Introduction

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DNA topoisomerase II inhibitors used in human cancer chemotherapy constitute a group of structurally unrelated compounds that share a common property, the capacity to induce a significant increase of the number of covalent enzyme-DNA complexes (cleavable complexes) present on the cell genome at a given time, thus triggering a cell response eventually leading to cell death by apoptosis (Liu, 1989; Froelich-Ammon and Osheroff, 1995). However, probably as a consequence of their structural diversity, DNA topoisomerase II inhibitors have been shown to be active in vitro through different mechanisms. It is generally admitted that these compounds are involved in the formation of a drug-enzyme-DNA ternary complex. Some drugs enter this complex by interacting predominantly with the protein (etoposide) or the DNA (ellipticine), but it is likely that most DNA-intercalating drugs bind to both the DNA, through intercalation of the chromophore at the enzyme-DNA interface, and the enzyme, through side chains, which are usually essential to their pharmacological activity. Some drugs [4'- (9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), etoposide] can inhibit the religation of the cleaved DNA, whereas others (ellipticine, genistein, quinolones) are presumed to accelerate the forward rate of complex formation (Froelich-Ammon and Osheroff, 1995).

The relatively simple structure of ellipticine has prompted chemists to design various structural modifications to synthesize compounds endowed with increased activity and specificity (Le Pecq et al., 1974; Auclair, 1987a). More than 70 molecules were thus obtained, but only one, 2-N-methyl-9-hydroxy-ellipticinium (NMHE), was finally used in human clinics with a moderate efficacy (Paoletti et al., 1980; Rouessé et al., 1993). Olivacine, a natural ellipticine isomer, differing by the shift of the methyl group from the 11 to the 1 position, was not nearly as extensively studied, although it displayed a significant antitumor activity on various experimental tumors (Pierré et al., 1997).

Recently, a new series of 6H-pyrido[4,3b]carbazole derivatives, characterized by a basic N-dialkylaminoalkyl carboxamido side chain grafted onto an olivacine chromophore, was synthesized (Jasztold-Howorko et al., 1994). Some of these compounds displayed a remarkable activity against various experimental tumors. The most active of these compounds, S16020-2 (NSC-659687) (Fig. 1), has demonstrated a broad range of antitumor activity against a panel of murine and human tumor xenografts, being particularly active against the Lewis lung carcinoma and the human non-small cell lung carcinoma NCI-H460, two highly aggressive and chemoresistant models. In these models, NMHE was inactive and S16020-2 was at least as active as adriamycin taken as a reference in preclinical studies. Recently, two orthotopic models were used to confirm the potential activity of S16020-2 against experimental lung cancer. Murine Lewis lung carcinoma and human A549 tumor cells were grafted by the i.v route, the latter into SCID mice, to obtain lung metastases, which progressively invade the lungs resulting in the death of the animals. S16020-2 totally inhibited the growth of lung metastases and cured 89% of Lewis lung carcinoma-bearing mice and increased by 50% the survival of A549-bearing mice (Guilbaud et al., 1997). In addition, the drug retained its activity on several sublines expressing the multidrug resistance (MDR) phenotype (Guilbaud et al., 1996; Pierré et al., 1998). All these observations made S16020-2 a very promising antitumor agent. Because of its favorable pharmacokinetic characteristics and acceptable toxicity in different species (Pierré et al., 1997), S16020-2 is presently in clinical trials.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Structures of olivacine and ellipticine derivatives.

The molecular basis for the remarkable antitumor activity of S16020-2 is currently unknown. In vitro, S16020-2 displays strong similarities with NMHE (Le Mée et al., 1998). S16020-2 intercalates between adjacent DNA base pairs with an affinity comparable to that of NMHE. This new olivacine derivative is a specific topoisomerase II inhibitor that stimulates the cleavable complex formation in a concentration range close to that of NMHE and has no detectable effect on the DNA religation rate. However, S16020-2 differs markedly from any other topoisomerase II poison by its absolute ATP requirement to stimulate the enzyme-mediated DNA cleavage. In contrast with the results obtained in vitro, S16020-2 was at least 30-fold more cytotoxic than NMHE on the DC-3F cell line, and in these cells, the formation of the cleavable complexes was detected at concentrations 500-fold lower than with NMHE.

We now report the selection and properties of S16020-2-resistant cells derived from the Chinese hamster fibrosarcoma cell line DC-3F. Comparison of these cells with DC-3F/9-OH-E cells, a DC-3F variant resistant to 9-OH-E, led us to propose a new model for the mechanism of action of S16020-2.

