Introduction

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L-Phenylalanine mustard (melphalan) is a rationally designed alkylating agent active against ovarian cancer, myeloma, breast cancer, and rhabdomyosarcoma. Because melphalan is the active agent, requiring no further metabolic activation, the drug has become a model for studying the mechanisms of alkylator resistance. Melphalan resistance can be mediated by a number of mechanisms, including altered drug transport, increased drug detoxification, or increased removal/repair of DNA interstrand cross-links (Redwood and Colvin, 1980; Batist et al., 1989; Bailey et al., 1992), acting either singly or in combination. We have demonstrated previously that in lymphoid cells, a major mechanism for melphalan resistance is an increased rate of removal of DNA-interstrand cross-links (Bezwoda and Pu, 1997). Such increased rates of interstrand-DNA cross-link removal may be caused either by increased activity of DNA repair enzymes or by alteration of DNA topology.

In eukaryotic cells, two major topoisomerases (I and II) catalyze changes in the topological conformation of DNA by the concerted breakage of single or double strands. Topoisomerase II action is important in DNA replication, transcription, recombination, and mitosis (Wang, 1987). Topoisomerase II is localized to AT-rich DNA regions, where it forms a significant part of the mitotic chromosomal scaffold (Earnshaw et al., 1985; Nelson et al., 1986). Two isoenzyme forms of topoisomerase II,  and , are expressed in mammalian cells. The two forms differ with respect to molecular size (170 kDa versus 180 kDa, respectively), cleavage site, thermal stability, and catalytic capacity (Drake et al., 1987, 1989). The enzyme described first, topoisomerase II, is expressed preferentially in proliferating cells (Heck and Earnshaw, 1986; Hsiang et al., 1988) and is cell-cycle regulated (Heck et al., 1987). The topoisomerase II enzyme, on the other hand, seems to be expressed at equivalent levels in proliferating and quiescent cells (Woessner et al., 1990, 1991).

In this study, we investigated topoisomerase I and II expression and activity in melphalan-resistant HL 60 cells (R-HL60) and in parental HL60 cells. The results show that elevated levels of topoisomerase II are associated with resistance to melphalan and that such R-HL60 cells are more sensitive than parental HL60 cells to the topoisomerase II inhibitor doxorubicin.

	Experimental Procedures

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Materials. Melphalan was obtained as >99.5% pure 4-[bis(2 chloroethyl)amino]-L-phenylalanine from Wellcome (Dartford, UK). Melphalan solutions were prepared daily in 70% ethanol containing an equimolar concentration of hydrochloric acid. To minimize the hydrolysis, further dilutions were made in aqueous medium immediately before use. RPMI 1640 was purchased from Highveld Biologicals (Johannesburg, South Africa). Doxorubicin and camptothecin were obtained from Topogen (Columbus, OH).

Cell Lines and Tissue Culture. HL60 cells and resistant HL-60 (R-HL60) cells were grown in RPMI 1640 that contained 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Incubations were performed at 37°C in a humidified atmosphere with 5% CO₂. R-HL60 cells were selected by intermittent exposure of surviving cells to increasing concentrations of melphalan. HL60 cells (2 × 10⁵ per 50-ml flask) were treated for 1 h with the IC₅₀ concentration of the drug. The cultures were observed daily and allowed to grow until they reached the initial cell density or greater. Melphalan concentrations for subsequent exposure were increased primarily on the basis of recovery time. For short recovery times (1-2 weeks), a 1.0- to 1.5-fold increase in concentration was used. For recovery times of 2 to 4 weeks, the increase was smaller (10-50%). After 6 months of intermittent exposure, a stable subline (R-HL60) demonstrating a 4-fold increase in the IC₅₀ melphalan concentration was grown out.

