Selection of Human Leukemic CEM Cells for Resistance to the DNA Topoisomerase II Catalytic Inhibitor ICRF-187 Results in Increased Levels of Topoisomerase IIa and Altered G2/M Checkpoint and Apoptotic Responses

ICRF-187 is a bisdioxopiperazine anticancer drug that inhibits the catalytic activity of DNA topoisomerase (topo) II without stabilizing DNA-topoII cleavable complexes. To better understand the mechanisms of action of and resistance to topoII catalytic inhibitors, human leukemic CEM cells were selected for resistance to ICRF-187. The clones CEM/ICRF-8 and CEM/ ICRF-18 are approximately 40and 69-fold resistant to ICRF187, and 12and 67-fold cross-resistant to ICRF-193, respectively, but are sensitive to other topoII catalytic inhibitors (merbarone and aclarubicin), as well as collaterally sensitive to the DNA-topoII complex-stabilizing drug etoposide (VP-16). Both the number of VP-16induced DNA-topoII complexes formed and the amount of in vitro topoII catalytic activity are enhanced in the drug-resistant cells. The ICRF-187-resistant clones contain ;5-fold increase in topoIIa protein levels and ;2.2-fold increase in topoIIa mRNA levels. Furthermore, CEM/ ICRF-8 expresses ;3.5-fold increase in topoIIa promoter activity, suggesting that up-regulation of topoIIa in this clone occurs at the transcriptional level. Treatment of the drug-resistant or -sensitive cells with equitoxic doses of merbarone or teniposide results in a G2/M arrest. In marked contrast, when treated with equitoxic ICRF-187 doses, the drug-resistant clones exhibit either a transient arrest or completely lack the G2/M checkpoint compared with the drug-sensitive cells. This aberrant cell cycle profile is associated with a 48-h delay in drug-induced apoptotic cell death, as revealed by fluorescentend labeling of DNA and poly (ADP-ribose) polymerase cleavage. In summary, resistance to ICRF-187 in CEM cells is associated with increased levels of catalytically active topoIIa and altered G2/M checkpoint and apoptotic responses. DNA topoisomerase II (topoII) is a nuclear enzyme that resolves DNA supercoiling and catenation by the breakage, strand-passage, and rejoining of double-stranded DNA (Champoux, 1990), thereby relieving topological constraints that occur during essential cellular processes such as DNA replication, transcription, cell division, and repair (Nelson et al., 1986; Brill et al., 1987). TopoII can also serve as a structural component of mitotic chromosome scaffolding (Uemura et al., 1987), playing an essential role in chromatin condensation during prometaphase and in sister chromatid segregation during anaphase (Adachi et al., 1991). DNA topoII is a target for a number of clinically useful antitumor agents, in part because it is essential for cell survival. To date, there are two general classes of topoII inhibitors that interfere with enzyme catalysis at distinct points of the enzyme reaction. DNA topoII inhibitors, such as teniposide (VM-26), etoposide (VP-16), and the anthracyclines (daunorubicin and doxorubicin), stabilize cleaved DNA-topoII complexes (Chen et al., 1984; Robinson and Osheroff, 1991). In contrast to the complex-stabilizing topoII inhibitors, merbarone, aclarubicin, and the bisdioxopiperazines (e.g., ICRF-187 and -193) block the catalytic activity of the enzyme. Specifically, the bisdioxopiperazines have been reported to stabilize topoII in a closed-clamp configuration around the DNA (for reviews, see Andoh, 1998; Andoh and Ishida, 1998), whereas agents such as merbarone have been implicated recently in blocking the topoII-mediated DNA cleavage reaction (Fortune and Osheroff, 1998). Because This work was supported in part by research Grants CA40570 and CA30103 from the National Cancer Institute (to W.T.B.), a research fellowship from the American Medical Association and Research Foundation (to S.E.M.), Cancer Center of the University of Illinois at Chicago, and research Grant CA31566 from the American Lebanese Syrian Associated Charities (ALSAC; to S.C.R.). S.E.M. was the (1998) Florence A. Carter Fellow of the American Medical Association. ABBREVIATIONS: topo, topoisomerase; VM-26, teniposide; VP-16, etoposide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; FBS, fetal bovine serum; PARP, poly (ADP-ribose) polymerase; PCR, polymerase chain reaction; RT, reverse transcriptase; BrdU, 5-bromo-2deoxyuridine; FITC, fluorescein isothiocyanate; CPT, camptothecin. 