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Decreased Nucleotide Excision Repair Activity and Alterations of Topoisomerase II Are Associated with the in Vivo Resistance of a P388 Leukemia Subline to F11782, a Novel Catalytic Inhibitor of Topoisomerases I and II

Division of Experimental Cancer Research, Pierre Fabre Research Center, Castres, Cedex, France

	ABSTRACT

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Purpose: The purpose of the study was to investigate the mechanisms associated with antitumor activity and resistance to F11782, a novel dual catalytic inhibitor of topoisomerases with DNA repair-inhibitory properties.

Experimental Design: For that purpose, an F11782-resistant P388 leukemia subline (P388/F11782) has been developed in vivo and characterized.

Results: Weekly subtherapeutic doses of F11782 (10 mg/kg) induced complete resistance to F11782 after 8 weekly passages. This resistant P388/F11782 subline retained some in vivo sensitivity to several DNA-topoisomerase II and/or I complex-stabilizing poisons and showed marked collateral sensitivity to cisplatin, topotecan, colchicine, and Vinca alkaloids, while proving completely cross-resistant only to merbarone and doxorubicin. Therefore, resistance to F11782 did not appear to be associated with a classic multidrug resistance profile, as further reflected by unaltered drug uptake and no overexpression of resistance-related proteins or modification of the glutathione-mediated detoxification process. In vivo resistance to F11782 was, however, associated with a marked reduction in topoisomerase II protein (87%) and mRNA (50%) levels, as well as a diminution of the catalytic activity of topoisomerase II. In contrast, only minor reductions in topoisomerases IIß and I levels were recorded. However, of major interest, nucleotide excision repair activity was decreased 3-fold in these P388/F11782 cells and was more specifically associated with a decreased (67%) level of XPG (human xeroderma pigmentosum group G complementing protein), an endonuclease involved in this DNA repair system.

Conclusions: These findings suggest that both topoisomerase II and XPG are major targets of F11782 in vivo and further demonstrate the original mechanism of action of this novel compound.

	INTRODUCTION

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DNA topoisomerase inhibitors represent a major class of anticancer agents with documented activities against a broad spectrum of human malignancies (1) . However, the emergence of tumor cell drug resistance remains a major clinical problem and the frequent cause of failure in the long-term efficacy of cancer therapy. Therefore, one goal of experimental oncology is to understand the underlying mechanisms responsible for this phenomenon, with the hope that more effective therapies can be devised.

Extensive experimental research has shown that cellular resistance mechanisms to topoisomerase inhibitors are generally multifactorial (2, 3, 4, 5) . Three major categories have been identified: (a) pretarget events, including drug uptake, metabolism, and intracellular distribution; (b) drug-target interactions; and (c) post-target events, including macromolecular syntheses, DNA repair, cell cycle progression, and regulation of cell death (4) . Earlier studies identified a novel epipodophylloid dual inhibitor of topoisomerases I and II, namely, F11782 (2'',3''-bispentafluorophenoxyacetyl-4'',6''-ethylidene-ß-D-glucoside of 4'-phosphate-4'-demethylepipodophyllotoxin, N-methyl glucamine salt) with different mechanistic properties from most specific inhibitors of topoisomerases I or II (6) . Indeed, F11782 exhibits distinct mechanisms of action against human topoisomerases; it appears to act by preventing the binding of topoisomerases to DNA through a preferential interaction with the enzyme (6) , but it is also capable of trapping topoisomerase II in an ATP-independent noncovalent salt-stable complex that does not depend on the ability to cleave DNA (7) . Furthermore, F11782 does not intercalate into DNA, and it does not stabilize DNA-topoisomerase cleavable complexes. However, F11782 was also found to create DNA damage in intact cells that differs from that mediated by classical topoisomerase poisons by increasing with time and being less efficiently repaired (8 , 9) . Subsequent studies provided evidence that F11782 is also a potent inhibitor of nucleotide excision repair (NER; Ref. 10 ). Furthermore, F11782 has major antitumor activity against a panel of experimental tumor models with different biological properties and chemosensitivities (11 , 12) . Therefore, this original mechanistic profile of F11782, coupled with the definite and broad spectrum of antitumor activity of F11782, prompted consideration of F11782 for clinical development.

