to equitoxic drug levels, the extent of DNA cleavage produced by doxorubicin was markedly lower than that produced by etoposide, DNA lesions produced by doxorubicin persisted and even increased following drug removal. In contrast, an almost complete disappearance of etoposide-induced DNA breaks was noted 1 hr after drug removal. Resealing of strand breaks was faster in N592 than in H146 cells. These findings suggest that reversal of these lesions plays a major role in cell survival rather than the occurrence of DNA breaks immediately following drug exposure. This observation is consistent with the view that inhibition of DNA re-ligation rather than stimulation of DNA cleavage is the critical step for drug action. The different response of these cell lines to cytotoxic action of the topoisomerase inhibitors is associated with a differential drug effect on DNA integrity (detected as DNA double-strand breaks and DNA-protein cross-links). However DNA lesions were comparable when cells were exposed to equitoxic drug levels. The observation that etoposide-induced DNA breaks were similar in isolated nuclei from both cell lines suggests that drug-target interaction is modulated in a different manner in the intact cell. As indicated by doxorubicin uptake and retention, cellular drug pharmacokinetics do not account for the different drug response of the studied SCLC lines, presumably, reflecting a different extent of DNA break formation and/or a different cytotoxic consequence of DNA damage.     

The effect of 9-β-D-arabinofuranosyl-2-fluoroadenine and 1-β-D-arabinofuranosylcytosine on the cell cycle phase distribution,topoisomerase II level,mitoxantrone cytotoxicity,and DNA strand break production in K562 human leukemia cells

 Antimetabolites and topoisomerase (topo) II-reactive drugs are frequently combined in the therapy of acute leukemia. The two types of agents are thought to be synergistic in their actions against malignant blasts but the mechanism for this synergism is incompletely described. This study sought to determine whether the combination of two rather than one antimetabolite with the topo II-reactive intercalator mitoxantrone would be greater than the effect of the single antimetabolite ara-C on mitoxantrone’s cytotoxic actions. We also aimed to determine a mechanism for synergism should it occur. The model system used was K562 human leukemia cells. The second antimetabolite selected was F-ara-A, the active form of fludarabine. The resultant combination (F-ara-A, ara-C, and a topo II-reactive drug) is one currently being tested against acute myelogenous leukemia in clinical trials. F-ara-A itself had little effect on the cytotoxicity or the topo II-mediated DNA cleaving actions of mitoxantrone, while ara-C potentiated these actions as it does those of other topo II-reactive drugs. Surprisingly F-ara-A enhanced the actions of ara-C on mitoxantrone-associated cytotoxicity by at least an order of magnitude. The effect of the addition of F-ara-A to ara-C on mitoxantrone-induced DNA cleavage was considerably smaller, but present. Antimetabolite treatment did not increase the amount of topo II within cells measured directly by immunoblotting or indirectly by quantifying the maximum number of topo II – DNA complexes stabilized by mitoxantrone. Rather, the antimetabolites altered the distribution of the cells in the cell cycle. Antimetabolite treatment caused a large increase in S-phase cells, a phase in which cells are more sensitive to topo II-reactive drugs than the associated topo II-mediated DNA cleavage would predict. Therefore, it is likely that this shift in the distribution of the cells within the cell cycle accounts for both the enhanced cytotoxicity of mitoxantrone in antimetabolite pretreated cells and the discrepancy between the magnitude of antimetabolite action on topo II-mediated DNA cleavage. Received: 11 April 1995/Accepted: 20 October 1995     

A variant of the HL-60 cell line expressing a high level of PTP1C exhibits increased susceptibility to differentiation by phorbol 12-myristate 13-acetate

A variant of the HL-60 cell line, HL-60/MCSFR4D2, has been found to express twice the amount of PTP1C as compared to the parental HL-60 cell line by immunoblotting and immunoprecipitation. Differentiation of the variant cells after phorbol 12-myristate 13-acetate (PMA) treatment was examined by the appearance of adherence. In 1% fetal calf serum (FCS), 20% of HL-60/MCSFR4D2 cells exhibited adherence after treatment with 0.5 ng/ml PMA for 48 h, 60% exhibited adherence after treatment with 1.0 ng/ml PMA and 80% exhibited adherence after treatment with 5.0 ng/ml PMA, while HL-60 cells exhibited only a slight response. Furthermore, antisense PTP1C oligonucleotides decreased the PMA-induced adherence of HL-60/MCSFR4D2 cells. These results suggest that the high-expression of PTP1C in HL-60 cells may be involved in the enhancement of susceptibility to macrophage-like differentiation by PMA.     