	Materials and Methods

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Cell Lines and Culture Conditions. The parental Chinese hamster lung fibroblast cell line DC-3F, the S16020-2-resistant subline DC-3F/S16, the 9-OH-E-resistant subline DC-3F/9-OH-E (Salles et al., 1982), clone 24 (Khélifa et al., 1999), and clone 11 cells (Dereuddre et al., 1997) were maintained as monolayer cultures in Eagle's minimal medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 50 µg/ml streptomycin (Dereuddre et al., 1997). The cells were incubated at 37°C in a humidified incubator with 5% CO₂ in air. The resistant cells DC-3F/S16 and DC-3F/9-OH-E were permanently grown in the presence of 100 nM S16020-2 and 0.6 µg/ml 9-OH-E, respectively. Before each experiment, the cells were grown for two or three passages in the absence of drug.

Drugs and Chemicals. S16020-2 and S30761, the ellipticine analog of S16020-2 with the side chain grafted at position 1, were provided by Institut de Recherches Servier (Suresnes, France); NMHE was purchased from Pasteur-Mérieux (Lyon, France), doxorubicine (Adriblastine) from Farmitalia (Rueil Malmaison, France), vincristine (Oncovin) from Lilly France S.A. (St. Cloud, France), and actinomycine D (Lyovac Cosmegen) from MSD (Whitehouse Station, NJ). These drugs were dissolved in H₂O. 9-OH-E was kindly provided by Dr. E. Lescot (Institut Gustave Roussy, Villejuif, France) and dissolved in HCl 10³ M. Etoposide and m-AMSA, obtained from Sigma Aldrich Chimie (Fallavier, France), were dissolved in dimethylsulfoxide. Stock solutions at 0.01 M were prepared for each drug. [³H]S16020-2 (39 Ci/mmol) was synthesized by the CEA (Saclay, France).

Population-Doubling Time. DC-3F cells (10⁴) and DC-3F/S16 cells (5 × 10⁴) were plated in 60-mm-diameter Petri dishes and incubated at 37°C. The cells were counted in duplicate every 12 h for 4 days. The data were analyzed by plotting the logarithm of the cell number as a function of time, and the population generation time was calculated from the exponential part of the growth curve.

Tumorigenicity. For determination of tumorigenicity, 1 or 2 × 10⁶ cells were injected s.c. into the left posterior flank of 6- to 8-week-old female nu/nu mice. The mice were examined biweekly for development of tumors. Cells were scored as tumorigenic if a palpable nodule appeared and grew progressively at the injection site within 10 weeks. Otherwise the mice were kept for at least 3 months after injection before being scored as nontumorigenic.

Assay of Drug Sensitivity. All of the experiments were carried out on exponentially growing cells. Two methods were used for determination of drug sensitivity: 1) The 72-h assay. The sensitivity of the DC-3F and DC-3F/S16 cells to the different drugs was determined by cell counting after 72-h drug exposure (Salles et al., 1982). Wells (16 mm) of a 24-well Falcon dish, containing 1 ml of minimal essential medium with graded drug concentrations, were inoculated with 1 ml of cell suspension containing either 10⁴ parental cells or 2 × 10⁴ resistant cells. After 72 (±2) h of drug exposure, the number of cells per well was determined with a model ZM Coulter Counter (Coultronic France, Margency, France). 2) The colony formation assay. This assay determines the cloning efficiency of the cell survival fraction after 3-h drug exposure. Parental cells or resistant cells (2 to 10⁵ or 4 to 10⁵, respectively) were grown into 60-mm-diameter Petri dishes (Falcon, Becton-Dickinson, Lincoln Park, NJ) for 18 h at 37°C before drug treatment. After 3 h of exposure to the drugs, the cells were trypsinized, and appropriate dilutions were made to seed 250 or 500 treated cells into 60-mm-diameter Petri dishes. Colonies were stained and counted 6 to 8 days later.