To investigate the mechanisms associated with antitumor activity and resistance to F11782, an F11782-resistant P388 leukemia subline (P388/F11782) has been developed in vivo and characterized. Data reported here describe the establishment of the F11782-resistant P388/F11782 subline; its in vivo profile of sensitivity/cross-resistance to a series of antitumor agents, including topoisomerase inhibitors, DNA cross-linking agents, antimetabolites, and tubulin-interacting agents; and some of its biochemical properties. More specifically, potential modification of drug uptake and resistance-related protein expression, as well as alterations in topoisomerase expression, activity, sequence and NER, were investigated in the F11782-resistant P388/F11782 subline.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Drug Preparations.
For in vivo evaluations, F11782, recently termed tafluposide [Pierre Fabre (Fig. 1) ], was solubilized in a solution containing 4.75% glucose, 5% Tween 80, and 90.25% water. The following group of drugs tested were obtained from various sources as indicated: 1,3-bis(2-chloroethyl)-1-nitroso-urea [BCNU (Bristol-Myers Squibb)]; cisplatin, colchicine, cyclophosphamide monohydrate, doxorubicin hydrochloride, and 5-fluorouracil (Sigma); mitoxantrone dihydrochloride (Lederle); and etopophos, TAS-103 dichlorhydrate or 6-[[(2-dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno(2,1-c)quinolin-7-one, teniposide, topotecan acetate, vinblastine sulfate, vinflunine, or 20',20'-difluoro-3',4'-dihydrovinorelbine sulfate and vinorelbine ditartrate from Pierre Fabre were all solubilized in a saline solution (0.9% NaCl). Camptothecin and methotrexate (both from Janssen), etoposide (Pierre Fabre), irinotecan (Yakult-Honsha), and merbarone (Calbio/VWR) were solubilized in a saline solution containing 5% Tween 80. Solubilization of test compounds was performed immediately before use, and administration was performed at 10 ml/kg. For in vitro evaluations, F11782, etoposide, and ICRF-193 (Pierre Fabre) were solubilized in DMSO to achieve a final concentration of 1% DMSO in the final reaction volume.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Chemical structure of F11782.

In Vivo Establishment of the P388/F11782 Subline, Resistant to F11782.
Viral-free parental P388 leukemia cells were maintained by serial weekly i.p. implantation into DBA/2 mice. For establishment of the F11782-resistant subline, the parental P388 cells were implanted i.p. at 10⁶ cells/mouse into a group of five mice on day 0. These mice were then treated with 10 mg/kg F11782 administered i.p. on day 1. When regrowth was evident, after approximately 1 week, leukemic cells from these F11782-treated mice were reimplanted in vivo, and the mice were again dosed i.p. the following day with 10 mg/kg F11782. The F11782-resistant P388/F11782 subline was thus developed over successive in vivo transplant generations.

Experimental Chemotherapy.
All experiments were conducted in compliance with French regulations and Centre de Recherche Pierre Fabre ethical committee guidelines, based on the United Kingdom Coordinating Committee on Cancer Research guidelines for the welfare of animals in experimental neoplasia, as detailed previously (13) . A total of 10⁶ P388 or P388/F11782 cells were implanted i.v. into CDF1 mice on day 0 (except for merbarone evaluations, for which the i.p. implanted P388 model was used because merbarone was inactive against the i.v. implanted P388 model). After their randomization in treatment cages, test compounds were administered to mice i.p. as a single dose, the day after tumor implantation. In each chemotherapy trial, mice were checked daily, and any adverse clinical reactions were noted, and deaths were recorded. Mice were weighed twice weekly during treatment and once weekly thereafter.

Evaluation of Antitumor Activity.
An increase of life span (ILS) was defined as follows: ILS (%) = T/C - 100, with the T/C (%) ratio representing (median survival of treated mice/median survival of control mice) x 100. The optimal ILS corresponds to the maximal value or to the maximal ILS achieved. According to National Cancer Institute criteria for the P388 model, 20% ILS < 75% is judged as being the minimum level for activity, and an ILS of 75% is judged as corresponding to a high level of antileukemic activity (14) .

The areas under the survival curve as a function of dose (i.e., Area_S and Area_R) have been calculated using the trapezium method (Sigma Plot; Jandel Corp.) for the parental (F11782-sensitive) P388 and F11782-resistant P388/F11782 leukemic cells, respectively. The relative resistance (RR), reflecting the chemotherapeutic response of the resistant subline vis-à-vis the sensitive one, was defined as follows: RR = Area_R/Area_S, with a RR value of 0 representing total resistance of the F11782-resistant subline to the compound tested, a RR value of 1 corresponding to similar sensitivity of both lines, and a RR value of >1 reflecting collateral enhanced sensitivity of the F11782-resistant subline.

Drug Uptake.
P388 and P388/F11782 cells were removed from the peritoneal cavities of tumor-bearing mice and maintained in suspension culture in RPMI 1640 supplemented with 10% heat-inactivated horse serum and 4 mM L-glutamine, all purchased from Seromed, together with 100 units/liter penicillin and 1.25 µl/ml fungizone (Invitrogen). For in vitro uptake studies, 10 nmol of [¹⁴C]F11782 (0.488 µCi) were added to a cell suspension of P388 or P388/F11782 cells (10⁵ cells/ml) incubated at 37°C. At predetermined time intervals, aliquots were removed and subjected to rapid cooling on ice. After centrifugation (2000 x g, 4°C, 2 min), the resultant cell pellet was washed twice with ice-cold PBS. Associated radioactivity was then quantitated using a scintillation spectrometer (Packard).

