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【关键词】 Vivo
The bisdioxopiperazines such as (+)-(S)-4,4'-propylenedi-2,6-piperazinedione (dexrazoxane; ICRF-187), 1,2-bis(3,5-dioxopiperazin-1-yl)ethane (ICRF-154), and 4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione (ICRF-193) are agents that inhibit eukaryotic topoisomerase II, whereas their ring-opened hydrolysis products are strong iron chelator. The clinically approved analog ICRF-187 is a pharmacological modulator of topoisomerase II poisons such as etoposide in preclinical animal models. ICRF-187 is also used to protect against anthracycline-induced cardiomyopathy and has recently been approved as an antidote for alleviating tissue damage and necrosis after accidental anthracycline extravasation. This dual modality of bisdioxopiperazines, including ICRF-187, raises the question of whether their pharmacological in vivo effects are mediated through interaction with topoisomerase II or via their intracellular iron chelating activity. In an attempt to distinguish between these possibilities, we here present a transgenic mouse model aimed at identifying the contribution of topoisomerase II to the effects of bisdioxopiperazines. A tyrosine 165 to serine mutation (Y165S) in topoisomerase II, demonstrated previously to render the human ortholog of this enzyme highly resistant toward bisdioxopiperazines, was introduced at the TOP2A locus in mouse embryonic stem cells by targeted homologous recombination. These cells were used for the generation of transgenic TOP2AY165S/+ mice, which were demonstrated to be resistant toward the general toxicity of both ICRF-187 and ICRF-193. Hematological measurements indicate that this is most likely caused by a decreased ability of these agents to induce myelosuppression in TOP2AY165S/+ mice, highlighting the role of topoisomerase II in this process. The biological and pharmacological implications of these findings are discussed, and areas for further investigations are proposed.
The nuclear enzyme topoisomerase II is found in all living organisms and possesses the essential activity of cleaving and rejoining DNA by forming a transient protein concealed double-strand break, through which an intact DNA double-strand helix is transported by a gating mechanism requiring ATP hydrolysis (Roca and Wang, 1994; Baird et al., 2001). This activity assists in a number of DNA metabolic processes such as DNA replication, transcription, chromosome condensation and decondensation, and chromosome segregation (Wang, 2002). Higher vertebrates have two isoforms of this enzyme, and β. The topoisomerase II isoform is nuclear, and its expression is highly cell cycle-dependent, peaking at G2M (Falck et al., 1999), whereas topoisomerase IIβ is located both in the nucleus and cytoplasm and displays a rather constant level during the cell cycle (Austin and Marsh, 1998). Topoisomerase II is essential for cell proliferation (Akimitsu et al., 2003), whereas topoisomerase II β knockout mice die shortly after birth because of developmental defects in the central nervous system (CNS) (Yang et al., 2000).
The bisdioxopiperazines such as ICRF-193, ICRF-154, and ICRF-187 (dexrazoxane) are small-molecule catalytic inhibitors of eukaryote topoisomerase II. These agents target an N-terminal ATP-operated clamp in topoisomerase II, thereby trapping topoisomerase II as a closed clamp on DNA (Roca et al., 1994; Morris et al., 2000). This closed-clamp configuration of topoisomerase II on DNA has been found to cause cytotoxicity (van Hille and Hill, 1998; Jensen et al., 2000a) and to inhibit transcription in mammalian cells (Xiao et al., 2003). The bisdioxopiperazines have also been demonstrated to enhance the levels of DNA breaks in vitro and in cells, although this remains controversial, because this effect depends on the experimental conditions (Huang et al., 2001; Hajji et al., 2003).
Besides having some anticancer activity (Bakowski et al., 1979; Von Hoff et al., 1981), the bisdioxopiperazines (especially ICRF-187) are effective as pharmacological modulators of topoisomerase II poisons such as etoposide and teniposide in preclinical animal models. ICRF-187 can antagonize the effect of these drugs outside the CNS (Hofland et al., 2005b), thereby allowing for a more effective targeting of CNS tumors (Holm et al., 1998). Furthermore, ICRF-187 [dexrazoxane (Savene)] has been found to be effective as an antidote against accidental anthracycline extravasation (Langer et al., 2000; Mouridsen et al., 2007). Finally, ICRF-187 (Cardioxane, Zinecard) is also used as a cardioprotectant in anthracycline treatment (Cvetkovi and Scott, 2005).
We have characterized previously a tyrosine 165 to serine mutation (Y165S) in human topoisomerase II, originally identified in a highly bisdioxopiperazine-resistant human small cell lung cancer cell line that was heterozygous for this mutation (Wessel et al., 2002). This cell line actively transcribed both the Y165S and wt topoisomerase II, suggesting a dominant phenotype of this resistance-conferring mutation, which was further confirmed by functional coexpression of Y165S and wt topoisomerase II proteins in yeast. In addition, recombinant Y165S topoisomerase II was found not to bind radioactively labeled ICRF-187 (dexrazoxane) in vitro (Renodon-Cornière et al., 2003). Finally, crystallographic data obtained with yeast topoisomerase II show that the homologous residue in yeast topoisomerase II Tyr144 contributes directly to ICRF-187 binding by forming an ATPase lid interacting extensively with the dioxopiperazine rings of ICRF-187 (Classen et al., 2003).