Cell Number. Cell numbers were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, based on active mitochondrial reduction of MTT. The assay used incorporated the modifications of Twentyman and Luscombe (1987). Assays were performed using 5×10⁴ cells in 96-well microtiter plates in 200 µl of culture medium. Cells were exposed to melphalan at concentrations ranging from 0 to 40 µM for 96 h and to doxorubicin or camptothecin at concentrations ranging from 0 to 5.0 µM for 96 h. Cells were then washed with fresh medium, followed by addition of 20 µl of MTT (5 mg/ml) in 200 µl of medium and incubated for 4 h. Medium was then aspirated and formazan solubilized in dimethyl sulfoxide. Absorbance was read at 540 nm within 30 min in an enzyme-linked immunosorbent assay plate reader. Each experiment was performed in triplicate at each of the drug concentrations. After treatment with each of the drugs, the surviving fraction of cells compared with control (untreated) cells was calculated for each drug concentration. Cell number was plotted versus drug concentration, and IC₅₀ values were calculated from dose- response curves as the concentration of drugs that reduced the number of viable cells to 50% of control.

Determination of Interstrand-DNA Cross-Links. HL60 cells were suspended in RPMI 1640 at 2 × 10⁶ cells/ml. Cells were exposed to various concentrations of melphalan at 37°C. Interstrand-DNA cross-link formation was determined by an ethidium bromide fluorescence assay based on the method of De Jong et al. (1991a). After hypotonic lysis (10 mM/l Tris, pH 7.4, 1 mM EDTA, and 0.2% Triton X100) the contents of each well were precipitated in cryotubes for 24 h at 20°C in 50% isopropanol and 0.5 M NaCl. The precipitates were pelleted by centrifugation, air dried, resuspended in Tris/EDTA (TE) buffer and treated with 1 U/tube of DNase-free RNase at 37°C for 1 h followed by proteinase K (0.5 mg/ml) treatment (25 µl/tube) for another hour. Aliquots of the resulting lysates were denatured by heating at 100°C for 5 min and then rapidly cooled to 23°C. Samples were then added to 3 ml of ethidium bromide (10 µg/ml) in 20 mM K₂HPO₄, and 0.4 mM EDTA buffer, pH 12.0, in tubes wrapped in aluminum foil to prevent light-induced cleavage of DNA by ethidium bromide. Fluorescence was measured both before and after denaturation in 1-cm² cuvettes at an excitation wavelength of 525 nm and an emission wavelength of 580 nm in an LS50 variable wavelength spectrofluorometer (Perkin Elmer, Norwalk, CT). The percentage of interstrand cross-linked DNA was determined by measuring the difference in fluorescence of denatured control cell lysates and denatured control cell samples according to the formula: C_t% = (ft  fc/1  fc) × 100, where C_t% = the percentage of interstrand cross-linked DNA on treated cells, ft = fluorescence intensity after heat denaturation divided by fluorescence intensity before heat denaturation in treated cells, and fc = the same fluorescence measurement in control cells.

Preparation of Nuclear Extracts. A procedure for isolation of nuclear and cytosolic fractions was developed based on the methods of Chow et al. (1985) and Matsumoto et al. (1993). In brief, cells in the exponential phase of growth were treated with detergent buffer (1% Nonidet P-40, 30 mM HEPES, 200 mM sucrose, 40 mM NaCl, 5 mM MgCl₂, and 5 mM EGTA, pH 8.0), with constant swirling for 10 to 15 min. The cell suspension was centrifuged at 400g for 10 min and the supernatant was saved as the cytosolic fraction. The sedimented nuclei were then washed three times by resuspending the pellet in buffer A (50 mM HEPES, 10% sucrose, and 10 mM -mercaptoethanol, pH 7.5) and repelleting. To the final pellet was added an equal volume of buffer B [50 mM HEPES, 10% sucrose, 10 mM -mercaptoethanol, and 0.7 M NaCl (final NaCl concentration, 0.35 M)]. After extraction for 60 min at 4°C, the mixture was centrifuged at 100,000g for 60 min, and the supernatant was saved as the nuclear extract. The cytosolic and nuclear extracts were dialysed against buffer C (50% glycerol, 50 mM Tris · HCl, 0.5 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, and 260 mM NaCl, pH 7.5) for 6 h. A combination of six protease inhibitors, including soybean trypsin inhibitor (10 µg/ml), leupeptin (50 µg/ml), pepstatin (1 µg/ml), aprotinin (20 µg/ml), benzamidine (1 mM), and phenylmethylsulfonyl fluoride (1 mM), were prepared just before each experiment and added to each of the above-mentioned buffers just before each experiment. The protein concentrations in the extracts were determined by the method of Bradford (1976).