Northern Blot Analyses of Topoisomerases II, IIß, and I mRNAs.
P388 or P388/F11782 cells were collected directly from the peritoneal cavities of tumor-bearing mice, centrifuged for 5 min at 100 x g at room temperature through 0.32 M sucrose in PBS to eliminate any contaminating RBCs, and then washed twice in 10 ml of cold PBS. Then, total RNA derived from either P388 or P388/F11782 cells was isolated using TRIzol reagents (Invitrogen), according to the manufacturer’s instructions. mRNA was further isolated from total RNA using a poly(dT) bead-based extraction kit (Dynal). Northern blotting was then performed using a NorthernMax (Clinisciences) kit. Briefly, mRNA was separated in a 1% formaldehyde-denaturated agarose gel and transferred to a positively charged nylon membrane (Clinisciences). After RNA fixation to membranes by UV cross-linking, membranes were hybridized at 42°C overnight using topoisomerase II, IIß, and I and glyceraldehyde-3-phosphate dehydrogenase probes labeled with [-³²P]dCTP by radioactive PCR using the following primers: 5'-GAT-CGG-ATC-CAA-ATA-GAT-GGC-AAA-ATA-GTC and 5'-GAT-CGA-ATT-CTC-TCG-TTT-CTT-TGT-TTG-CAC for topoisomerase II; 5'-GAT-CGG-ATC-CAG-ATA-CAG-GGG-AAA-ATT-ACT and 5'-GAT-CGA-ATT-CTT-AAA-ATC-AGG-GCC-TGA-AGG for topoisomerase IIß; 5'-GAT-CGG-ATC-CCA-TAA-ATG-GAA-GGA-AGT-CCG and 5'-GAT-CGA-ATT-CTG-AAG-TAC-AGT-GCT-ACA-GCT for topoisomerase I; and 5'-GAT-CGG-ATC-CGA-GAA-TGG-GAA-GCT-TGT-CAT-C and 5'-GAT-CGA-ATT-CGT-TCA-CAC-CCA-TCA-CAA-ACA-T for glyceraldehyde-3-phosphate dehydrogenase, which was used as a house-keeping gene to normalize RNA levels between samples. mRNA content was quantified using a PhosphorImager (Bio-Rad).

Western Blot Analyses of Topoisomerases II, IIß, and I and XPG and XPF Proteins.
P388 or P388/F11782 cells were collected directly from the peritoneal cavities of tumor-bearing mice, centrifuged for 5 min at 100 x g at room temperature through 0.32 M sucrose in PBS, and then washed twice in 10 ml of cold PBS containing a mixture of protease inhibitors (Boehringer Mannheim). The resultant cell pellets were resuspended in 1 ml of lysis buffer (20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 mM KF, and a mixture of standard protease inhibitors) and then frozen at –80°C. Before use, cell preparations were thawed at 4°C. For the detection of XPG and XPF (human xeroderma pigmentosum group G and F complementing proteins, respectively), cell samples were further sonicated and passed five times through a 27-gauge needle. Cell lysates were then boiled in SDS sample buffer (20% glycerol, 2% SDS, 0.025 mg/ml bromphenol blue, 0.125 M Tris base, and 0.7 M ß-mercaptoethanol). Whole cell lysates were then subjected to SDS-PAGE (8% acrylamide gels) and transferred onto nitrocellulose membranes. After blocking nonspecific sites (5% fat-free milk), the nitrocellulose sheet was probed overnight with an anti-topoisomerase II (2011-1; dilution range, 1:4000 to 1:500; TopoGen), an anti-topoisomerase IIß (BT1002; dilution, 1:5000; Biotrend), an anti-topoisomerase I (2012; dilution, 1:1000; TopoGen), an anti-XPG (SC-8791; dilution, 1:100; Santa Cruz Biotechnology), an anti-XPF (SC-10159; dilution, 1:100; Santa Cruz Biotechnology), or an anti-actin (SC-1616; dilution, 1/200; Santa Cruz Biotechnology) antibody, followed by a 1-h incubation with appropriate secondary antibody conjugated to peroxidase (Jackson Immunoresearch Laboratories) before enhanced chemiluminescence detection (Pierce, Interchim, France). Membranes were scanned using a MolecularImager system (Bio-Rad).

Catalytic Activity of Topoisomerases.
Topoisomerases were extracted from P388 and P388/F11782 cells as follows. Cells removed from the mouse peritoneal cavity were processed as described above and pelleted. The cell pellet was gently resuspended in lysis buffer [20 mM Tris, 1 mM EGTA, 25 mM KCl, 5 mM MgCl₂, 250 mM sucrose, and 0.5% NP40 (pH 7.2)] and incubated for 10 min on ice, followed by a 2-min centri-fugation at 6000 rpm in a microfuge (Eppendorf 5804R; Merck Eurolab). The pellet (containing nuclei) was resuspended in 50 µl of extraction buffer [20 mM Tris, 1 mM EGTA, 2 mM EDTA, 2 mM DTT, and 400 mM NaCl (pH 7.2)] and incubated for 30 min on ice, followed by a 15-min centrifugation at 14,000 rpm in the same microfuge. The supernatant (containing extracted topoisomerases) was removed, and its protein content was determined by the procedure of Bradford (15) before the addition of 50% (v/v) glycerol and storage at –80°C.

The catalytic activity of topoisomerase II-containing crude extracts was evaluated using a decatenation assay. The total reaction volume was 20 µl, containing 50 mM Tris (pH 8.0), 120 mM KCl, 0.5 mM DTT, 0.5 mM ATP, 10 mM MgCl₂, 30 µg/ml BSA, 0.2 µg kinetoplast DNA (TopoGen), the required amount of topoisomerase II-containing crude extract to completely decatenate 0.2 µg of kDNA, and either the test compound or the solvent alone. After incubation at 37°C for 30 min, the reaction was stopped by the addition of 5 µl of a solution maintained at 4°C containing 50 mM EDTA, 50% glycerol, and 0.25 mg/ml bromphenol blue. Samples were separated through a 1% agarose gel. After staining with ethidium bromide, gels were photographed and counted under UV illumination.