0026-895X/00/020296-12$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 57:296–307 (2000). 296 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from these drugs do not stabilize DNA-topoII complexes (i.e., they do not induce DNA strand breaks), they are termed “catalytic inhibitors” of topoII (Andoh and Ishida, 1998). Inactivation of topoII by inhibitors, particularly the bisdioxopiperazines, is associated with abnormalities in chromosome condensation and sister chromatid segregation during the mitotic phase of the cell cycle, resulting in enhanced chromosomal breakage and cell death (Uemura et al., 1987; Adachi et al., 1991). These results are consistent with the fact that topoII plays an active role in kinetochore assembly, is a major structural component of the chromosome axis during mitosis, and is localized over interphase chromosomes (Wartburton and Earnshaw, 1997). Inhibition of topoII by bisdioxopiperazines such as ICRF-193 also results in a G2/M checkpoint that is sensitive to the decatenation state of DNA; this checkpoint is believed to be distinct from a DNA damageinduced G2 checkpoint (Downes et al., 1994). Bisdioxopiperazine-induced cell death is believed to be mediated through a programmed cell death pathway because treatment of murine thymocytes with ICRF-193 or ICRF-154 or CEM cells with ICRF-187 results in enhanced DNA laddering and reduced cell viability (Kizaki and Onishi, 1997; Khelifa and Beck, 1999). These types of topoII catalytic inhibitors may thus serve as useful tools to elucidate the biochemical mechanisms involved in the G2/M checkpoint and apoptotic cell death pathways in response to abnormal chromosomal processing. ICRF-187, originally developed as an antitumor agent (Creighton et al., 1969), is now used for the protection of cells against doxorubicin-induced cardiotoxicity (Speyer et al., 1992) and is a powerful nontoxic protector against VP-16induced toxicity in treatment of brain tumors and metastases (Holm et al., 1996). To better understand the mechanisms of cellular response to bisdioxopiperazines, we selected human leukemic CEM cells for resistance to ICRF-187 and biochemically and pharmacologically characterized some of the ICRF187-resistant clones. In the present study, we characterized two of our novel ICRF-187-resistant cell lines in terms of their drug responsiveness, karyotypes, DNA-topoII complexforming and -decatenation activities, topoIIa expression levels, and cell cycle and cell death responses. We report herein that the resistance of these cells differs from that of previously reported ICRF-187-resistant Chinese hamster cell lines (Hasinoff et al., 1997; Sehested et al., 1998) and ICRF-187resistant human small-cell lung cancer cells (Wessel et al., 1999) and is associated with increased topoIIa protein levels and altered G2/M checkpoint and apoptotic responses. Materials and Methods Cell Lines and Drugs. Human leukemic CEM cells, a merbarone-resistant subline CEM/B1 (Kusumoto et al., 1996), and the novel ICRF-187-resistant sublines CEM/ICRF-8 and CEM/ICRF-18 were cultured in minimal essential medium for suspension cells (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS; Sigma Chemical Co., St. Louis, MO), and 2 mM L-glutamine (Life Technologies, Gaithersburg, MD). All cell lines were incubated at 37°C in a humidified chamber containing 5% CO2/95% air. ICRF187-resistant cells were selected from the original parent CCRFCEM cell line by continuous incubation with increasing concentrations of ICRF-187, generously provided by Pharmacia and Upjohn (Kalamazoo, MI) and Dr. Ellen Friche (Riggs Hospital, Copenhagen, Denmark). Forty-four CEM/ICRF resistant clones were isolated from the polyclonal populations at different concentrations of ICRF-187 by limiting dilution using 96-well plates. Two of these 44 CEM/ICRF clones that retain resistance in the absence of ICRF-187 were used for further characterization. Other drugs, including VM-26, VP-16, 