Drug Uptake and Efflux Studies. Cells were grown in 35-mm wells containing 4 ml of growth medium for 24 h to a cell density of 5 × 10⁵ to 1 × 10⁶ cells/well. The growth medium was then removed and replaced with 1 ml of fresh medium containing ³H-labeled S16020-2 at a concentration of 50 nM (specific activity adjusted to 2 Ci/mmol). At various times thereafter, the cells were washed three times with 1 ml of PBS at 4°C and lysed overnight with 1 ml of 2% Triton X-100 at room temperature. The cells lysates were withdrawn, and each well was washed with 1 ml of PBS. Each lysate and wash was collected in a scintillation vial and mixed with 10 ml of Ultima Gold (Packard Instruments Co., Meriden, CT) for liquid scintillation counting. Cell counting was carried out on duplicate samples grown in the same conditions with a model ZM Coulter Counter.

cDNA Probes. The SP1' probe, obtained from a human cDNA library (Khélifa et al., 1994), selectively recognizes the 170-kDa form of the enzyme, whereas the PH1 probe, a PstI/HindIII fragment isolated from the pBhamTOP2 plasmid, selectively detects the 180-kDa form (Dereuddre et al., 1997). The actin DNA probe was kindly provided by Dr. F. Dautry (Institut de Recherche Scientifique sur le Cancer, Villejuif, France). These probes were labeled by random priming with -³²P using the Multiprime labeling kit from Amersham (Buckinghamshire, UK).

Anti-DNA Topoisomerase II Antibodies. The rabbit polyclonal antibody designated A6 was previously described (Khélifa et al., 1994). This antibody raised against a 837-amino acid fragment of the human topoisomerase II, recognizes the - and -enzymes from both human and hamster origins.

Northern Blots. RNA was extracted by the guanidine thiocyanate technique (Chirgwin et al., 1979). Polyadenylated RNAs were purified from total RNA using the mRNA purification kit from Pharmacia LKB Biotechnology (Saclay, France) and following the manufacturer's instructions. mRNAs (4 µg) were fractionated by electrophoresis in 1.2% (w/v) agarose gels containing 7% formaldehyde and transferred to a Hybond-N nylon membrane (RPN3050, Amersham, Buckinghamshire) in 150 mM ammonium acetate (Khélifa et al., 1994). With the PH1 probe, prehybridization was performed for 2 h at 42°C in 50% formamide, 5× SSC (SSC is 0.15 M NaCl, 15 mM sodium citrate), 50 mM phosphate buffer, pH 6.8, 5× Denhardt's solution, 1% SDS. Hybridization was then performed for 20 h in the same buffer, with 1× Denhardt's solution, containing the ³²P-labeled cDNA probe. The membrane was washed twice at room temperature in 0.1% SDS, 2× SSPE (SSPE is 10 mM NaH₂PO₄, 0.13 M NaCl, 1 mM EDTA) and twice at 50°C for 30 min in 0.1% SDS/0.5× SSPE. With the SP1' probe, prehybridization and hybridization were performed in the buffers described above containing 40% formamide. The second wash of the membrane was performed in 0.1% SDS, 1× SSPE.

Nuclear Extracts. Nuclear extracts from DC-3F and DC-3F/S16 cells were prepared from 7 to 8 × 10⁷ exponentially growing cells as previously described (Riou et al., 1991). Freshly prepared protease inhibitor phenylmethylsulfonyl fluoride (1 mM), benzamidine (1 mM), aprotinin (1 µg/ml), and soybean trypsin inhibitor (10 µg/ml) were added to all buffers. Protein concentration in the nuclear extracts was determined as previously described (Bradford, 1976). The extracts were used within 2 days.

Western Blot. Freshly prepared nuclear extracts (100 µg) were fractionated on 7.5% SDS-polyacrylamide gels. The proteins were transferred (4 mA/cm²) to a nitrocellulose membrane (BioTrace NT, Gelman Sciences, East Hills, NY). Membranes were incubated with the polyclonal anti-topoisomerase II antibody A6. Bound antibody was visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG and enhanced chemiluminescence detection (Amersham, Buckinghamshire).