Topoisomerase I and II Assays. Measurement of the topoisomerase II catalytic activity in cytosol and nuclear extracts was performed using the topoisomerase II decatenating method (Topogen), which is a modification of the method of Marini et al. (1980). Photographic negatives of the ethidium bromide-stained agarose gels were scanned with a REP Scanning Densitometer (Helena Laboratories, Beaumont, TX) and the quantity of liberated minicircles was measured as a percentage of total kinetoplast DNA (kDNA). Results are expressed in arbitrary units and the ratio of activity in R-HL60 cells to that in parental HL60 cells was used for comparison.

Western Blot Analysis. Proteins from the nuclear extract and cytosol fractions of HL60 and R-HL60 cell lines were electrophoresed (100 µg of protein/lane) in 7.5% SDS-polyacrylamide gels and transferred to nitrocellulose paper by the method of Harker et al. (1991). The nitrocellulose strips were preincubated in blocking buffer (3% BSA-5% nonfat dry milk in PBS) overnight at 4°C. Transferred, immobilized topoisomerase proteins were detected using purified mouse antihuman DNA topoisomerase II p170 (Topogen) and antitopoisomerase II p180 (PharMingen, San Diego, CA) monoclonal antibody, or rabbit polyclonal, monospecific, antihuman topoisomerase I antibody (Topogen). Blots were incubated with primary antibody for 4 h at 37°C, then washed with a wash buffer (1% BSA in PBS containing 0.2% Tween 20). The bound antibodies were visualized with alkaline phosphatase-linked sheep antimouse or antirabbit IgG, using 5-bromo-4 chloro-3-indolyl phosphate and nitroblue tetrazolium chloride substrates. Each experiment included controls for nonspecific binding using nonimmune rabbit serum in place of specific antibody and a negative control without primary antibody. The relative amounts of topoisomerase II, II, and I proteins seen on Western blots were quantified densitometrically. Specified regions of the film images of the Western blot were digitized by the scanner and the area and image intensities were calculated. The image intensity was calibrated against internal computer standards and values are expressed as arbitrary units relative to the standards. A linear relationship was present between the value of the integration units and the amount of protein extract loaded on the gel in the range (30-250 µg of protein) (data not shown). Protein extracts (100 µg) were used for all comparative experiments.

Preparation of HL60 Native DNA. A procedure for isolation of DNA from HL60 cells was developed based on the method of Gross-Bellard et al. (1972). Briefly, HL60 cells were suspended in 1 volume of digestion buffer (100 mM NaCl, 10 mM Tris-Cl, 25 mM EDTA, 0.5% SDS, and 0.1 mg/ml proteinase K, pH 8.0). Samples were incubated in a shaking incubator in tightly capped tubes for 12 to 18 h at 50°C. An equal volume of phenol/chloroform/isoamyl alcohol was used for extraction, followed by centrifugation for 10 min at 1700g (3000 rpm in Sorvall H1000B rotor; Sorvall Instruments, Newton, CT). This organic extraction was repeated twice. The aqueous layer was transferred to a new tube and .5 volumes of 7.5 M ammonium acetate and 2 volumes of 100% ethanol were added. The solution was centrifuged for 2 min at 1700g. Organic solvents and salt were removed by two dialyses against 100 volumes of TE buffer for 24 h. Residual RNA was removed by adding 0.1% SDS and 1 µg/ml DNase-free RNase and incubating for 1 h at 37°C. The organic extraction and purification was repeated and the resultant purifed DNA was resuspended in TE buffer at 1 mg/ml.

Preparation of Cross-Linked DNA. HL60 DNA was suspended at 100 µg/ml in TE buffer. Samples in a thin layer were irradiated at room temperature with UV light (360-nm peak) 12 cm below four FL-15BLB fluorescent lamps. The incident fluence rate was 30 W/m², measured with a Model J221 Blak-Ray ultraviolet meter (Ultraviolet Products, San Gabriel, CA). The solution was extracted with phenol/chloroform/isoamyl alcohol. After ethanol precipitation, the DNA was resuspended in TE buffer at 1 mg/ml. The formation of cross-links was determined by ethidium bromide fluorescence assay mentioned above. Cross-linked DNA accounted for 80% of total DNA after UV treatment.