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     

Doxorubicin-DNA adducts induce a non-topoisomerase II-mediated form of cell death

Doxorubicin (Adriamycin) is one of the most commonly used chemotherapeutic drugs and exhibits a wide spectrum of activity against solid tumors, lymphomas, and leukemias. Doxorubicin is classified as a topoisomerase II poison, although other mechanisms of action have been characterized. Here, we show that doxorubicin-DNA adducts (formed by the coadministration of doxorubicin with non-toxic doses of formaldehyde-releasing prodrugs) induce a more cytotoxic response in HL-60 cells than doxorubicin as a single agent. Doxorubicin-DNA adducts seem to be independent of classic topoisomerase II-mediated cellular responses (as observed by employing topoisomerase II catalytic inhibitors and HL-60/MX2 cells). Apoptosis induced by doxorubicin-DNA adducts initiates a caspase cascade that can be blocked by overexpressed Bcl-2, suggesting that adducts induce a classic mode of apoptosis. A reduction in the level of topoisomerase II-mediated double-strand-breaks was also observed with increasing levels of doxorubicin-DNA adducts and increased levels of apoptosis, further confirming that adducts exhibit a separate mechanism of action compared with the classic topoisomerase II poison mode of cell death by doxorubicin alone. Collectively, these results indicate that the presence of formaldehyde transfers doxorubicin from topoisomerase II-mediated cellular damage to the formation of doxorubicin-DNA adducts, and that these adducts are more cytotoxic than topoisomerase II-mediated lesions. These results also show that doxorubicin can induce apoptosis by a non-topoisomerase II-dependent mechanism, and this provides exciting new prospects for enhancing the clinical use of this agent and for the development of new derivatives and new tumor-targeted therapies.     

2-deoxyglucose inhibits chemotheapeutic drug-induced apoptosis in human monocytic leukemia U937 cells ith inhibition of c-Jun N-terminal kinase 1/stress-activated protein kinase activation

Human monocytic leukemia U937 cells undergo apoptosis when treated with antitumor drugs, such as etoposide, camptothecin and mitomycin C. The molecular mechanism of the drug-induced apoptosis is not well understood. In this study, we found that 2-deoxyglucose (2DG), an analog of D-glucose and an inducer of glucose-regulated stress, inhibited anticancer drug-induced but not tumor necrosis factor-alpha-induced apoptosis of U937 cells. 2DG did not reduce initial cellular damage caused by etoposide, an inhibitor of topoisomerase II, suggesting that 2DG affected subsequent cellular responses involved in apoptosis. 2DG inhibited the etoposide-induced activation of c-Jun N-terminal kinase 1/stress-activated protein kinase (JNK1/SAPK) and the subsequent activation of CPP32, both of which are positive regulators for etoposide-induced apoptosis of U937 cells. Our results indicate that 2DG inhibits apoptosis by blocking the signals from cellular DNA damage for JNK1/SAPK activation. Int. J. Cancer 76:86–90, 1998.© 1998 Wiley-Liss, Inc.     

Spermine acts as a negative regulator of macrophage differentiation in human myeloid leukemia cells

The role of putrescine, spermidine and spermine in phorbol 12-myristate-13-acetate (PMA)-induced macrophage differentiation was examined in human HL-60 and U-937 myeloid leukemia cells. Unlike other polyamines, spermine affected this differentiation by acting as a negative regulator. This negative regulation was established by showing that the PMA-induced macrophage phenotype, but not PMA-associated replication arrest, was abrogated (a) by replenishing the PMA-evoked decrease in cellular spermine levels with this polyamine from an exogenous source and (b) by blocking PMA-induced expression of the polyamine catabolic enzyme N(1)-spermidine/spermine acetyltransferase (SSAT) with antisense oligonucleotides in the presence of low substrate level. The PMA-evoked reduction in cellular spermine appears to result from an increase in the activity of SSAT and a decrease in the activity of ornithine decarboxylase, the polyamine biosynthetic enzyme. To a degree, these changes are due to corresponding changes in the expression of the genes that code for these enzymes. When cell differentiation is initiated, SSAT expression is increased after PMA-evoked activation of protein kinase C-beta. The present studies raise the possibility that agents able to reduce spermine levels in patients' myeloid leukemia cells may enhance the activity of differentiation therapy drugs for this type of leukemia.