The catalytic activity of topoisomerase I-containing crude extracts was evaluated using a relaxation assay. The total reaction volume was 20 µl, containing 10 mM Tris-HCl (pH 7.9), 0.15 M NaCl, 1 mM EDTA, 0.1 mM spermidine, 0.1% BSA, 5% glycerol, 0.2 µg of pBR322 (Invitrogen), the amount of topoisomerase I-containing crude extract required to completely relax 0.2 µg of pBR322, and either the test compound or the solvent alone. After incubation at 37°C for 30 min, the reaction was stopped by the addition of 5 µl of a solution maintained at 4°C containing 50 mM EDTA, 50% glycerol, and 0.25 mg/ml bromphenol blue. Samples were separated and processed as described above.

Sequencing of Topoisomerase II Segments.
Total RNA was isolated from P388 or P388/F11782 cells, collected directly from the peritoneal cavities of tumor-bearing mice, and centrifuged for 5 min at 100 x g at room temperature through 0.32 M sucrose in PBS, using TRIzol reagent (Invitrogen). The reverse transcription was performed in a Genius PCR System (Techne) using a Superscript II one-step reverse transcription-PCR kit (Invitrogen) with oligo(dT)_12–18, according to the manufacturer’s instructions. The entire topoisomerase II cDNA was amplified and sequenced as follows: cDNA obtained from 5 µg of total RNA from P388 or P388/F11782 cells was used to perform a PCR with 1 µM primer pairs, 2 units of Taq DNA polymerase (Invitrogen) 0.2 mM deoxynucleotide triphosphate in PCR buffer [20 mM Tris-HCl (pH 8.4) and 50 mM KCl], and 1.5 mM MgCl₂. PCR was carried out in 50 µl using the Genius PCR System (Techne), with an initial denaturation for 4 min at 94°C, annealing for 1 min at 65°C, and extension for 2 min at 72°C; followed by 34 cycles of denaturation for 30 s at 94°C, annealing for 45 s at 65°C, and extension for 2 min at 72°C; and finished by 10 min at 72°C. To verify that PCR products were of the correct length, 5 µl of PCR products were electrophoresed in TBE buffer (100 mM Tris, 90 mM boric acid, and 1 mM EDTA) in a 1% agarose-0.5% ethidium bromide gel. Sequencing of the PCR products was performed using the GeneAmp PCR System 2400 (Perkin-Elmer) and the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer). Sequencing of the PCR products was performed twice on two different mRNA extraction using primers, with 25 cycles of denaturation for 10 s at 98°C, annealing for 5 s at 50°C, and extension for 4 min at 56°C.

NER Activity Measurement Using Unscheduled DNA Synthesis.
The method of Bootsma et al. (16) was used to monitor unscheduled DNA. Briefly, cells were removed from the peritoneal cavity of tumor-bearing mice and maintained in suspension culture, as described in "Drug Uptake." A cell suspension (3 x 10⁴ cells/ml) was incubated on histological slides at 37°C in 5% CO₂ in air for 18 h. Medium was then removed, and after a 4-h incubation at 37°C with or without a range of concentrations of 10^–6 to 10^–4 M mechlorethamine to damage DNA, cells were incubated in culture medium containing 10 µM 5-fluoro-2'-deoxyuridine and 10 mM hydroxyurea to inhibit replication and then labeled with 10 µCi/ml [³H]thymidine. Cells were washed three times for 20 min with culture medium containing 200 µM nonradiolabeled thymidine and then fixed with methanol-acetic acid (3:1) for 15 min and air dried. Slides were dipped for 10 s into an autoradiographic emulsion (EM-1; Amersham). After a 5-day exposure in the dark at 4°C, slides were developed (Kodak), and the nuclei were counterstained with Mayer’s hemalum solution (Merck). Cells were visualized under a Zeiss Axioplan microscope combined with a KY-F50 camera (JVC) using a x40 objective. For each image, nuclei were detected, and the number of nuclear silver grains, reflecting NER activity, was counted using Histolab software (Microvision).