Measurements of DNA Damage by Alkaline Elution. The methodology of DNA alkaline elution has been previously described (Kohn et al., 1981). DNA single-strand breaks (SSBs) were assayed by DNA-denaturing alkaline elution carried out under deproteinizing conditions. Cellular DNA in exponentially growing DC-3F and DC-3F/S16 cells was radioactively labeled for 20 h at 37°C by adding to the growth medium either [2-¹⁴C]thymidine (0.02 µCi/ml, 56 mCi/mmol; Amersham, France) or [methyl-³H]thymidine (0.1 µCi/ml, 20 mCi/mmol; Amersham, France) diluted in 10⁶ M unlabeled thymidine (internal standard cells). After the radioactive medium was removed, the cells were grown for at least 2 h in label-free medium before any experiment. [¹⁴C]Thymidine-labeled cells were treated with the drug at increasing concentrations for 1 h. Drug treatments were terminated by removal of the drug-containing medium and resuspension of the cells in ice-cold Hanks' balanced solution containing 0.02% EDTA. ¹⁴C-labeled cells were mixed with untreated ³H-labeled cells (internal standard) that had been irradiated, on ice, with 1000 rad equivalents. The alkaline elution technique was the low-sensitive assay to detect SSB up to 3000 SSB rad equivalents (Kohn et al., 1981). The elution rate of [³H]thymidine-labeled cells served to normalize the elution rate of DNA from cells labeled with [¹⁴C]thymidine.

Topoisomerase II-Mediated Cleavage Reactions. pSP65 DNA (0.2 µg) was incubated with 5 µg of nuclear extracts from the different cell lines, in the absence or presence of drug, in 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 15 µg/ml BSA, 1 mM ATP for 15 min at 37°C. The cleavage reaction (15 µl) was terminated by the addition of SDS and proteinase K to final concentrations of 0.4% and 0.1 mg/ml, respectively, and the mixture was incubated for an additional 30 min at 50°C. After the addition of 4 µl of loading buffer (0.4% bromphenol blue, 0.4% xylene cyanol, 50 mM EDTA, 50% glycerol), the products of the reaction were fractionated by electrophoresis on 1% agarose gels for about 15 h (2.5 V/cm) in TBE (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) containing 0.5 µg/ml of ethidium bromide. DNA bands were visualized by transillumination with UV light and quantified by scanning the gel with a Bioprofil Scan (Vilbert-Lourmat, Marne-la-Vallée, France).

Cloning of Topoisomerase II and  cDNAs from DC-3F/S16. Polyadenylated RNAs were purified as described above. The cDNA libraries were constructed using the Zap cDNA synthesis kit from Stratagene (La Jolla, CA), following the manufacturer's instructions. cDNAs were cloned in the uni-ZAP XR vector and packaged in -phagemide as previously described (Dereuddre et al., 1995). Approximately 4 × 10⁵ phages from each library were plated out and screened by plaque hybridization with the probes SP1' or HP1. 9 plaques hybridizing with SP1', and six plaques hybridizing with HP1 were picked up and purified by tertiary screening. cDNA fragments from single positive plaques were excised by digestion with XhoI and EcoRI. Six clones were found to contain inserts with sizes greater than 4.6 kb, the approximate size expected for a complete topoisomerase II cDNA. Topoisomerase II cDNA contains two EcoRI-sensitive sites, and two clones presented the expected digestion pattern. pBluescript phagemids were then excised from the uni-ZAP vector and transfected in XL1-Blue bacteria, following the Stratagene protocol. Restriction analysis showed that these fragments spanned the entire coding region of the topoisomerase II and  cDNAs, with approximately 95 and 160 bp upstream of the initiation codon, respectively.

Sequencing of Topoisomerase II and  cDNAs. DNA sequencing was performed and analyzed at ACT Gene-Euro Sequence Genes Services (Génopole, Evry, France), on an ABI 377 sequencer with an ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit and AmpliTaq polymerase FS (Perkin-Elmer/Applied Biosystems division, Foster City, CA).

	Results

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Selection of S16020-2-Resistant Cells. S16020-2-resistant DC-3F cells were selected by adding stepwise increasing drug concentrations to the cell growth medium. At each concentration (2, 5, 20, 50, and 100 nM), the cells were maintained until they recovered a normal growth pattern. After about 6 months of drug exposure in these conditions, a subline was established that was able to grow normally in the presence of S16020-2 at 100 nM with a doubling time (12 h) identical with that of the parental cells. From this subline, three independent clones were isolated that displayed identical properties, and one of them, designated DC-3F/S16, was chosen for further characterization.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Cytotoxicity of S16020-2 (, ) and NMHE (, ) on DC-3F cells (filled symbols) and DC-3F/S16 cells (open symbols). Exponentially growing cells were treated with the drug at the indicated concentrations for 3 h. Cells were trypsinized, counted, and seeded, and after 6 to 7 days colonies were stained and counted as described under Materials and Methods. The inset shows an enlargement of the survival curve of DC-3F cells treated with S16020-2. Bar, S.D. of at least three independent determinations.