Although the bisdioxopiperazines are known to kill cells by interacting with topoisomerase II, these agents are also strong iron chelators, and the role of this activity versus topoisomerase II inhibition has never been directly addressed in a higher vertebrate. One way of addressing this question is to engineer a transgenic mouse having altered topoisomerase II with reduced interaction with bisdioxopiperazines. Because of the strong phenotype of the topoisomerase II Y165S mutation, we selected this alteration in topoisomerase II to be engineered into the TOP2A locus in embryonic stem cells by homologous recombination (gene targeting) followed by blastocyst injection, generation of chimeric germ line transmitters, and back-crossing. Using this setup, we were able to develop heterozygous TOP2AY165S/+ mice carrying this dominant bisdioxopiperazine resistance conferring mutation at the correct genomic loci. We here demonstrate for the first time in the mouse that interaction with topoisomerase II is involved in mediating the effects of bisdioxopiperazines on general and hematological toxicity.
Drugs and Reagents. ICRF-187 [dexrazoxane (Cardioxane)] was obtained from Chiron Corporation (Amsterdam, The Netherlands); ICRF-193 was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA) or synthesized. Daunorubicin (Cerubidin) was from Aventis Birkerod (Hørsholm, Denmark). Etoposide and cisplatin was from Bristol-Myers Squibb Co. (Lyngby, Denmark). Restriction endonucleases were from New England Biolabs (Medinova Scientific A/S, Glostrup, Denmark), and [32P]dATP was from GE Healthcare (Hillerød, Denmark). All other reagents were from Sigma-Aldrich (Vallensbaek Strand, Denmark) unless otherwise indicated.
Construction of Gene Targeting Vector. A cDNA probe covering amino acids 131 to 325 of the mouse topoisomerase II coding sequence was amplified and cloned by PCR from a mouse testis cDNA library (Stratagene, AH Diagnostics, Århus, Denmark) by using the following oligonucleotide pairs: 5'-GAG CCA AAA ATG TCT TGT ATT AG-3' and 5'-GAG ATG TCT GCC GCC CTT AGA AG-3'. The resulting PCR fragment was labeled with 32P and used as probe for screening a mouse 129/SvEvTACCfBr spleen BAC library (Invitrogen A/S, Taastrup, Denmark), and 10 positive clones were obtained. From one of the isolated clones (BAC clone 182-D4), a 10.8-kb SacI-BamHI fragment (genomic position 2111–12888) containing exons 4 to 12 was subcloned into a pUC19 vector in which the HindIII site of the poly-linker had been inactivated previously. This construct is now referred to as the unmodified targeting vector. A 735-bp fragment containing SwaI-HindIII fragment of exon 6 was PCR-amplified using the oligonucleotides 5'-CTC TAT TTA AAT ATC GAT TCC TAG GTC ATT ACC AAG GCT TTT AGA ATG-3' and 5'-TCTTTAGTGGAGCCAGCGGT-3', in which the forward oligonucleotide contained an extra AvrII restriction site (underlined). The PCR product was cloned into the TOPO2.1 vector (Invitrogen A/S), and the Y165S mutation and a silent ScaI site were introduced by site-directed mutagenesis (Stratagene) by using the mutagenic oligonucleotide 5'-GGT CGA AAT GGC TCT GGA GCT AAA CTG TGT AAC ATA TTC AGT ACT AAA TTT ACT G-3' and its complementary oligonucleotide (Fig. 1E). The mutated DNA fragment was then reintroduced into the unmodified targeting vector as a SwaI-HindIII fragment. A self-excision neomycin-resistance marker (neoR) controlled by the phosphoglycerate kinase promoter was PCR-amplified from pPGK neo bpA (Addgene Inc., Cambridge, MA) vector using the oligonucleotides 5'-CTC TCT ATT TAA ATA TAA CTT CGT ATA GCA TAC ATT ATA CGA AGT TAT TGA TGC TCG TCA GGG GG-3' and 5'-CTC TCC TAG GAT AAC TTC GTA TAA TGT ATG CTA TAC GAA GTT ATT CCA CCG CGG TGG CGG-3' that both contained a loxP site (in boldface type) and SwaI and AvrII restriction sites in the forward and reverse primers, respectively (underlined). The neoR cassette was introduced into the modified targeting vector as a SwaI-AvrII fragment. Finally a diphtheria toxin resistance marker was linked to the 3'-end of the topoisomerase II sequence. The diphtheria toxin marker was PCR-amplified using the oligonucleotides 5'-TTT TGA GCT CGG ATC CAC CGC TCT AGA ACT ACG ATC C-3 and 5'-TTT TGA GCT CCG AAC AAC TCC GCC GCG-3' and ligated into the targeting vector using the SacI sites that were introduced in the oligonucleotides (underlined). All PCR-amplified DNA fragments were sequenced before being introduced into the targeting vector.