Iodination of Topoisomerase II. Topoisomerase II was iodinated by the Iodogen method (Salacinski et al., 1981), purified by contricon 100 (Amicon, Beverly, MA) and formulated in topoisomerase II storage buffer (15 mM sodium phosphate, pH 7.1, 700 mM NaCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 50% glycerol) at the concentration of 1 U/µl (1 U will decatenate 0.2 µg of kDNA in 30 min at 37°C). The specific activity of the radiolabeled material is 600 cpm/mU topoisomerase II.

Affinity of Topoisomerase II to DNA. HL60 cell DNA (5 µg) or HL60 cell UV-irradiated cross-linked DNA was incubated with various concentrations of ¹²⁵I-labeled topoisomerase II in Topo II assay buffer (50 mM Tris-Cl, 120 mM KCl, 10 mM MgCl₂, 0.5 mM dithiothreitol, and 30 µg BSA/ml, pH 8) at 37°C for 1 h. For some experiments, the ¹²⁵I-labeled topoisomerase II was in the assay buffer with 5 µM doxorubicin preincubation for 30 min. Reaction mixture were collected on DEAE-cellulose filters, washed with 5% trichloroacetic acid, and counted by a Packard (Meriden, CT) 5330 gamma-ray counter. There were duplicate determinations for each point. Nonspecific binding was defined as bound ¹²⁵I-labeled topoisomerase II that remained in the presence of a 100-fold excess of the unlabeled topoisomerase II, except for the competitive binding. The specific binding was determined by subtracting the nonspecific binding from total binding. Scatchard analysis was employed to determine the maximal binding (B_max)

Neutral Comet Assay of Double-Strand DNA Breaks. The single-cell comet electrophoresis assay was performed according to the method of Olive et al. (1991). Cells were suspended in PBS at a density of 5 × 10⁵/ml and treated with doxorubicin at a dose of 10 µM at 37°C for 3 h. After the treatment, 1.5 ml of 1% low-gelling-temperature agarose gel (Sigma, St. Louis, MO) at 40°C were added to a tube containing 0.5 ml of cold cell suspension. The contents were quickly pipetted onto a frosted microscope slide and allowed to gel for about 1 min on a cold surface. Then slides were immersed for 5 h at 50°C in 0.5% SDS and 30 mM EDTA, pH 8.3. After lysis, slides were thoroughly rinsed overnight in large volumes of 90 mM Tris/90 mM boric acid/2 mM EDTA buffer. Slides were placed in horizontal gel electrophoresis chambers containing 1 liter of 90 mM Tris/90 mM boric acid/2 mM EDTA buffer at 0.6 V/cm for 30 min. DNA was stained by immersing slides in 2.5 µg/ml propidium iodide for 30 min.

Statistical Methods. Differences between two cell lines were tested for statistical significance by Student's t test.

	Results

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Cytotoxicity and Interstrand DNA Cross-Link Formation. The effects of melphalan on growth and survival of HL60 cells and R-HL60 cells is shown in Fig. 1A. R-HL60 cells showed a 4-fold increase in the IC₅₀ concentration (27.8 ± 4.2 µM) of melphalan compared with parental HL60 cells (6.9 ± 1.8 µM; p < .01). The effects of doxorubicin (Fig. 1B), a topoisomerase II inhibitor, and camptothecin (Fig. 1C), a topoisomerase I inhibitor, were also studied. Statistical analysis showed that HL60 cells were significantly more sensitive to melphalan than R-HL60 cells (p < .05); however, parenteral HL60 cells were significantly more resistant to doxorubicin (doxorubincin IC₅₀, 1.3 ± 0.8 µM) than R-HL60 (doxorubincin IC₅₀, 0.25 ± 0.15 µM; p < .05). There was no difference in IC₅₀ dose for camptothecin when HL60 cells and R-HL60 cells were compared (camptothecin IC₅₀, 1.02 ± 0.25 µM versus 0.92 ± 0.30 µM, respectively). On the other hand, there was a significant (p < .01) difference in IC₅₀ dose for melphalan between R-HL60 (27.84 ± 4.2 µM) and R-HL60 cell with 0.05 µM doxorubicin pretreatment (5.3 ± 2.21 µM); there was no difference between HL60 (6.9 ± 1.78 µM) and HL60 with 0.05 µM doxorubicin pretreatment (6.1 ± 1.13 µM).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Viable cell fraction (as a proportion of control) after treatment with melphalan (A), doxorubicin (B), and camptothecin (C) against HL60 () and R-HL60 cells (). Values are the mean ± S.D. of five independent experiments.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Percentage of interstrand-DNA cross-link formation (Ct%) after 60-min melphalan exposure followed by 4-h drug-free incubation in HL60 cells () and R-HL60 cells (). Ct was significantly different (p < .05) when comparing R-HL60 and parental HL60 cells.