Cross-Resistance to Other Compounds. With the colony formation assay, we first examined the sensitivity of DC-3F/S16 cells to ellipticine derivatives. Figure 2 and Table 1 show that NMHE and 9-OH-olivacine were approximately 30-fold less cytotoxic than S16020-2 on DC-3F cells. Surprisingly, DC-3F/S16 cells were only 3- to 4-fold cross-resistant to these drugs. We also tested the sensitivity to 9-OH-E, which was almost as cytotoxic as S16020-2 on DC-3F cells (Table 1). Again, the cross-resistance of DC-3F/S16 cells to this compound was very low (approximately 12-fold). However, DC-3F/S16 cells were approximately 90-fold cross-resistant to S30761, a 9-OH-E derivative carrying the same substitutions as S16020-2 (S16020-2 side chain grafted at position 1 and a methyl at position 6). Table 1 also shows that DC-3F/9-OH-E cells, an ellipticine-resistant variant of DC-3F cells (Salles et al., 1982), were highly cross-resistant to S16020-2. This unexpected pattern of cross-resistance to drugs, which belong to closely related chemical series and display very similar in vitro properties, suggested that different mechanisms of action might be involved in the toxicity of these compounds.

Tumorigenicity of DC-3F/S16 Cells. A decreased tumorigenicity is frequently observed in drug-resistant cell lines, including DC-3F/9-OH-E cells, the ellipticine-resistant variant of DC-3F cells (Remy et al., 1984). S16020-2-sensitive and -resistant cells were tested for their ability to form tumors after s.c. injection into nude mice. As previously reported (Remy et al., 1984), DC-3F cells were highly tumorigenic, with 100% tumor take within 1 week. Inoculation of 2 × 10⁶ DC-3F/S16 cells provoked tumor take in approximately 70% of the animals within 10 days. After inoculation of 1 × 10⁶ cells, the tumor take was reduced to 30% of the mice, whereas the latency was increased up to 1 month. These results show that development of resistance to S16020-2 was associated with a decreased oncogenic potential, although to a lesser extent than in DC-3F/9-OH-E cells, which completely lost their tumorigenicity.

Cellular Accumulation and Efflux of S16020-2. Cellular accumulation of S16020-2 by parental and resistant cells was measured by exposing cells to the ³H-labeled drug at 5 × 10⁸ M for the indicated times. Figure 3A shows that, after a rapid increase during the first 20 min, the drug uptake reached a plateau that remained stable for 3 h. This plateau was slightly higher (12%) for the resistant than for the sensitive cells. S16020-2 efflux kinetics were measured on the sensitive and resistant cells previously loaded for 30 min with the drug. The efflux kinetics (Fig. 3A) were identical for both cell lines: 50% of the drug was lost during the first 30 min, and the efflux progressively slowed down for the next 3.5 h. At that time, approximately 10% of the initial amount of drug was still present in each cell line.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Uptake and efflux kinetics of [3H]S16020-2 by the parental () and S16020-2-resistant cells () at 37°C. A, cells were incubated with the drug for the indicated times. For efflux kinetics, cells, loaded with the drug for 30 min, were then incubated in drug-free medium for the indicated times. The amount of intracellular drug was determined as described under Materials and Methods. Values are means of two individual experiments, each done in triplicate. Bar, S.D. B, uptake of S16020-2 at different external concentrations by sensitive and resistant cells. The cells were incubated for 30 min with the drug at the indicated concentrations. The amount of intracellular drug was determined as in A.

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Stimulation of DNA Cleavage by DNA Topoisomerase II Poisons. Stimulation of topoisomerase II-mediated DNA cleavage is a critical feature in the mechanism of action of many topoisomerase II inhibitors, and such a stimulation by S16020-2 was previously demonstrated in an in vitro assay using purified DNA topoisomerase II (Le Mée et al., 1998).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Stimulation by S16020-2, NMHE, m-AMSA, and etoposide of DNA cleavage induced by nuclear extracts from DC-3F and DC-3F/S16. Nuclear extracts (5 µg) from parental (filled symbols) and resistant (open symbols) cells were incubated with 0.2 µg of pSP65 DNA in the presence of the indicated concentrations of S16020-2 (A), NMHE (B), m-AMSA (C), and etoposide (D). After agarose gel electrophoresis separation, the amount of linear DNA was quantified. The results of two independent experiments are shown.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   DNA SSBs induced by S16020-2 (A), NMHE (B), m-AMSA (C), and etoposide (D) in DC-3F and DC-3F/S16 cells. DC-3F (filled symbols) and DC-3F/S16 cells (open symbols) were exposed to the indicated drug concentrations for 1 h at 37°C. The drug was then removed by washing the cells twice with Hanks' balanced salt solution at 0°C. Cells were scraped, and DNA SSBs were measured by DNA-denaturing alkaline elution. Note that abscissa in A is logarithmic.