Fig. 1. Targeting strategy for the Y165S knock in into the TOP2A gene. A, genomic organization of the topoisomerase II genomic locus in the mouse. B, design of the targeting vector used for mutagenesis of the TOP2A gene. C, graphic presentation of the targeted TOP2A allele after correct homologous integration of targeting vector DNA. D, structure of the targeted TOP2A allele after removal of the neomycin resistance cassette by Cre-lox recombination. E, nucleotide changes introduced into the coding sequence of topoisomerase II at the targeted TOP2A allele upon homologous recombination. F, Southern blot of genomic DNA isolated from TOP2AneoR/+ (lane H) and wt embryonic stem cells (lane W) hybridizing to the 5'-probe. G, Southern blot of genomic DNA isolated from TOP2AneoR/+ (lane H) and wt embryonic stem cells (lane W) hybridizing to the 3'-probe. H: topoisomerase II levels in crude extracts isolated from TOP2A+/+, TOP2AneoR/+, and TOP2AY165S/+ embryonic stem cells. I, topoisomerase IIβ levels in crude extracts isolated from TOP2A+/+, TOP2AneoR/+, and TOP2AY165S/+ embryonic stem cells. J, actin levels in crude extracts isolated from TOP2A+/+, TOP2AneoR/+, and TOP2AY165S/+ embryonic stem cells.
Gene Targeting by Homologous Recombination. Murine CJ7 embryonic stem cells (Swiatek and Gridley, 1993) (Fig. 1A) were electroporated with BsaBI-linearized targeting vector (Fig. 1B) and screened for resistance toward G418 at a concentration of 300 µg/ml. DNA was isolated and digested with AvrII according to Ramírez-Solis et al. (1993) and subsequently analyzed by Southern blotting using a 32P-labeled 5'-end probe that binds at the genomic position 1768 to 2044 bp (Fig. 1F). Three clones that showed correct integration at the 5'-end were further confirmed with a probe that binds the 3'-end of the integration at genomic position 13240 to 13646 (Fig. 1C). Finally, the phosphotransferase expression cassette was excised from the TOP2A loci in correctly targeted ES clones (Fig. 1D) by transient expression of Cre recombinase, and correct excision was confirmed by PCR using the following primers: 5'-ATG AGC AAA GTC GTG TTC CC-3' and 5'-CTT TGC TGT CCA TCC AGG TT-3'. This PCR reaction amplifies the region that contains the single loxP site, which remains after excision of the neomycin resistance cassette. Consequently, in this PCR reaction, the wild-type allele produces a 292-bp product, whereas the mutated allele produces a 326-bp product.
Generation of Transgenic Knock-In Mice. Three positive ES cell clones were individually injected into B6D2F2 blastocysts, which were then introduced into pseudopregnant NMRI female mice that gave birth to chimeric animals. Male chimeras were mated with C57BL/6 female mice, and germ line transmission was obtained from two independent ES cell clones. Heterozygous mutant mice were identified by PCR using the primer pair described above. In initial crossings between heterozygous TOP2AY165S/+ male and female 129B6F1 animals, no homozygous TOP2AY165S/Y165S animals could be obtained. TOP2AY165S/+ and TOP2A+/+ animals appeared at the ratio of 2:1 as expected in case of the TOP2AY165S/Y165S allele being embryonic lethal. As a consequence, in our present studies, all in vivo and ex vivo experiments were performed using heterozygous TOP2AY165S/+ female B6129S hybrids obtained by crossing heterozygous TOP2AY165S/+ 129B6 male mice with wild-type C57BL/6 female mice.
Western Blot Analysis. Twenty micrograms of protein obtained from whole-cell extracts were denatured and reduced by boiling in SDS sample buffer containing dithiothreitol, separated by SDS-polyacrylamide gel electrophoresis in NuPAGE 4–12% Bis-Tris Gels (Invitrogen A/S), and transferred to nitrocellulose membranes (Invitrogen A/S) according to standard procedures. The following antibodies were used: Rabbit polyclonal antiactin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit polyclonal antitopoisomerase II and IIβ (Biotrend, Copenhagen, Denmark). Detection was performed by using a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare), and an ECL plus Western Blotting Detection system (GE Healthcare).
Decatenation Assay. Topoisomerase II catalytic activity was measured by kinetoplast DNA (kDNA) decatenation by using 3H-labeled kDNA isolated from Crithidia fasciculata (American Type Culture Collection, Manassas VA) as described previously (Wessel et al., 1999), by using a 96-well format filter-based assay (Jensen et al., 2002).
Embryonic Stem Cells Viability Assay. The sensitivity of TOP2A+/+, TOP2AneoR/+, and TOP2AY165S/+ embryonic stem after exposure toward increasing concentrations of ICRF-193 or etoposide was assessed as described previously (Kobayashi et al., 2001).