Kinetics of Interstrand-DNA Cross-Linking. To better define the kinetics of the formation and removal of interstrand-DNA cross-links, C_t values were determined at 0, 4, and 24 h after a 60-min incubation with 10 µM melphalan. After melphalan exposure for 60 min, the cells were washed and reincubated in drug-free medium for the stated times. Viability and metabolic activity of cells was monitored through this incubation and remained >90% at all test times.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Interstrand-DNA cross-links (Ct%) after 60-min exposure to 10 µM melphalan (MLN) and a 24-h drug-free incubation in: A, HL60 cells (); B, HL60 cells with doxorubicin pretreatment (6 h) (); C, R-HL60 (); D, R-HL60 with doxorubicin pretreatment (6 h) (). Time 0 represents the start of incubation with melphalan, which was continued for 1 h, followed by washing and then drug-free incubation for another 24 h.

ct

Topoisomerase II Activity and Expression in HL60 cells and R-HL60 Cells. The strand-passing activity of topoisomerase II was measured by decatenation of kDNA. This reaction, resulting in release of double-stranded minicircles from the catenated kDNA network, is ATP-dependent and specific for topoisomerase II (Fig. 4A). Figure 4A shows the results obtained using 0.35 M NaCl nuclear extracts at various dilutions. Enhanced decatenation activity was observed in R-HL60 cells. In a set of five independent experiments, a mean increase of 4.28-fold in catalytic activity was found in nuclear extracts of R-HL60 (topoisomerase II catalytic activity, 100.7 ± 39 U) compared with parental HL60 cells (topoisomerase II catalytic activity, 23.5 ± 10.8 U; p < .01).

View larger version (47K): [in this window] [in a new window]   Fig. 4.   A, topoisomerase II activity in nuclear extracts from HL60 cells and R-HL60 cells. Lane 1, kDNA control; lane 2, decatenated kDNA control; lanes 3 and 5, HL60 nuclear extracts (containing 50 ng and 100 ng of protein/lane); lanes 4 and 6, R-HL60 nuclear extracts (containing 50 ng and 100 ng of protein/lane). B, topoisomerase II protein levels, by Western blot analysis of whole-cell lysates from HL60 cells and R-HL60 cells. Lane 1, the purified p170 kDa topoisomerase II maker (Topogen); lanes 2 to 4, HL 60 cell extracts; lanes 5 to 7, R-HL60 cell extracts. Each sample contained 100 µg of total cellular protein per lane. The blots were stained with a combination of monoclonal antitopoisomerase II p170 antibody and monoclonal antitopoisomerase II p180 antibody (PharMingen).

Affinity of Topoisomerase II for DNA. Figure 5A shows the ability of the unlabeled topoisomerase to compete with ¹²⁵I-labeled topoisomerase II for binding to DNA. The unlabeled topoisomerase II competed efficiently with ¹²⁵I-labeled topoisomerase II in a dose-dependent manner. The time-course analysis indicated that the maximal specific binding of ¹²⁵I-labeled topoisomerase II to DNA was reached after 60 min of incubation at 37°C (Fig. 5B). Scatchard analysis revealed that the affinity of topoisomerase II for HL60 UV-irradiated, cross-linked HL60 DNA was increased ~2.47-fold compared with that of native HL60 DNA. Doxorubicin decreased topoisomerase II binding to both native DNA and cross-linked DNA by 3.39-fold and 2.94-fold, respectively (Table 2 and Fig. 6, A and B).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   A, competitive binding of unlabeled topoisomerase II to HL60 native DNA and HL60 UV-irradiated, cross-linked DNA. DNA was incubated with 10 mU/ml 125I-labeled topoisomerase II for 1 h at 37°C in the topoisomerase II assay buffer. Specific binding was determined by subtracting the nonspecific binding (in the presence of 1 U/ml unlabeled topoisomerase II) from total binding. Values shown represent the percentage of specific binding of 125I-labeled topoisomerase II alone. B, time course of binding of 125I-labeled topoisomerase II to HL60 native DNA and HL60 cross-linked DNA. DNA was incubated with 10 mU/ml 125I-labeled topoisomerase II with or without a 100-fold excess of unlabeled topoisomerase II for the indicated times at 37°C. Points represent means ± S.D. These determinations were repeated three time with similar results.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Scatchard analysis of the binding of 125I-labeled topoisomerase II to HL60 native DNA (A) and HL60 UV-irradiated, cross-linked DNA (B). DNA was incubated with different concentrations of 125I-labeled topoisomerase II (10 mU/ml to 1 U/ml) with (, ) or without (, ) pretreatment with 5 µM doxorubicin for 30 min at 37°C in the presence or absence of a 100-fold excess of the unlabeled topoisomerase II for 1 h at 37°C. The results in each panel represent data from one of three experiments, each of which gave similar results.