Analysis of DNA Topoisomerases II and  Expression in the DC-3F/S16 Cells. The above results, showing the cross-resistance of the DC-3F/S16 cells to various topoisomerase II inhibitors and the alterations in the capacity of these compounds to induce the cleavable complex formation, suggested that quantitative and/or qualitative alterations of the DNA topoisomerase II activities might be involved in the resistance phenotype of DC-3F/S16 cells.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Expression of topoisomerase II and  in DC-3F/S16 cells. A, immunoblot analysis. Nuclear proteins (100 µg) extracted by 0.35 M NaCl from S16020-2-sensitive and -resistant cells were separated on SDS-polyacrylamide gel, transferred to nylon membranes, and probed with the A6 antibody. B, Northern blot analysis. Topoisomerase II transcripts were fractionated by agarose gel electrophoresis (1.2%) and transferred to a nylon membrane. The RNAs were sequentially hybridized with the SP1' (), HP1 (), and -actin probes.

Cloning and Sequencing of DNA Topoisomerases II and  cDNAs from DC-3F/S16 Cells. To further investigate the molecular basis of the reduced formation of DNA single-strand breaks in S16020-2-resistant cells, the complete cDNAs encoding topoisomerases II and  in DC-3F/S16 cells were cloned and sequenced. The sequence of the isoform  cDNA was the same in the sensitive and resistant cells. In contrast, sequencing of the cDNA-encoding isoform  revealed several differences compared with the sequence previously reported by Chan et al. (1993) for the sequence of the topoisomerase II cDNA from a Chinese hamster ovary cell line. The open reading frame, also extending from nucleotide 1 to 4579, contained seven silent mutations, spread all over the coding region, and three additional mutations in the noncoding region (Table 3). Furthermore, a point mutation was identified at position 1456 that converts a codon AGA to a GGA, resulting in a missense mutation that changes Arg⁴⁸⁶ to Gly (Table 3). This mutation was confirmed in three independent clones by sequencing of the corresponding region.

View this table: [in this window] [in a new window]   TABLE 3 Topoisomerase II allele polymorphism in DC-3F/S16 The accession number to the complete sequence of the DC-3F/S16 topoisomerase II cDNA in the EMBL/EBI database is Y16595.

View larger version (66K): [in this window] [in a new window]   Fig. 7.   Topoisomerase II allele polymorphism. From the genomic DNA, 815-bp fragments, containing the BamHI site associated with the mutation at position 3933 in the cDNA of the mutated allele, were amplified with appropriate primers. After the sample was digested with the restriction enzymes BamHI and AcsI, the products were analyzed by agarose gel electrophoresis. Lanes 1 and 4, undigested fragments, amplified from DC-3F and DC-3F/S16 cells; lanes 2 and 3, BamHI and AcsI fragments from DC-3F cells; lanes 5 and 6, BamHI and AcsI fragments from DC-2F/S16 cells; lane 7, size markers. The sizes of the fragments, labeled a to d, are 815, 716, 486, and 230 bp, respectively. A 99-bp fragment, generated by either BamHI or AcsI digestion, was not visible in these experimental conditions.