	RESULTS

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In Vivo Development of Resistance to F11782 Associated with Alterations in Topoisomerase II.
Mice bearing P388 leukemia cells implanted in the peritoneal cavity were treated with a single suboptimal dose (10 mg/kg) of F11782 via the i.p. route. After approximately 7 days, ascites fluid from treated mice was transplanted into new hosts i.p., and the treatment process was repeated. During this period, the level of resistance achieved in the F11782-treated ascitic cells was evaluated by implanting them i.v. and then administering F11782 i.p. at the therapeutic dose of 160 mg/kg to these tumor-bearing mice (Fig. 2) . The chemosensitivity of i.v. implanted P388 cells to 160 mg/kg F11782 was reflected by an ILS of 200% (Fig. 3A) . Successive treatments and transplantations resulted in the progressive development of resistance to F11782, with complete resistance to this dose of 160 mg/kg being achieved in vivo after 8 weeks, as reflected by an ILS of 0% (Fig. 3A) . Furthermore, these F11782-resistant cells (designated P388/F11782 cells) also proved completely resistant to a single dose of 320 mg/kg F11782, which was previously identified in mice bearing P388 cells as the maximal tolerated dose, corresponding to its optimal antitumor activity (ILS = 320%; Ref. 11 ). Under continuous in vivo F11782 selection pressure, this resistance has remained stable (Fig. 3A) , and the in vivo growth rate of this P388/F11782 subline was judged to be similar to that of the parental line.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Diagrammatic representation of the in vivo establishment of the P388/F11782 subline. Parental P388 cells were implanted i.p. into mice on day 0. These mice were then treated with 10 mg/kg F11782, a suboptimal dose of F11782 administered i.p. on day 1. When regrowth was evident, after approximately 1 week, ascitic cells from these F11782-treated mice were reimplanted in vivo, and the mice were again dosed i.p. the following day with 10 mg/kg F11782. The F11782-resistant P388/F11782 subline was thus developed over successive in vivo transplant generations. During this period, the level of resistance achieved in the F11782-treated ascitic cells was evaluated by implanting them i.v. and then administering F11782 i.p. at the therapeutic dose of 160 mg/kg to these tumor-bearing mice. Levels of the topoisomerases were also monitored during this in vivo establishment of F11782 resistance.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Monitoring of the in vivo development of resistance to F11782. A, P388 cells were exposed in vivo to subtherapeutic doses (10 mg/kg) of F11782, as described in "Materials and Methods." The evaluation of response to treatment with F11782 was then carried out over time (in weeks) after the initiation of resistance induction, so as to monitor the development of resistance, as assessed by the percentage of increase in life span. The dose of F11782 used in these evaluations was 160 mg/kg, which resulted in an increase in life span of 200% in mice bearing the parental P388 cells. B, levels of topoisomerase II (•), IIß (), and I () were evaluated in these F11782-treated cells throughout the in vivo establishment of F11782 resistance. Topoisomerase levels in P388/F11782 whole cell lysates were calculated as a percentage of the levels of P388 whole cell lysates, assessed using Western blotting. C, Western blot analyses of the levels of topoisomerases II, IIß, and I protein in P388 and P388/F11782 cells, assessed on weeks 1 and 17 after the start of the in vivo establishment of F11782 resistance.

View larger version (53K): [in this window] [in a new window]   Fig. 4. Northern blot analyses of the levels of topoisomerases II (A), IIß (B), and I (C) mRNA in P388 and P388/F11782 cells.

In Vivo Evaluation of Therapeutic Responses of the F11782-Resistant P388/F11782 Subline to a Series of Antitumor Agents.
The activities of several members of each of the major classes of antitumor agents were investigated in vivo against the stable P388/F11782 subline (after 17 transplant generations). The results, expressed as optimal ILS values obtained at the optimal dose of each compound, are listed in Table 1 . Although the P388/F11782 cells were totally resistant to F11782, they retained some sensitivity to the topoisomerase II poisons etoposide, etopophos, teniposide, and mitoxantrone, with optimal ILS values ranging from 43% to 100%, judged as representative of definite antitumor activity (ILS > 20%) according to National Cancer Institute criteria (14) . A high level of sensitivity of P388/F11782 cells to the three topoisomerase I poisons tested was also observed, with optimal ILS values of 114–186%. P388/F11782 cells also retained sensitivity to TAS-103 (optimal ILS = 43%), the dual inhibitor of topoisomerases I and II. In contrast, P388/F11782 cells showed complete cross-resistance to the catalytic inhibitor of topoisomerase II, merbarone, as reflected by an optimal ILS value of 0%. Unfortunately, the evaluation of the chemosensitivity of this F11782-resistant P388/F11782 subline to dioxopiperazines was not possible because these compounds failed to show any in vivo activity against the parental P388 cell line. Furthermore, P388/F11782 cells remained highly sensitive to the three DNA cross-linking agents evaluated, namely, cisplatin, cyclophosphamide, and BCNU, with optimal ILS values of 200–900%, as well as to the four tubulin-interacting agents (optimal ILS of 100–129%). Some sensitivity was also expressed to both 5-fluorouracil and methotrexate (optimal ILS values of 100% and 43%). Finally, it is notable that the F11782-resistant P388/F11782 subline was also completely cross-resistant to doxorubicin, as reflected by an optimal ILS value of 0%.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Comparative in vivo responses of P388 (A) and P388/F11782 (B) cell lines to F11782. The effects of single i.p. doses of F11782 on the survival [assessed by maximal T/C ratios; T/C = (median survival of drug-treated group/median survival of control group) x 100] of mice bearing either P388 or P388/F11782 cells were compared. The shaded section under each survival curve corresponds to the calculated area under the curve [AreaS and AreaR for sensitive and resistant sublines, respectively; relative resistance (AreaR/AreaS) ratio = 0.0]. Each dose has been evaluated in a total of seven tumor-bearing mice, involving at least three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Comparative in vivo responses of P388 (A) and P388/F11782 (B) cell lines to cisplatin [A1 and B1; relative resistance (RR) AreaR/AreaS ratio = 4.5], etopophos (A2 and B2; RR ratio = 0.6), and doxorubicin (A3 and B3; RR ratio = 0.0). See the Fig. 5 legend.