Ex Vivo Granulocyte-Macrophage Colony-Forming Unit Assay. Mice were sacrificed with 70% CO2/O2, and bone marrow cells were obtained by flushing the femoral bone cavity with Iscove's modified Dulbecco's medium with 10% fetal bovine serum at 37°C. Cells were then cooled at 1°C per minute until -180°C in a freezing solution containing 7.5% dimethyl sulfoxide. All cells were kept under liquid nitrogen and were prepared identically for use; after washing twice in 37°C medium, they were left for 1 h to rest at 37°C and then stained with 0.1% nigrosin to test for cell viability. Clonogenic CFU-GM assays were carried out as described previously (Hofland et al., 2005b).
Animals and Housing. Experiments were conducted according to institutional and national guidelines for the care and use of laboratory animals and were approved by the Danish Experimental Animal Inspectorate, Ministry of Justice. Female B6129SF1 TOP2AY165S/+ and TOP2A+/+ mice, 10 to 16 weeks old and weighing 20 to 28 g, were obtained from contract breeding at Taconic (Ry, Denmark). The mice were allowed to become acclimatized for at least 1 week before being included into experiments. They were kept in standard type III plastic cages (Scanbur B-K, Koege, Denmark) on wood-chip bedding with nest material, wooden chewing blocks, and plastic houses for environmental enrichment (Brogaarden, Gentofte, Denmark). They were kept in a 12-h light/dark cycle with ad libitum access to tap water and standard laboratory diet (Altromin; Brogaarden). Treatments were given during the light-cycle period.
Treatment of Mice with ICRF-187 and ICRF-193. Female B6129SF1 TOP2AY165S/+ and TOP2A+/+ animals were used in these experiments. Because in pilot experiments single-dose administration of the bisdioxopiperazines ICRF-193 and ICRF-187 proved insufficient to induce toxicity, these agents were administered for 3 consecutive days by the intraperitoneal route (days 0, 1, and 2) in a volume of 0.2 ml of isotonic saline for ICRF-187 or in 0.05 ml of dimethyl sulfoxide for ICRF-193. In general toxicity experiments (survival studies), the following daily doses of ICRF-187 were given: 50, 125, 250, 350, 450, 500, and 550 mg/kg body weight. For hematological studies, three daily doses of 250 or 450 mg/kg ICRF-187 were administered. This represents a nonlethal and a lethal dose, respectively. In toxicity experiments with ICRF-193, three daily doses of 10, 20, 25, and 30 mg/kg were administered in general toxicity studies. For hematological measurements 10, 20, and 30 mg/kg ICRF-193 was used, representing one partially lethal dose and two lethal doses (in wt animals).
Histopathology. Groups of TOP2A+/+ and TOP2AY165S/+ male and female mice were treated with a single daily dose of 550 mg/kg dexrazoxane or 30 mg/kg ICRF-193 i.p. for 3 consecutive days. The mice were sacrificed the day after last treatment by cervical dislocation, and 1-cm samples were excised in the same anatomical location in each animal from duodenum (right after the stomach), mid of jejunum, and ileum (right before colon). The small intestinal samples were immersed in phosphate-buffered 4% formaldehyde for 3 days and were then mounted in paraffin, and 5-µm longitudinal sections were cut and stained with hematoxylin and eosin stain.
Hematological Toxicity. Mice were sacrificed on day 3, and the right femur bone was dissected free from muscle and excised immediately after sacrifice. The ends of the femur bone were clipped off to expose the bone marrow cavity. The marrow cells were flushed out with phosphate-buffered saline in a syringe with a 27-gauge needle using four turns of flushing with the same fluid (fluid volume, 1 ml), two flushes from each end to ensure good recovery and dispersion of the bone marrow cells. After flushing, the emptied femur bone was weighed, and the total bone marrow cell counts normalized to the femur bone weight. The cell count of the marrow flush fluid was measured using the CA530VET hematology analyzer set up for mouse sample analysis (Boule Medonic, Stockholm, Sweden). This device uses electrical impedance for sizing and counting cells in fluid (blood), and counts obtained for cells of a size corresponding to peripheral white blood cells express the number of bone marrow stem cells recovered from the femur. Counts obtained with the hematology analyzer were found to correlate well with bone marrow cell counts performed manually in a Fuchs-Rosenthal counting chamber (r2 = 0.68, n = 15, p < 0.001; data not shown).
Peripheral blood counts [white blood cells (WBCs), red blood cells (RBCs) and platelets (PLTs)] were also determined using the CA530VET hematology analyzer. Before sampling, mice were placed for approximately 2 min in a Plexiglas cage under a heating lamp to produce vasodilation and facilitate bleeding. The tail vein was then punctured using a 27-gauge needle, and a blood drop was collected into a 20-µl K2EDTA-coated capillary tube.
In Vivo Toxicity Using Humane Endpoints. Mice were also allocated to survival studies after treatments, and the survival endpoint was defined as the number of days until the mice showed signs of toxicity according to humane criteria defined below. The mice were observed twice daily, weighed two or three times weekly, and assigned an activity score from 1 to 5 (Hofland et al., 2005a). To receive a score of 5, the mouse exhibits a normal active and curious behavior. It moves about and stands upright at the sides of the cage. For a score of 4, the mouse is not quite as active. It does not stand up as often and prefers to stay in the corners of the cage. For a score of 3, the mouse is less active, and when it moves, it often stops and sits. It stays in the nest corner. For a score of 2, the mouse moves only when touched, and only for a short distance. It preferably hides in the nest corner. Finally, for a score of 1, the mouse is moribund. Activity scores of 5 or 4 were accepted. Mice having an activity score of 3 were monitored closely, and if activity decreased below a score of 3, they were sacrificed. Mice that suffered a weight loss of more than 20%, regardless of an otherwise acceptable activity score, were euthanized. In these experiments, the number of mice in each group ranged from six to seven.
