(CANCER RESEARCH 49. 1110-1117. March 1. 1989] Novobiocin- and Phorbol-12-myristate-13-acetate-induced Differentiation of Human Leukemia Cells Associated with a Reduction in Topoisomerase II Activity1 Andreas Constantinou,2 Cynthia Henning-Chubb, and Eliezer Huberman Biological, Environmental, and Medical Research Division, Argonne National Laboratory, Argonne, Illinois 60439 ABSTRACT Studies were conducted to determine the possible involvement of DNA topoisomerase II (Topo II) in the induction of differentiation in two human promyeloc)tic 111 (ill leukemia cell variants that are either sus ceptible or resistant to differentiation induced by phorbol-12-myristate- 13-acetate (I'M \). a protein kinase C activator. The acquisition of maturation markers and changes in the activity, level, and phosphoryla- tion of Topo II were determined after treatment with either novobiocin, a Topo II inhibitor, or I'MA. Novobiocin at 50-150 MMinduced differ entiation in the 111-20? 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 n\\ for 6 h or at 2 HIMfor 30 min resulted in a 4-fold or higher reduction in Topo II activity in the differentiation-susceptible III -2(1? cells but not in the differentiation-resistant HL-525 cells. A differential response in Topo II activity was also observed after treatment with I'M \. 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 phosphor) lai ion. l-(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. INTRODUCTION Topoisomerases are ubiquitous enzymes that change the con formation of DNA molecules. Their mode of action involves a transient breakage of one DNA strand (type I topoisomerase) or two DNA strands (type II topoisomerase) followed by rota tion and rejoining of the strands (1, 2). In eukaryotes, these enzymes have been implicated in the replication and processing of DNA, as well as in the transcription of specific genes (3-9). Studies with mammalian cells have shown that the activity of Topo II3 is higher in replicating cells than in cells exhibiting a mature phenotype (10-16), thus suggesting that a reduction in Topo II activity may be associated with either inhibition of growth or the induction or maintenance of the differentiated state. The present studies were initiated to distinguish between these possibilities and to determine specifically if a reduction in Topo II activity is a prerequisite for the induction of cell Received 8/23/88: revised 11/28/88: accepted 12/1/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by the United States Department of Energy, under Contract W-31-109-ENG-38. 2 Recipient of National Research Service Award CM 10662 from the NIH. 3The abbreviations used are: Topo II, type II topoisomerase: m-AMSA, 4'-(9- acridinylamino)methanesulfon-/n-anisidide, amsacrine: DDW, double-distilled water; H-7, l-(5-isoquinolinesulfonyl)-2-methylpiperazine; NBT, nitroblue tetra- zolium; NSE, nonspecific esterase; PKC, protein kinase C; PMA, phorbol-12- myristate-13-acetate; SDS, sodium dodecyl sulfate; Topo I. type 1 topoisomerase. differentiation in the human promyelocytic leukemia HL-60 cells (17). In one approach, we tested the ability of novobiocin and related agents that inhibit Topo II activity (1,2, 18-21) to induce differentiation in HL-60 cells. For comparison, we in cluded camptothecin, an inhibitor of Topo I (22). In another approach, we tested the ability of PMA, a potent differentiation inducer (23-25), to reduce the activity of Topo II in the HL-60 cells shortly (within 30 min) after treatment. Because the bio logical activity of PMA is believed to involve specific protein phosphorylation induced by PKC (26), we examined the alter ations in the activity, level, and phosphorylation of Topo II after treatment of the cells with either novobiocin or PMA in the presence and absence of H-7, an inhibitor of protein kinases, including PKC (27). Induction of cell maturation and changes in Topo II level, activity, and phosphorylation were tested in cell-line HL-205, a differentiation-susceptible HL-60 cell var iant, and in cell-line HL-525, an HL-60 cell variant that is resistant to induction of differentiation by PMA but not by other types of inducers (28). Our results indicate that novobiocin caused the HL-205 cells but not the HL-525 cells to acquire a mature myeloid pheno type. Furthermore, in the HL-205 cells but not the HL-525 cells, novobiocin and PMA were able to reduce the activity of Topo II and to alter the level of phosphorylated Topo II within 30 min after treatment. All of these changes were diminished by pretreatment of the cells with H-7. On the basis of these results, we suggest that induction of differentiation in HL-60 cells by some chemical agents is associated with a reduction in Topo II activity, which is facilitated by a protein kinase(s), perhaps PKC. MATERIALS AND METHODS Chemicals and Reagents. m-AMSA was obtained from the National Cancer Institute, and novobiocin from Boehringer Mannheim. Cou- mermycin was purchased from Sigma Chemical Co., PMA from Chem icals for Cancer Research; and H-7 from Seikagaku America, Inc. These chemicals, stored as stock solutions at —¿20°C, were used as follows. Novobiocin at 10 mM in sterile DDW, m-AMSA at 2.5 mM in 50% dimethyl sulfoxide in DDW, PMA at 1.6 mM in dimethyl sulfox- ide, and H-7 at 10 mM in DDW. When a combination of H-7 and another drug was used, H-7 was added l h prior to the second drug. Bacteriophage P4 Vir\ del 10 was provided by R. Calendar (University of California, Berkeley, CA). The MoP-9 monoclonal antibody was provided by A. Dimitriou-Bona (Mt. Sinai School of Medicine, New York, NY), the B52.1 monoclonal antibody was provided by G. Trin- chieri (Wistar Institute of Anatomy and Biology, Philadelphia, PA), and two different rabbit IgG antibodies against mouse cell Topo II were provided by F. Drake (Smith Kline Beckman Co.). All other antibodies were purchased from either Ortho Pharmaceutical Corp., Coulter Im munology, or Becton Dickinson. A Topo II control sample was the active fraction of the Mono Q column partially purified from HL-60 cells as described by Drake et al. (29). Protein kinase C from rat brain was purified in our laboratory by J. Hardwick. Cells, Culture Conditions, and Differentiation Markers. The HL-205 cell variant was isolated from HL-60 cells, while the HL-525 cell variant was obtained after cloning HL-60 cells that had been subcultured 102 times in the presence of PMA (28). Both cell variants were stable for 1110 Research. on January 3, 2016. © 1989 American Association for Cancer cancerres.aacrjournals.org Downloaded from