View larger version (15K): [in this window] [in a new window]   Fig. 7. In vitro uptake of [14C]F11782 by P388 () or P388/F11782 (•) cells, with respect to time.

A K155N Mutation of Topoisomerase II Is Associated with Resistance to F11782.

View larger version (60K): [in this window] [in a new window]   Fig. 8. Electrophoregram tracing of antigens sequence of topoisomerase II cDNA from 530–508 bp from P388 and P388/F11782 cells, showing the homozygous nucleotide 514 CA point mutation in P388/F11782 cDNA leading to a K155N conversion. Arrows, nucleotide 514, which is C only in P388 cDNA and A only in P388/F11782 cDNA.

View larger version (15K): [in this window] [in a new window]   Fig. 9. Nucleotide excision repair activity in P388 () and P388/F11782 () cells, quantitated by measuring unscheduled DNA synthesis. Cells were incubated for 4 h in the absence or presence of 10–6 to 10–4 M mechlorethamine to damage DNA, together with 10 µM 5-fluoro-2'-deoxyuridine and 10 mM hydroxyurea to inhibit DNA replication, and labeled with 10 µCi/ml [3H]thymidine to evaluate nucleotide excision repair activity. Results are expressed as the mean number of silver nuclear grains/cell (±SD). Each experiment was performed at least three times, and 100 cells were analyzed for each experimental point.

View larger version (31K): [in this window] [in a new window]   Fig. 10. Western blot analyses of the levels of XPG (A) and XPF (B) protein in P388 and P388/F11782 cells.

	DISCUSSION

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The present study has shown that P388 cells treated in vivo by repeated subtherapeutic doses of F11782 developed total resistance to F11782 after eight transplant generations. The resultant P388/F11782 subline exhibits a markedly reduced level of topoisomerase II protein and mRNA, which was associated with a decrease of topoisomerase II catalytic activity, yet only minimal modifications in the levels of topoisomerases IIß and I. Furthermore, P388/F11782 cells exhibited a K155N mutation in the topoisomerase II isoform, and topoisomerase II extracted from P388/F11782 cells proved resistant to F11782 while retaining sensitivity to etoposide and ICRF-193. Interestingly, the decreased NER activity in these F11782-resistant cells (10) is now shown more specifically to be associated with a reduced level of the endonuclease XPG. Furthermore, resistance to F11782 does not seem to involve classic multidrug resistance mechanisms, as reflected by unaltered drug uptake and a lack of any overexpression of resistance-related proteins or modification of the glutathione-mediated detoxification process. Indeed, this P388/F11782 subline exhibits an original in vivo cross-resistance profile because it is totally cross-resistant to only 2 of the large series of 19 agents tested, namely, doxorubicin and merbarone, while retaining some sensitivity to topoisomerase poisons and demonstrating moderate or marked collateral sensitivity to 10 other agents including DNA cross-linking compounds, topoisomerase I poisons, one antimetabolite, and several tubulin-interacting agents.