Statistical Analysis and Graphical Presentation. The graphs were produced using the Prism 4 software (GraphPad Software Inc., San Diego, CA). Statistical analysis was performed using the software SAS 9.1 (SAS Institute, Cary, NC). Hematology values and bone marrow counts were analyzed using analysis of variance and t tests on preplanned comparisons or when the residuals from analysis of variance were not normally distributed using nonparametric Wilcoxon test. Survival data were analyzed using log-rank statistics. The level of significance was set to a probability of p = 0.05 or below.
Introduction of a Heterozygous Y165S Mutation at the TOP2A Loci in Embryonic Stem Cells. To investigate the function of the topoisomerase II Y165S mutation in vivo, the mutation was introduced into mouse CJ7 embryonic stem cells by targeted homologous integration. The murine TOP2A gene is located on chromosome 11 and consists of 34 exons encompassing 30.1 kb of DNA (Fig. 1A). A targeting construct was designed to replace the tyrosine codon 165 with a serine (Y165S) by homologous recombination (Fig. 1, A and B). This construct contains a 10.4-kb stretch of the murine TOP2A gene spanning exons 4 to 12. Here, the Y165S mutation is located in codon 6 (Fig. 1B). After being linearized, this construct was introduced in mouse embryonic CJ7 stem cells as described under Materials and Methods. Correctly targeted ES cells were selected in medium containing G418 (Fig. 1C).
Identification of Cell Clones with Correctly Integrated Homologous Recombination. Figure 1F demonstrates the presence of the correct prognostic 7.7-kbp fragment hybridizing to the 3'-probe (lane H), whereas Fig. 1G demonstrates the presence of the correct prognostic 11-kbp fragment hybridizing to the 3'-probe (lane H). In both F and G, the expected 16-kbp fragment prognostic for the untargeted TOP2A+/+ allele was also detected (lane W), demonstrating that only one allele has been altered by homologous recombination in these cells. Cells showing correct homologous integration were finally transfected with a Cre recombinase-expressing plasmid to excise the neomycin marker (Fig. 1D). Correct removal of the neoR cassette was finally assessed by PCR (data not shown).
Fig. 2. Results of survival assays and decatenation experiments performed with TOP2A+/+, TOP2AneoR/+, and TOP2AY165S/+ embryonic stem cells and crude extracts of these. A, inhibition of topoisomerase II catalytic activity in crude extracts from TOP2A+/+ embryonic stem cells by increasing concentrations ICRF-187 and ICRF-193. B, inhibition of topoisomerase II catalytic activity in crude extracts from TOP2AneoR/+ embryonic stem cells by increasing concentrations ICRF-187 and ICRF-193. C, lack of inhibition of topoisomerase II catalytic activity in crude extracts from TOP2AY165S/+ embryonic stem cells by increasing concentrations ICRF-187 and ICRF-193. D, ES cell survival assays of TOP2A+/+, TOP2AneoR/+, and TOP2AY165S/+ embryonic stem cells toward ICRF-193. E, ES cell survival assay of TOP2A+/+, TOP2AneoR/+, and TOP2AY165S/+ embryonic stem cells toward etoposide. In A to C, error bars represents S.E.M. of three experiments. In D and E, error bars represents S.E.M. of 3 to 10 experiments.
TOP2A+/+ and TOP2AY165S/+ Embryonic Stem Cells Have Comparable Expression Levels of Topoisomerase II. The levels of topoisomerase II and IIβ expressed in normal TOP2A+/+ CJ7 ES cell, in TOP2AneoR/+ (before removal of neomycin resistance marker) cells, and in TOP2AY165S/+ cells were determined by Western blotting using actin levels as loading controls. The results of these experiments are depicted in Fig. 1, H, I, and J. It is evident that comparable expression levels of topoisomerase II is detected in TOP2A+/+ and TOP2AY165S/+ cells (compare in Fig. 1H). This result suggests that the Y165S mutant topoisomerase II protein is expressed at normal levels in TOP2AY165S/+ cells and that Y165S and wt topoisomerase II protein have similar stabilities when expressed from the TOP2A locus in CJ7 ES cells. In contrast, the level of topoisomerase II was reduced to approximately 50% in TOP2AneoR/+ cells (Fig. 1H). Apparently, the neoR allele represents a TOP2A null allele. The levels of topoisomerase IIβ were similar in the three cell lines as expected.