Effect of the Topoisomerase II Inhibitor, Doxorubicin, on Interstrand-DNA Cross-Link Formation in HL60 and R-HL60 Cells. A 6-h exposure to a minimally cytotoxic concentration (<9% growth inhibition of HL60 and 11% growth inhibition of R-HL60 cells after 96 h of exposure) of doxorubicin (0.05 µM) resulted in interstrand-DNA cross-link formation of 0.6% and 1.6% in HL60 and R-HL60 cells, respectively. Interstrand-DNA cross-link formation was significantly enhanced in R-HL60 cells pre-exposed to 0.05 µM doxorubicin for 6 h followed by a 1-h incubation with melphalan (10 µM) and then assayed at both 4 (p < .05) and 24 h (p < .01) compared with parenteral HL60 cells (Fig. 7). Pre-exposure to 0.05 µM doxorubicin decreased the IC₅₀ value for melphalan concentration for R-HL60 cells from 27.84 ± 4.2 to 5.3 ± 2.21 µM.

View larger version (42K): [in this window] [in a new window]   Fig. 7.   The effect of doxorubicin (D) pretreatment on melphalan (M)-induced interstrand-DNA cross-link formation. D, 0.05 µM doxorubicin for 6-h; M, 10 µM melphalan for 1 h; D  M, preincubation with 0.05 µM doxorubicin for 6 h and then 10 µM melphalan treatment for 1 h. Cells were then washed and CT% was measured after 4- and 24-h drug-free incubations. Values are the mean ± S.D. of 10 experiments, each performed in triplicate.

Effect of Doxorubicin on Double-Strand DNA Breakage in HL60 and R-HL60. DNA is able to migrate toward the anode in an electric field when cells are embedded in agarose and lysed. Individual cell DNA was visualized by fluorescence microscope. Each cell has the appearance of a "comet," with brightly fluorescent head and tail, and an intensity that is related to the amount of double-stranded DNA breakage sustained by the cell. The neutral comet assay measures only double-strand breaks (Olive et al., 1991). HL60 cells and R-HL60 cells were examined for DNA double-strand breaks induced by doxorubicin. The average tail moment of R-HL60 cells increased ~1.7-fold compared with that of HL60 cells (Fig. 8).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Doxorubicin-induced double-stranded DNA Breaks of HL60 cells and R-HL60 cells. Double-stranded DNA breaks were measured by the neutral comet assay. Comets were analyzed by calculating the "tail moment" for each cell. In A-D, representative histograms from 100 comets are shown. A, HL60 cells without doxorubicin pretreatment; B, R-H L60 cells without doxorubicin pretreatment; C, HL60 cells pretreated with 10 µM doxorubicin; D, R-HL60 cells pretreated with 10 µM doxorubicin; E, the means ± S.D. (error bars) of tail moments for 100 comets are shown.

