[CANCER RESEARCH 50, 6723-6730. October 15. 1990] Induction of the Differentiation of WEHI-3B D+ Monomyelocytic Leukemia Cells by Inhibitors of Topoisomerase II1 Germana Rappa, Aurelio Lorico,2 and Alan C. Sartorelli3 Department of Pharmacology and Developmental Therapeutics Program. Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510 [G. R., A. C. S.], and Department of Experimental Therapeutics, Bristol-Myers Company, Wallingford, Connecticut [A. L.] ABSTRACT Topoisomerase II has been suggested to have a role in the early events of differentiation. This possibility was evaluated by measuring the effects of inhibitors of topoisomerase II on the induction of the differentiation of WEHI-3B D* monomyelocytic leukemia cells. Differentiation of this cell line was induced along the granulocytic pathway by treatment with the topoisomerase II inhibitors novobiocin (150-300 MM),teniposide (20- 50 H\I),etoposide (0.1 MM),elsamicin (0.5 MM),and doxorubicin (40 IIM). Maturation was assessed by the morphological appearance of mature forms of the granulocytic lineage, an increase in cell surface Fc receptors, the ability to reduce nitroblue tetrazolium, and the loss of proliferative capacity. In contrast, the non-topoisomerase H-reactive agent cisplatin and the topoisomerase I-reactive drug camptothecin did not cause the maturation of WEHI-3B I)+ cells. Aclacinomycin A and retinole acid, which are known efficacious inducers of the differentiation of this cell line, affected topoisomerase II extracted from WEHI-3B D+ cells in vitro, causing concentration-dependent inhibition of the strand-passing activity of the enzyme. Treatment of WEHI-3B D+ cells with novobiocin at 150 MMfor 3 h or with teniposide at 50 IIMfor 24 h resulted ina 2- to 3-fold increase in etoposide-induced protein-DNA cross-links. Nuclear proteins in 0.35 M NaCl extracts from cells treated with novobiocin at 150 MMfor 3 h or with teniposide at 50 mi for 24 h showed a slight increase in topoisomerase II activity compared to untreated cells. No changes in topoisomerase II levels, as measured by immunoblotting, were detected after treatment of WEHI-3B D* cells with 150 MMnovobiocin or 50 HMteniposide during the first 2 days of treatment. At day 3 of treatment, however, a decrease in topoisomerase II was observed in cells treated with either drug, possibly due to decreased cellular proliferation consequent to cell differentiation. The findings support the conclusion that topoisomerase II may have a role in the induction of granulocytic differentiation of WEHI-3B D* leukemia cells. INTRODUCTION Several agents with antineoplastic activity, such as VP-16,4 VM-26, anthracyclines, aminoacridines, and ellipticines, have been shown to interact with the nuclear enzyme topoisomerase II, causing stabilization of the reaction intermediate between the enzyme and DNA (1-4). The biochemical events that result from complex stabilization to produce cytotoxicity have not been elucidated. Other chemicals, such as novobiocin, have been shown to inhibit prokaryotic and, at relatively high con centrations, eukaryotic topoisomerase II, possibly by blocking Received 5/1/89; accepted 6/27/90. 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 investigation was supported in part by USPHS grant CA-02817 from the National Cancer Institute and by a fellowship from the Italian Association for Cancer Research (G. R.). 2On leave of absence from the Department of Experimental Oncology, C.R.O., Aviano, Italy. 3To whom requests for reprints should be addressed, at the Department of Pharmacology, Yale University School of Medicine. 333 Cedar Street, New Haven, CT 06510. * The abbreviations used are: VP-16, etoposide; VM-26, teniposide; TPA, 12- O-tetradecanoylphorbol-13-acetate; DMSO, dimethyl sulfoxide; PBS, phosphate- buffered saline (137 mM NaCI-2.6 min KC1-8 mM Na2HPO.,-1.4 itiM KHjPO4); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TCA, tri- chloroacetic acid; NBT, nitroblue tetrazolium. the ATPase function of the enzyme (4-6). Topoisomerase II appears to have diverse functions in euka ryotic cells. Its double-strand passing activity is probably critical for DNA replication (7-9) and chromosome disjunction at mitosis (9-12). Moreover, there are several lines of evidence that suggest that topoisomerase II is involved in the regulation of gene expression, including the possibility that the supercoil- ing status of DNA may regulate the binding of transcriptional factors to promotors (13, 14). In support of this concept, topoisomerase H-mediated supercoiling has been shown to be an important element of gene transcription in both Xenopus and polyomavirus systems (15, 16). In addition, topoisomerase II cleavage sites have been found in flanking sequences, which are possibly regulatory sequences, of a number of genes (17, 18). Topoisomerase II has been proposed to have a role in early events of the differentiation process. Thus, Sahyoun et al. (19) have found that topoisomerase II can be activated in vitro by protein kinase C, the cellular target of the differentiating agent TPA. Retinoic acid, an inducer of granulocytic differentiation, has been reported to stimulate transient relaxation of DNA supercoiling and to cause a small amount of protein-linked DNA breaks, which are characteristic of topoisomerase reac tions, within 1-2 h of treatment (20, 21). In HL-60 leukemia cells, a decrease in drug-induced, topoisomerase II-mediated cleavage, which occurred within 24 h of exposure to TPA, was recently reported by Zwelling et al. (22). In addition, differen tiation of HL-60 cells initiated by TPA or novobiocin was shown by Constantinou et al. (23) to be associated with inhibi tion of topoisomerase II activity. The WEHI-3B D+ murine monomyelocytic leukemia (24), which causes a fulminant leukemia when injected into mice, closely resembles the clinical presentation of acute myelocytic leukemia, extensively infiltrating host hematopoietic tissue (25). Changes in protooncogene expression during early stages of differentiation have been found in this cell line (26). Contin uous exposure of WEHI-3B D+ cells in vitro to the anthracy clines, aclacinomycin A or doxorubicin, or to retinoic acid induced the appearance of a mature granulocytic phenotype (27). Furthermore, the antileukemic action of aclacinomycin A against a subline of WEHI-3B D* cells, transfected with a gene conveying neomycin resistance which permits identification of mature cells as being derived from leukemic precursors, trans planted into BALB/c mice was shown to be due at least partially to the terminal maturation of the leukemic cells (28). The biochemical events responsible for initiating the com mitment to undergo differentiation by the structurally varied compounds capable of inducing maturation are not known. It is conceivable that effects at the level of topoisomerase II may have a role in the differentiation process. Thus, although the cytotoxic activity of doxorubicin has been attributed to DNA intercalation, to the formation of free radicals, and to mem brane-mediated effects (29), topoisomerase II has also been proposed as the main biochemical target of this antibiotic (1). 6723 Downloaded from http://aacrjournals.org/cancerres/article-pdf/50/20/6723/2441600/cr0500206723.pdf by guest on 23 February 2022