The Topoisomerase II Catalytic Activity of Crude Extract Isolated from TOP2AY165S/+ Embryonic Stem Cells Is Resistant to Inhibition by Bisdioxopiperazines. To functionally assess whether the heterozygous TOP2AY165S/+ allele affected the sensitivity of topoisomerase II toward bisdioxopiperazines in CJ7 ES cells, we isolated crude extract from three different cell lines TOP2A+/+, TOP2AneoR/+, and TOP2AY165S/+. In this assay unprocessed kDNA network is retained on a filter, whereas processed kDNA minicircles pass through when washed (Jensen et al., 2002). Because the kDNA is radioactively labeled, the amount of radioactivity on the filter represents the degree of inhibition of topoisomerase II catalytic activity. The results of these experiments are depicted in Fig. 2, A to C. It is evident that the decatenation activity of extract from TOP2A+/+ (Fig. 2A) and TOP2AneoR/+ (Fig. 2B) cells is sensitive to inhibition by both ICRF-187 and ICRF-193. In contrast, the decatenation activity of crude extract from TOP2AY165S/+ cells is highly resistant toward inhibition by both of these bisdioxopiperazines (Fig. 2C). When another catalytic topoisomerase II inhibitor, aclarubicin, was used, there was no difference between these three cell lines (data not shown). This result confirms that the Y165S mutation is dominant and specific with regard to bisdioxopiperazine resistance as demonstrated previously in OC-NYH small cell lung cancer cells (Wessel et al., 2002). This result also points to the notion that the Y165S protein is not expressed in TOP2AneoR/+ cells or that it is expressed at very low levels.
Fig. 3. Clonogenic sensitivity of bone marrow stem cells isolated from TOP2A+/+ and TOP2AY165S/+ mice toward different drugs as assessed in CFU-GM assay. A, ICRF-187; B, ICRF-193; C, etoposide; D, cisplatin; E, camptothecin; F, daunorubicin. Error bars represent S.E.M. of two determinations.
The Sensitivity of TOP2AY165S/+ Embryonic Stem Cells toward ICRF-193 Is Greatly Reduced. We next turned toward assessing whether these decatenation results could be translated into cellular sensitivity toward bisdioxopiperazines. We exposed TOP2A+/+, TOP2AneoR/+, and TOP2AY165S/+ cells toward increasing concentrations of the bisdioxopiperazine compound ICRF-193 (Fig. 2D) and the unrelated topoisomerase II targeting compound etoposide (Fig. 2E) and assessed cell survival. TOP2AY165S/+ cells were highly resistant toward ICRF-193 compared with TOP2A+/+ cells, thus confirming the decatenation results and attesting to the fact that the Y165S mutation is dominant with regard to bisdioxopiperazine resistance (Fig. 2D). The TOP2AneoR/+ cells were also less sensitive than TOP2A+/+cells. This result can be explained by the fact that TOP2A+/neoR cells contain lower levels of topoisomerase II than TOP2A+/ + cells. A similar correlation between topoisomerase II content and bisdioxopiperazine sensitivity has been reported previously for mouse embryonic stem cells (Kobayashi et al., 2001). In contrast, TOP2AY165S/+ cells were only moderately resistant toward the topoisomerase II poison etoposide that interacts with topoisomerase II in a different way (Fig. 2E). Together, these results establish that the Y165S mutation is dominant and specific for bisdioxopiperazine resistance. Finally, the observation that TOP2AneoR/+ cells are slightly resistant to etoposide is also in line with their 50% reduction of topoisomerase II levels (Kobayashi et al., 2001). In our hands, the CJ7 embryonic stem cells were generally quite insensitive toward ICRF-193. Although the exact reason for this is not known, the ICRF-193-resistant phenotype of the TOP2AY165S/+ cells is still evident.
Bone Marrow Stem Cells Isolated from Heterozygous TOP2AY165S/+ B6129SF1 Hybrids are Highly Resistant toward Bisdioxopiperazines. Ex vivo experiments were performed with bone marrow cells isolated from the femur of TOP2AY165S/+ and TOP2A+/+ female B6129SF1 hybrids. CFU-GM clonogenic assays were carried out as described under Materials and Methods. The results of these experiments are depicted in Fig. 3, A to F. From Fig. 3, it is evident that bone marrow stem cells from TOP2AY165S/+ female B6129SF1 hybrids are highly resistant toward the bisdioxopiperazines ICRF-187 (Fig. 3A) and ICRF-193 (Fig. 3B) compared with similar TOP2A+/+ cells. In contrast, TOP2AY165S/+- and TOP2A+/+-derived cells showed similar sensitivity toward etoposide (Fig. 3C), cisplatin (Fig. 3D), camptothecin (Fig. 3E), and daunorubicin (Fig. 3F).
Fig. 4. Kaplan-Meyer survival plots of TOP2A+/+ and TOP2AY165S/+ mice after treatment with different dose levels of ICRF-187 given daily on days 0, 1, and 2 of the study. A, 350 mg/kg ICRF-187; B, 450 mg/kg ICRF-187; C, 500 mg/kg ICRF-187; and D, 550 mg/kg ICRF-187. E, Kaplan-Meyer survival plots of pooled data from A to D. In A to D, the number of animals in each group ranged from six to seven. E, the number of mice in each group was 26. p Values from log-rank analysis are shown on the figure for each dose level.