The fact that F11782-resistant cells showed modifications in the level of expression, sequence, catalytic activity, and drug sensitivity of topoisomerase II suggests that, in vivo, this constitutes the major/preferential target of F11782. Indeed, these findings were supported by the recent study of Jensen et al. (7) , which demonstrated that cell lines resistant to bisdioxopiperazines, due to topoisomerase II mutations, were also resistant to F11782. This research group also showed that F11782 displays a dual mechanism of action on human topoisomerase II, reducing its affinity for DNA while also stabilizing the protein bound in the form of an ATP-independent salt-stable complex. Levels of topoisomerase are commonly decreased in topoisomerase poison-resistant cell lines, and these generally show cross-resistance to other topoisomerase poisons (2 , 4 , 5) . The resistance phenotype of non-complex-stabilizing catalytic inhibitors of topoisomerase has more recently been reviewed and revealed certain common features and also differences relative to the complex-stabilizing topoisomerase poisons (2) . For example, whereas a leukemic CEM cell line resistant to the catalytic topoisomerase II inhibitor merbarone showed a decreased level of topoisomerase II and proved cross-resistant to topoisomerase poisons (21) , an increase in topoisomerase II protein level, associated with collateral sensitivity to etoposide, was observed in a CEM subline resistant to ICRF-187, another topoisomerase II catalytic inhibitor (22) . However, although all of the non-complex-stabilizing compounds are commonly termed "catalytic inhibitors," they do not interfere with topoisomerase catalytic activity by the same mechanisms, and thus different resistance mechanisms might well be expected to operate. P388/F11782 cells, which are resistant to F11782, a catalytic inhibitor of topoisomerases, in vivo retain some sensitivity or show only partial cross-resistance to the topoisomerase II poisons etoposide, etopophos, teniposide, and mitoxantrone. This reduced sensitivity to these topoisomerase poisons might be due to decreased complex formation as a consequence of the decreased topoisomerase II level in these F11782-resistant cells. Furthermore, it has been postulated that topoisomerase IIß could be a pharmacological target for etoposide (23) and mitoxantrone (24) . In this case, mitoxantrone, etoposide, and its derivatives, etopophos and teniposide, could retain some activity against the F11782-resistant cells by targeting topoisomerase IIß, which appears little modified in these cells. Furthermore, resistance of P388/F11782 cells was also found to be associated with a K155N mutation in the topoisomerase II isoform, and whereas the catalytic activity of topoisomerase II extracted from F11782-resistant cells was resistant to F11782, it retained sensitivity to etoposide. Indeed, this mutation conferring resistance to F11782 occurred in the ATP binding domain of the topoisomerase II isoform, at sites distinct from those mainly identified as responsible for resistance to complex stabilizing agents, such as etoposide or amsacrine, i.e., around the Walker B or catalytic domains of topoisomerase II (25 , 26) . Therefore, overall, these data suggest that although F11782 is structurally related to etoposide and has topoisomerase II as a common target, its resistance profile and associated mechanisms appear quite different. In contrast, the functional mutations Y49F and Y165S in topoisomerase II, identified in bisdioxopiperazine-resistant small cell lung cancer and Chinese hamster ovary cells, respectively, proved to confer resistance to F11782 (7) . This suggests that F11782 might have an interaction site in a topoisomerase II domain also targeted by the dioxopiperazine compounds. However, whereas the catalytic activity of topoisomerase II extracted from P388/F11782 was resistant to F11782, it retained sensitivity to the dioxopiperazine compound ICRF-193. Therefore, these various mutations seem to induce different alterations of the interaction of dioxopiperazine compounds and F11782 with topoisomerase II. These findings are in agreement with previous studies suggesting that, despite showing some similarities, F11782 and dioxopiperazines appear to function as quite distinctive catalytic inhibitors of topoisomerase II (7 , 27) . One of the most critical differences is that F11782-induced salt-stable complexes were observed even in the absence of a nucleotide triphosphate (7) . Therefore, as suggested by Jensen et al. (7) , F11782 might induce a conformational change resembling the ATP-bound form of the enzyme, which could also partly explain the inhibition of DNA binding by F11782. However, because the level of salt-stable complexes that can be obtained with F11782 is much less than the level of complexes that can be formed by the dioxopiperazines (7) , it can be postulated that F11782 might interact with both free and DNA-bound topoisomerase II, resulting in an inhibition of the binding of topoisomerase II to DNA or to the trapping of the enzyme on DNA. Furthermore, F11782-resistant P388/F11782 cells demonstrated total cross-resistance to only two compounds, merbarone (a catalytic inhibitor of topoisomerase II) and doxorubicin. Alterations of topoisomerase II from P388/F11782 cells (i.e., reduced level and/or mutation of topoisomerase II) might contribute to this resistance. However, in the case of doxorubicin, if its major recognized mode of action involved intercalation into DNA, resulting in impairment of topoisomerase II activity, other mechanisms, including free radical formation, direct membrane effects, or DNA ligase impairment, have also been postulated (28) . Therefore, resistance induction in P388 cells by F11782 might have modified other parameters important for these other aspects of doxorubicin activity. Overall, these data suggest that F11782 interferes with topoisomerase II in a manner different from that of the majority of topoisomerase II-targeting agents. P388/F11782 cells also showed some collateral sensitivity to the topoisomerase I poisons topotecan and irinotecan. Although only minor alterations in topoisomerase I were noted in these cells, NER activity was decreased. Because it has been suggested that NER would be involved in topoisomerase I poison-induced DNA damage repair (5 , 29) , it can be postulated that the decreased NER activity of these P388/F11782 cells contributes to enhanced sensitivity to topoisomerase I poisons.

The increased sensitivity of P388/F11782 cells to certain Vinca alkaloids has not been elucidated. It can only be postulated that F11782 might have induced modifications of certain cellular signals critical for the transformation of Vinca alkaloid-induced microtubule damage into antitumor activity.