Topoisomerase I Activity and Expression in HL60 and R-HL60 Cells. The possibility that the increase in topoisomerase II activity might be balanced by decreased topoisomerase I activity was also investigated. Quantification of relaxed DNA forms in six independently prepared extracts of R-HL60 and HL60 showed a ratio of 1.27 ± 0.32 (p > .05), which indicates similar topoisomerase I activity in both cell lines. The level of topoisomerase I protein was determined in five independent extracts by Western blot analysis. Densitometric analysis showed no significant difference in the level of topoisomerase I protein expressed in the two cell lines. The ratio of topoisomerase I proteins in R-HL60 extracts to HL60 cells was calculated to be 1.13 ± 0.16 (p > .05).

	Discussion

Top Summary Introduction Experimental Procedures Results Discussion References

Antineoplastic drugs that alkylate DNA are used extensively in cancer chemotherapy, either alone or in combination with other classes of chemotherapeutic drugs. One of the principal factors limiting the effectiveness of alkylating agents is cellular resistance to the alkylating effect. There is considerable evidence from a number of in vitro models, including both human and animal tumor cell lines (Frei et al., 1985; Louie et al., 1985; Robson et al., 1985) that the cytotoxicity of these drugs results directly from interstrand DNA cross-link formation (Ross et al., 1978; Hansson et al., 1987; Erickson et al., 1997; Ewig and Kohn, 1997). The alkylator-resistant phenotypes are multifactorial and include decreased uptake, increased glutathione content, and increased DNA repair activity (Bungo et al., 1990; Richon et al., 1990). In addition to these previously defined mechanisms, the results of the current study demonstrate that alkylator resistance is critically determined by the capacity to repair interstrand-DNA cross-links, which in turn is determined by increased topoisomerase II activity, resulting from an increase in topoisomerase II protein expression.

Melphalan-resistant HL60 cells showed increased topoisomerase II activity and protein expression proportional to the degree of melphalan resistance. These findings, taken in conjunction with the studies of the kinetics of DNA cross-link formation in the sensitive and resistant cell lines, suggest that enhanced DNA repair attributable to increased topoisomerase II activity with increased binding of topoisomerase II to cross-linked DNA in R-HL60 is responsible for the resistance to alkylating agents. These melphalan-resistant (R-HL60) cells were more sensitive to the cytotoxic effects of the topoisomerase II inhibitor doxorubicin, exposure to which resulted in increased double-strand DNA breakage in R-HL60 cells. Inhibition of topoisomerasse II activity by minimally cytotoxic concentrations of doxorubicin was able to significantly increase the amount of interstrand-DNA cross-link formation after melphalan exposure in R-HL60 cells, thus reversing the alkylator-resistant phenotype.

Increased topoisomerase II activity and binding thus seem to be the rate-limiting step for DNA repair. Although we could not exclude, in the present study, that additional DNA repair mechanisms were also increased, the restoration of melphalan sensitivity by topoisomerase II inhibition suggests that this is the most important mechanism. In addition, specificity of the increase of topoisomerase II level and activity was further suggested by the observation that there was no increase in topoisomerase I expression or alteration in sensitivity to the topoisomerase I inhibitor camptothecin in R-HL60 cells compared with parental HL60. These results are consistent with earlier studies demonstrating that increased topoisomerase II expression was associated with resistance to mechlorethamine and cisplatin and that this resistance was associated with increased topoisomerase II binding to cisplatin-damaged DNA (Eder et al., 1995). A possible correlation between topoisomerase II activity and cellular resistance to alkylating agents in the mechlorethamine resistant Raji-HN₂ cell line and in a cisplatin-resistant human lung cancer cell line (Tan et al., 1987; De Jong et al., 1991a) has also been described.

Topoisomerase II has been identified as the putative cellular target of many clinically active antineoplastic agents, including the aminoacridines, anthracyclines, and epipodophyllotoxin (Glisson and Ross, 1987; Liu, 1989). Sensitivity to these agents in vitro seems to correlate with cellular topoisomerase II levels. Previous studies have shown that topoisomerase II inhibitors can also partially reverse resistance to the alkylating agents (Tan et al., 1987; De Jong et al., 1993), although the mechanism of this effect has not been fully defined.

The results of the current study provide further insights into the mechanisms of alkylator resistance and provide a mechanistic framework for the clinical use of alkylating agents in combination with topoisomerase II inhibitors such as doxorubicin and epipodophyllotoxins. The findings provide a rationale for the clinical use of combination chemotherapy regimens based on alkylating agents and topoisomerase II inhibitors.