TOP2AY165S/+ Mice Are Resistant to the General Toxicity of Bisdioxopiperazines. The bisdioxopiperazines including ICRF-187 (dexrazoxane) are known to target eukaryotic topoisomerase II in vitro and in cells, and the latter has been directly associated with cytotoxicity, because cell lines containing mutations in topoisomerase II isoform (Y50F, R162Q, and Y165S) have shown high-level resistance toward these compounds (Sehested et al., 1998; Wessel et al., 1999, 2002). However, little is known about the role of this interaction for the mechanism of action of bisdioxopiperazines in mammalians. To study this, we exposed TOP2AY165S/+ and TOP2A+/+ mice to high doses of ICRF-187 given i.p. using groups of mice ranging from six to seven in each experiment, and we looked at the survival in accordance with the humane endpoint evaluation criteria outlined under Materials and Methods. The doses were given on days 0, 1, and 2. In these experiments, we used daily ICRF-187 doses ranging from 50 to 550 mg/kg. Doses of less than 350 mg/kg body weight did not cause any toxicity and are not discussed further. Daily doses of 350, 450, 500, and 550 mg/kg ICRF-187 were effective in exerting toxicity, and the outcome of these experiments is depicted in Fig. 4. At the two lowest dose levels, there was little effect of topoisomerase II, status because many mice survived treatment and were censored (Fig. 4, A and B). At the 500 and 550 mg/kg dose levels, borderline significance was reached because TOP2AY165S/+ mice tended to survive longer with p values of 0.054 and 0.076, respectively. However, when data presented in Fig. 4, A to D, were pooled to obtain larger group sizes, the TOP2AY165S/+ allele was found to be a significant determinant for ICRF-187 resistance (p = 0.019, n = 27 in each group; Fig. 4E). This result demonstrates that TOP2AY165S/+ mice are resistant to the general toxicity of ICRF-187.
We also performed survival studies with the bisdioxopiperazine analog ICRF-193 that is approximately 10 times more potent against topoisomerase II than ICRF-187 (Hasinoff et al., 1995). In these experiments, doses of 10, 20, and 30 mg/kg/day were used. TOP2AY165S/+ mice were highly resistant toward the toxicity of ICRF-193 compared with normal mice at two different dose levels, namely 20 and 30 mg/kg/day for 3 days (Fig. 5, B and C; p = 0.009). At the lowest dose level, 10 mg/kg, there was a tendency of the TOP2AY165S/+ mice to have a better survival than wild-type mice (Fig. 5 A, p = 0.14). When these results (A–C) were pooled, TOP2AY165S/+ mice were resistant to ICRF-193 toxicity (p < 0.0001, n = 18 in each group; Fig. 5D). This result shows that TOP2AY165S/+ mice are extremely resistant toward ICRF-193 general toxicity.
Fig. 5. Kaplan-Meyer survival plots of TOP2A+/+ and TOP2AY165S/+ mice after treatment with different dose levels of ICRF-193 given daily on days 0, 1, and 2 of the study. A, 10 mg/kg ICRF-193; B, 20 mg/kg ICRF-193; C, 30 mg/kg ICRF-193. D, Kaplan-Meyer survival plots including all mice treated with ICRF-193 in A to C. In A to C, the number of animals in each group was six. In D, the number of mice in each group was 18. p Values from log-rank analysis are shown on the figure for each dose-level.
TOP2AY165S/+ Mice Are Resistant toward Bisdioxopiperazine-Induced Bone Marrow Cell and Peripheral White Blood Cell Depression. Bisdioxopiperazines like ICRF-187 are known to cause myelosuppression at dose levels required for clinical activity (Bakowski et al., 1979; Von Hoff et al., 1981). Although bisdioxopiperazines are known to target topoisomerase II in vitro and in cell lines (Hasinoff et al., 1995; Ishida et al., 1995; Sehested et al., 1998; Wessel et al., 1999, 2002; Kobayashi et al., 2001), a direct role of the topoisomerase II isoform in bisdioxopiperazine-induced myelosuppression has never been established. We therefore assessed whether there was any difference in the effect of ICRF-187 on bone marrow cell counts and peripheral blood cell counts (WBCs, RBCs, and PLTs) between TOP2A+/+ and TOP2AY165S/+ mice. Daily doses of 250 or 450 mg/kg ICRF-187 were given i.p. on days 0, 1, and 2 to groups of six to seven mice. The hematological measurements were performed on day 3. Although there was no significant difference in the values for bone marrow cell counts obtained from TOP2A+/+ and TOP2AY165S/+ mice in the absence of drug (Fig. 6A, p = 0.68), there was significantly less suppression of bone marrow cell counts in the TOP2AY165S/+ mice compared with the TOP2A+/+ mice at the 250 mg/kg dose level (Fig. 6A; p = 0.0001) and a tendency to less suppression in mutant mice at the 450 mg/kg dose level (Fig. 6A; p = 0.06). We also looked at suppression of peripheral white blood cell counts (Fig. 6B). Here, the TOP2AY165S/+ genetic background was found to significantly reduce the effect of ICRF-187 on depressing the white blood cell numbers (Fig. 6B; p = 0.04 in comparisons at both dose levels).