Of major interest, P388/F11782 cells were shown to exhibit a decreased level of XPG protein, an endonuclease involved in NER, reflected by a global decrease of NER activity in these F11782-resistant cells. Furthermore, it has been shown that F11782 was a potent inhibitor of NER and, more specifically, of the DNA incision step (10) . Therefore, these results suggest that in vivo, in addition to topoisomerase II, XPG also appears to be a target of F11782. The decreased NER activity in P388/F11782 cells was associated with a marked collateral sensitivity to cisplatin, which is known to induce DNA damage repaired mainly by NER (20) . Furthermore, F11782-resistant P388/F11782 cells also showed collateral sensitivity to two other DNA cross-linking agents, cyclophosphamide and BCNU. Consistent with these results, synergistic antitumor activity had been identified in vivo in the parental P388 model, when single i.p. doses of F11782 were combined with either cisplatin or mitomycin C, another DNA cross-linking agent (30) . Certain components of the NER system have been shown to play a role in cellular sensitivity to DNA cross-linking agents. More specifically, overexpression of XPA or the endonuclease ERCC1 was identified in patient ovarian cancer tissues resistant to cisplatin (31) . On the other hand, cells deficient in ERCC1 or XPF were found to be hypersensitive to cisplatin, cyclophosphamide, melphalan, or mitomycin C (32, 33, 34) . More recently, it has been shown that cellular resistance to ecteinascidin 743, a DNA-damaging agent, resulted in only acquired XPG deficiency, whereas deficiency in any of the other XP proteins led to ecteinascidin 743 resistance (35) . These XPG-deficient cells did not overexpress multidrug resistance transporters and did not show cross-resistance to doxorubicin, Taxol, and etoposide (36) . However, thus far, XPG deficiency has not been associated with resistance to topoisomerase inhibitors, although resistance to topoisomerase inhibitors has already been associated with modifications of cellular response to alkylating agents. For example, increased topoisomerase activities have been shown to contribute to the resistance of both nitrogen mustard- and cisplatin-resistant cells, and similarly, cells with decreased topoisomerase II levels show increased sensitivity to cisplatin, carmustine, mitomycin C, and nitrogen mustard (4) . Therefore, it would be interesting to investigate whether resistance to topoisomerase in such cell lines could be associated with modifications of XPG or other components of the NER system. The XPG endonuclease appears to have multiple functions that are not completely elucidated. It is a phosphoprotein that is hyperphosphorylated on DNA damage (37) . In addition to the endonuclease activity of XPG in NER, XPG has nonenzymatic functions via protein-protein or protein-DNA interactions. Indeed, XPG appears to coordinate the function of several proteins implicated in different DNA repair mechanisms (37) . More specifically, XPG has an essential nonenzymatic role in transcription-coupled repair, a process aimed at removing, by either NER or base excision repair, DNA lesions that blocks transcription (38) . Additional research studies are therefore expected to advance our understanding of the multiple functions of XPG and its involvement in response to anticancer treatment.

As discussed above, previous studies have suggested a causal relationship between topoisomerase II expression and resistance/sensitivity to DNA cross-linking agents (4) . Indeed, it is considered that DNA topoisomerase might influence the formation of interstrand cross-links through regulation of DNA structure. Cells with lower topoisomerase activities are likely to have a higher degree of superhelical density, and it has been shown that bifunctional cisplatin lesions are formed at a higher rate on supercoiled plasmids than on relaxed ones (39) . Therefore, the markedly decreased level of topoisomerase II in P388/F11782 cells might also contribute, at least in part, to the enhanced sensitivity of these cells to DNA cross-linking agents.

Furthermore, recent studies tend to suggest that certain DNA endonucleases and topoisomerases may have structural homology and might evolve from a few common ancestors (40) . Interestingly, comparison of the amino acid sequences between various topoisomerases and human or murine XPG endonuclease showed that the K155N mutation identified in the P388/F11782 cells occurred in a highly conserved domain of these enzymes (Fig. 11) . In addition, site-directed mutagenesis studies have revealed that this domain is critical for XPG functions (41) . Therefore, these findings support our data indicating that topoisomerase II and the endonuclease XPG are two major targets of F11782, as initially identified in vitro (6 , 10) and subsequently shown here in vivo.

View larger version (24K): [in this window] [in a new window]   Fig. 11. Comparison of amino acid sequences between murine topoisomerase II (MM TopoII), human topoisomerase II (HS TopII), human topoisomerase IIß (HS TopIIß), Drosophilia melanogaster topoisomerase II (DM TopoII), Saccharomyces pombe topoisomerase II (SP TopoII), S. cerevisiae topoisomerase II (SC TopoII), murine XPG endonuclease (MM XPG), and human XPG endonuclease (HS XPG). Sequence alignment with hierarchical clustering was performed using the Mutalin software available on the web site of Pole Bio-informatique Lyonnais (http://pbil.univ-lyon1.fr). The K155N conversion found in P388/F11782 topoisomerase II cDNA is indicated (arrow).

In conclusion, this study has shown that P388/F11782 cells selected in vivo for resistance to F11782 either showed no cross-resistance or proved only partially cross-resistant to other topoisomerase-targeted agents while exhibiting collateral sensitivity to a range of other antitumor agents, including DNA cross-linking agents, topoisomerase I poisons, antimetabolites, and tubulin-interacting agents. Furthermore, alterations of topoisomerase II and XPG in these F11782-resistant cells suggest that these two enzymes are major targets of F11782 in vivo. Therefore, these findings further demonstrate the original mechanism of action of this novel dual catalytic inhibitor of topoisomerases with DNA repair-inhibitory properties. These data also suggest that resistance to F11782 might be overcome by cotreatment with several compounds from different pharmacological classes and provide useful background information vis-à-vis the potential incorporation of F11782 into combination chemotherapy regimens.

	ACKNOWLEDGMENTS

 
We thank Eric Chazottes, Caroline Dejean, and Christel Ricome for skilled technical assistance.

	FOOTNOTES

 
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.

Requests for reprints: Anna Kruczynski, Division of Experimen-tal Cancer Research, Pierre Fabre Research Center, 17 Avenue Jean Moulin, 81106 Castres, Cedex 06, France. Phone: 33-563714266/33-563714211; Fax: 33-563714299; E-mail: anna.kruczynski{at}pierre-fabre.com

Received 10/29/02; revised 12/30/03; accepted 1/29/04.

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