7-ethyl-10-hydroxycamptothecin (SN-38), CPT, merbarone, and aclarubicin, were obtained from sources described previously (Kusumoto et al., 1996). ICRF-193 was provided by Dr. John Nitiss (St. Jude Children’s Research Hospital, Memphis, TN) and vinblastine was from either Sigma or Eli Lilly and Co. (Indianapolis, IN). Growth Inhibition and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) Assays. For growth inhibition assays, exponentially growing cells were incubated with several different concentrations of drug for 48 h. The ratio of drug-treated to control cells was determined, and the concentration of drug required to inhibit cell growth by 50% (IC50) was calculated. Fold-resistance was calculated by dividing the IC50 value of the resistant cells by the IC50 value of the drug-sensitive parental cell line. Growth inhibition of CEM cells and their drug-resistant sublines was shown to correlate with clonogenic survival (Kusumoto et al., 1996). ICRF-187-induced cytotoxicity was also measured by the MTT assay. Exponentially growing cells (CEM, CEM/ICRF-8, and CEM/ ICRF-18) were plated at 500 cells/well in 96-well microtiter plates (100 ml/well). ICRF-187 was added to the cells at various concentrations in a final volume of 200 ml/well, and the cells were incubated at 37°C for 96 h. After drug exposure, 25 ml of MTT compound (Sigma; 4 mg/ml in SMEM without FBS) were added to each well and the cells were incubated at 37°C for 4 h. The plates were centrifuged in a swinging bucket rotor (1000g, 5 min), and the cells were incubated with 200 ml of dimethyl sulfoxide for 15 min at 25°C. The metabolic activity of the cells was measured by quantifying the conversion of the yellow MTT to a purple metabolite, MTT-formazan. Absorbance was read at 540 nm using a microplate reader. Four replicates were measured for each drug concentration and the experiments were done in triplicate. The IC50 value was calculated as the concentration of drug that killed 50% of the cells. Chromosome Analysis. Karyotype analysis was performed as described previously (Kusumoto et al., 1996). Approximately five metaphases were examined for each cell line and the karyotype was written according to the International System for Human Cytogenetic Nomenclature (ISCN 1995). DNA-Protein Complex Formation Assay in Intact Cells. TopoII-DNA covalent complex formation in intact cells was measured as described previously (Kusumoto et al., 1996). Briefly, cellular DNA and protein were labeled by incubating exponentially growing cells at 37°C with [C]leucine (0.2 mCi/ml) and [H]thymidine (0.6 mCi/ml) for approximately 16 to 24 h. The labeled cells were then incubated with various concentrations of VP-16 for 30 min, after which time the cells were disrupted, the DNA sheared, and DNAtopoII complexes measured as described previously (Kusumoto et al., 1996). To examine the effect of ICRF-187 on VP-16-mediated complex formation, cells were pretreated with ICRF-187 at the indicated concentrations for 2 h before the addition of VP-16. Results are expressed as the ratio of HDNA to C-protein, using the counts of protein precipitated as the internal control for all samples. Decatenation Activity Assay. TopoII catalytic activity was analyzed in nuclear cell extracts by decatenation of kinetoplast (k) DNA using a topoII activity assay kit according to the manufacturer’s instructons (TopoGEN, Columbus, OH). Briefly, appropriate dilutions of nuclear cell extracts were incubated with 200 ng of kDNA in a 13 reaction buffer (50 mM Tris-HCl, pH 8.0, 120 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM ATP, and 30 mg of BSA/ml) for 30 min at 37°C. The reactions were terminated with 0.1 volume stop buffer (0.025% bromphenol blue, 50% glycerol) and the decatenation products were separated in a 1% ethidium bromide-stained agarose gel at 100 V. Western and Northern Blot Analysis. Whole-cell lysates or nuclear cell extracts were prepared from logarithmically growing cells (5 3 10 cells/ml) as described previously (Mo and Beck, 1997). Resistance of Leukemic CEM cells to ICRF-187 297 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from Proteins (50 mg/well) were separated in 7.5% SDS-polyacrylamide