Role of DNA Topoisomerases II and  in the Resistance of DC-3F/9-OH-E Cells to S16020-2. In the Chinese hamster lung cell line DC-3F/9-OH-E, made resistant to 9-OH-E, the amount of topoisomerase II is 4- to 5-fold lower than in the parental DC-3F cell line. A mutation generating a stop codon in the topoisomerase II gene completely abolishes the expression of this isoform in DC-3F/9-OH-E cells. To analyze the contribution of these quantitative alterations, the DC-3F/9-OH-E cells were transfected with a eukaryotic expression vector containing either the human -isoform or the hamster -isoform (Dereuddre et al., 1997; Khélifa et al., 1999). The restoration of a normal topoisomerase II or  catalytic activity in the resistant cells had only very little effect on the toxicity of ellipticine derivatives. Because DC-3F/9-OH-E cells are highly cross-resistant to S16020-2, the sensitivity of - and -transfected DC-3F/9-OH-E cells to S16020-2 was also determined. Figure 8 shows that the cytotoxicity of S16020-2 in DC-3F/9-OH-E cells transfected with either topoisomerase II (clone 24)or  (clone 11) cDNA, determined by colony formation assay, remained exactly the same as in the untransfected cells. In contrast, there was a partial reversion of sensitivity to m-AMSA in clone 24 (-enzyme) and an almost complete reversion in clone 11 (-enzyme) (data not shown). Increased sensitivity of the transfected cells to these compounds was associated with an increased formation of the cleavable complex. In contrast, ellipticine derivatives remained unable to induce the cleavable complex formation in the - or -transfected DC-3F/9-OH-E cells, which is consistent with the topoisomerase II-mediated mechanism of action of these compounds.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Cytotoxicity of S16020-2 in DC-3F (), DC-3F/9-OH-E (), clone 11 (), and clone 24 () cells. Experimental details are as in Fig. 2.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The data presented in this paper, together with our previous in vitro studies (Le Mée et al., 1998), indicate that: 1) DNA topoisomerases II are intracellular targets of S16020-2; 2) interaction of S16020-2 with these enzymes is strongly influenced by the N-[2(dimethylamino)ethyl]carbamoyl side chain; and 3) despite common properties between S16020-2 and other olivacine derivatives, in vivo induction of the cleavable complex in the presence of this compound appears to involve a peculiar mechanism, the alteration of which would account for the properties of DC- 3F/S16 cells.

S16020-2 induces cleavable complex formation in vivo at concentrations that are approximately 500-fold lower than NMHE, whereas, in in vitro experiments using purified enzyme, both drugs displayed similar properties. We first hypothesized that this difference might result from a higher intracellular accumulation and/or a peculiar intracellular localization of the molecule, because of the relative hydrophobicity of its side chain (Auclair et al., 1987b). Because S16020-2 is not fluorescent, its intracellular localization will be difficult to analyze. Comparison of uptake and efflux kinetics of S16020-2 with those of NMHE (Charcosset et al., 1983; Delaporte et al., 1988) showed that S16020-2 uptake rate was faster than that of NMHE, whereas efflux rates were identical for both drugs. Determination of drug accumulation showed that, for an equal external concentration, the amount of intracellular S16020-2 was approximately 4-fold higher than that of NMHE. However, because S16020-2 is 30-fold more cytotoxic than NMHE, its estimated intracellular amount at equitotoxic concentrations was approximately 6-fold lower than that of NMHE (40 and 250 µM, respectively). Therefore, a higher intracellular accumulation cannot account for the in vivo activity of S16020-2. This indicates that different mechanisms are involved in the toxicity of S16020-2 and NMHE, a conclusion also strongly supported by analysis of cross-resistance patterns in DC-3F/9-OH-E and DC-3F/S16 cells.

Analysis of Cross-Resistance Patterns. DC-3F/9-OH-E cells, selected up to the highest possible level of resistance to 9-OH-E, display a complex phenotype (Larsen and Jacquemin-Sablon, 1985). Among other traits, these cells are highly cross-resistant to all topoisomerase II inhibitors, including S16020-2. This resistance involves a 4- to 5-fold decreased expression of a mutated topoisomerase II and the complete loss of the -enzyme (Khélifa et al., 1994). Restoration of a normal - or -activity in DC-3F/9-OH-E cells, by transfection of the corresponding cDNAs, had similar effects on their drug sensitivity: formation of cleavable complexes and sensitivity to etoposide and m-AMSA were partially restored, whereas resistance to ellipticine derivatives, genistein, and S16020-2 was unchanged, these compounds remaining unable to induce the cleavable complex formation (Dereuddre et al., 1997; Khélifa et al., 1999). We interpreted these data as indicating that: 1) topoisomerase II alterations in DC-3F/9-OH-E cells were associated with cross-resistance to all topoisomerase II inhibitors; and 2) resistance to drugs that do not inhibit the religation step (ellipticines and genistein) involves a peculiar mechanism not presently understood. Because S16020-2 belongs to this latter group (Le Mée et al., 1998), the same mechanism should account for the resistance of DC-3F/9-OH-E cells to this compound.