gels, electrophoretically transferred onto nitrocellulose, and incubated with either antiserum Ab-284, specific to the amino terminus of human topoIIa (Boege et al., 1995), or a polyclonal anti-poly(ADPribose) polymerase (PARP) antibody (Upstate Biotechnology, Lake Placid, NY). Bound antibody was detected using the enhanced chemiluminescence detection method (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions. Autoradiographic signals were quantified by densitometric scanning using a GS-700 Imaging Densitometer and Molecular Analyst Software (Bio-Rad, Hercules, CA). Total RNA derived from the parental and drug-resistant cells was isolated using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Total RNA (30 mg/lane) was separated in a 1.2% formaldehyde-denatured agarose gel and transferred to a Hybond membrane (Amersham). The membrane was hybridized using a previously constructed topoIIa probe (Tsai-Pflugfelder et al., 1988) that was labeled with [a-P]dCTP using the random prime II kit (Stratagene, La Jolla, CA). Total RNA content was quantified using a GS-700 Imaging Densitometer (Bio-Rad). Luciferase Reporter Assay. Transient transfections were performed using plasmid p557 (Wang et al., 1997) that contains the full-length topoIIa promoter (nucleotides 2577 to 190) (Hochhauser et al., 1992) subcloned upstream to the luciferase reporter gene in a pGL2-Basic vector (Promega, Madison, WI). The p557 luciferase plasmid was cotransfected with pSV-b-galactosidase control vector (Promega) to normalize for the transfection efficiency. DNA was introduced into the cells by electroporation using the Gene Pulser II apparatus with an extender (Bio-Rad), according to the manufacturer’s instructions. After electroporation, the cells were incubated for approximately 17 h at 37°C and cell extracts were prepared using a 13 reporter lysis buffer (Promega). Luciferase activity was measured by a luminometer with an auto-injector (Model TD-20/20; Turner Designs, Sunnyvale, CA). Luciferase activities were normalized to b-galactosidase activities. Nucleotide Sequencing. Polymerase chain reaction (PCR) amplification of the topoIIa promoter region (nucleotides 2577 to 190) was performed as described previously (Mo et al., 1997) using a commercial kit (Ampli-Taq; Perkin-Elmer Corp., Foster City, CA). For amplification of defined regions within the topoIIa cDNA, singlestranded topoIIa cDNAs were synthesized from total RNA (5 mg) derived from CEM, CEM/ICRF-8, and CEM/ICRF-18 cells using Superscript II Reverse Transcriptase (RT; Life Technologies) in the presence of 39 gene-specific primers (described below). After the RT reaction, cDNAs were PCR-amplified according to standard conditions (Danks et al., 1993). Primer sequences used for the PCRamplification of motif B/dinucleotide binding site (nucleotides 1318– 1603) as well as the tyrosine 805 active site (nucleotides 2264–2518), have been described previously (Danks et al., 1993). Primer sequences used for the PCR-amplification of the proximal amino-terminal region of topoIIa (nucleotides 1–165) were the following: topoIIa-5.1 (59-ACCATGGAAGTGTCACCATTGCA) and topoIIa-3.1 (59-GGTGGATCCAGCAATATCAT). PCR clones were sequenced by the dideoxy chain termination method with the Sequenase kit version 2 (Amersham) using universal, reverse, or specific primers when necessary. The sequences obtained from the PCR clones were compared with the published sequence of the human topoIIa cDNA (Tsai-Pflugfelder et al., 1988). Cell Cycle Analyses. Cellular DNA content and cell cycle distribution were assessed by propidium iodide labeling. Approximately 1 3 10 cells (untreated or treated with drug) were harvested, washed with 13 PBS pH 7.4, fixed in 1 ml of 70% methanol, and incubated on ice for 30 min. The fixed cells were centrifuged (1200g, 5 min at 4°C), resuspended in 800 ml of 13 PBS, 200 ml of propidium iodide (0.1 mg/ml), and 5 ml of RNase (10 mg/ml) (Sigma), incubated in the dark (25°C, 30 min), and analyzed by flow cytometry. All cells were analyzed on a Becton Dickinson FACScan flow cytometer and the percentage of cell cycle distribution was determined by either the CellQuest or MODFIT programs (Verity Software House, Topsham,