Importance of the N-[2(Dimethylamino)ethyl]carbamoyl Side Chain. Alterations in drug capacity to increase the cleavable complex formation indicated that resistance of DC-3F/S16 cells to topoisomerase II inhibitors should be related to quantitative and/or qualitative alterations of either topoisomerase II or  or both. Northern and immunoblot analyses did not reveal any change in the expression of topoisomerase II. The amount of topoisomerase II transcripts was approximately 50% lower in resistant than in sensitive cells, whereas the aspect of the band in Western blot was more fuzzy and therefore difficult to quantify. Because previous studies indicated that m-AMSA may target primarily the -enzyme (Dereuddre et al., 1997; Herzog et al., 1998), transcriptional and/or eventual post-translational alterations of this isoform might account at least partially for the high cross-resistance of DC-3F/S16 cells to this compound. The only genetic modification identified in S16020-2-resistant cells is a point mutation converting Arg⁴⁸⁶ to a Gly in the topoisomerase II cDNA. Arg⁴⁸⁶ is part of a sequence of eight amino acids -Pro-Leu-Arg-Gly-Lys-(Leu/Ile/Met)-Leu-Asn- present in all eukaryotic and bacterial topoisomerases II. In HL60 cells (Lee et al., 1992) and in patients with small cell lung cancer (Kubo et al., 1996), replacement of Arg⁴⁸⁷ (corresponding to hamster Arg⁴⁸⁶) by a Lys has been associated with the acquisition of the resistance to m-AMSA and etoposide, whereas the same substitution had no effect in yeast (Wasserman and Wang, 1994). In the B subunit of Escherichia coli DNA gyrase, a mutation of Lys⁴⁴⁷ (position corresponding to Arg⁴⁸⁶ in the hamster enzyme) to glutamic acid confers resistance to nalidixic acid (Yamagishi et al., 1986). As previously suggested (Lee et al., 1992), the high degree of conservation and the drug resistance associated with the majority of the mutations of Arg⁴⁸⁶ suggest that the -Pro-Leu-(Arg/Lys)-Gly-Lys-(Leu/Ile/Met)-Leu-Asn- sequence should play an important part in the topoisomerase II catalytic cycle and be involved in the formation of the ternary complex with certain drugs. Molecules like S16020-2 or m-AMSA would bind to DNA by intercalation and to the enzyme through interaction of their side chains with the -Pro-Leu-(Arg/Lys)-Gly-Lys-(Leu/Ile/Met)-Leu-Asn- sequence. This latter interaction would be essential to their pharmacological activity because either a mutation in the target sequence or a chemical modification of the drug side chain resulted in a decreased activity. DC-3F/S16 cells display a very low cross-resistance to drugs, like olivacine, 9-OH-E, or NMHE, which do not carry the appropriate side chain to interact with the target sequence and, therefore, are not sensitive to the Arg⁴⁸⁶ mutation. We then predicted that DC-3F/S16 cells should be cross-resistant to S30761 (Fig. 1), an ellipticine derivative that carries the same side chain as S16020-2 and is almost as cytotoxic. Tables 1 and 2 show that this was indeed the case.

Mechanism of Cleavable Complex Induction. Biochemical and structural studies have led to a model that describes topoisomerase II as an ATP-modulated clamp with two molecular gates at opposite ends (Berger et al., 1996). The enzyme catalytic cycle is driven by a series of conformational changes triggered by cofactor binding. Because S16020-2 induces topoisomerase II-mediated DNA cleavage only in the presence of ATP, we hypothesize that it would only bind to the enzyme-DNA complex when it is in the ATP-bound conformation. In this conformation of the complex, the eight-amino acid target sequence would take a position proximal to the catalytic site, thus allowing S16020-2 to interact with both the enzyme and the DNA. The cleavage-religation equilibrium would then be shifted to the cleaved form, possibly through a deformation of the DNA molecule. Such a reaction would increase the number of cleavable complexes on the genome at a given time and would then account for the drug toxicity. After the T fragment passage, the religation of the G fragment requires that the enzyme recovers a conformation similar to that before ATP binding, a transition that might involve the hydrolysis of one ATP molecule (Harkins and Lindsley, 1998; Harkins et al., 1998). This conformation change would provoke the release of the drug then unable to interfere with the religation step. The strict ATP dependence of S16020-2 would be a property dependent on the side chain. However, a similar mechanism could be extended to other compounds that do not inhibit the religation step, except that they might enter the enzyme-DNA complex before the ATP binding.