Fig. 6. Hematological toxicity induced by different dose levels of ICRF-187 in TOP2A+/+ and TOP2AY165S/+ mice given the shown doses daily for 3 consecutive days. Bone marrow and blood samples were taken the day after the last dose. A, effect of ICRF-187 on bone marrow cell counts. B, effect of ICRF-187 on peripheral white blood cell counts. C, effect of ICRF-187 on red blood cell counts. D, effect of ICRF-187 on platelet counts. Error bars represent S.E.M. of data from 6 to 12 individual animals.
The effect of ICRF-193 on hematology values was also examined. Daily doses of 10, 20, and 30 mg/kg were given in the same 3-day scheme, and blood/bone marrow samples were taken on day 3. As seen for ICRF-187, the TOP2AY165S/+ genetic background reduced the effect of ICRF-193 on bone marrow cell depression at all three doses (Fig. 7A, p 0.02). The peripheral white blood cell counts were also lower in TOP2A+/+ mice compared with TOP2AY165S/+ mice after daily doses of 20 or 30 mg/kg for 3 days (Fig. 7B; p = 0.01 in both comparisons).
Fig. 7. Hematological toxicity induced by different dose levels of ICRF-193 in TOP2A+/+ and TOP2AY165S/+ mice given the shown doses daily for 3 consecutive days. Bone marrow and blood samples were taken the day after the last dose. A, effect of ICRF-193 on bone marrow cell counts. B, effect of ICRF-193 on peripheral white blood cell counts. C, effect of ICRF-193 on red blood cell counts. D, effect of ICRF-193 on platelet counts. Error bars represents S.E.M. of data from seven individual animals.
Intestinal Alterations Induced by ICRF-187 and ICRF-193 Concentrations Resulting in Selective Toxicity Toward wt Mice Are Insufficient to Explain Death. To examine the possibility that the mice died as a result of intestinal toxicity, we exposed groups of wt and TOP2AY165S/+ mice to three daily doses of 30 mg/kg ICRF-193 or 550 mg/kg ICRF-187 and performed histopathology on longitudinal sections of the duodenum, jejunum, and ileum by using hematoxylin and eosin straining. We used these concentrations because they had been found previously to confer selective toxicity to wt mice in the survival assay. Furthermore, as a control experiment, we performed hemotoxicity measurements and could demonstrate that the expected differential bone marrow and WBC depression did actually occur. The sections were then evaluated in a blinded fashion. Although an increase in apoptotic basal crypt cells and enlarged nuclei in parabasal cells was observed in both treated wt and TOP2AY165S/+ animals, no severe changes such as ulceration or shedding were found.
Thus, the extent of the cellular alterations was judged to be insufficient to cause severe intestinal toxicity. This result is in line with the fact that the mice (wt and TOP2AY165S/+) showed no signs of diarrhea during the toxicity assays. Together, these results indicate that intestinal toxicity was not the cause of the selective killing of wt mice by ICRF-193 and ICRF-187.
The bisdioxopiperazines were originally developed as agents showing moderate anticancer activity as single agents, with their main side effect being myelosuppression, which was seen at pharmacologically active concentrations (Bakowski et al., 1979; Von Hoff et al., 1981). Besides interacting with topoisomerase II, ICRF-187 is also a strong iron chelator (Hasinoff et al., 1998). It is the ring-opened hydrolysis product ADR-925 that has activity as iron chelator. ICRF-187 is approved with the indication of reducing anthracycline-induced cardiomyopathy [dexrazoxane (Cardioxane, Zinecard)]. It is believed that the iron chelating activity of its ring-opened hydrolysis product, ADR-925, is responsible for its cardioprotective effect through the removal of iron from its highly toxic complexes with anthracyclines (Hasinoff et al., 2003b), in turn protecting the cardiac myocytes against mitochondrial damage caused by redox-cycling of the anthracycline-iron complex (Hasinoff et al., 2003a). However, an effect through topoisomerase II inhibition has never been excluded. ICRF-187 is also approved as an antidote for accidental anthracycline extravasation (dexrazoxane; Mouridsen et al., 2007), and the role of iron chelation versus topoisomerase II inhibition in this process still remains to be fully elucidated. Finally, ICRF-187 has proven effective in modulating the pharmacological effects of topoisomerase II poisons in preclinical models (Holm et al., 1998; Hofland et al., 2005b), and the role for topoisomerase II versus topoisomerase IIβ in these processes also needs to be fully understood.
We here present an animal model developed with the aim of elucidating the role of topoisomerase II in the pharmacological effects of bisdioxopiperazines in vivo. In these studies, we also include the more potent (toward topoisomerase II) analog ICRF-193 (Hasinoff et al., 1995). We chose to look at the topoisomerase II isoform because we have described previously several mutations in this enzyme rendering it highly resistant to the actions of bisdioxopiperazines (Sehested et al., 1998; Wessel et al., 1999, 2002; Jensen et al., 2000b). In our present work, we take advantage of a Y165S mutation in human topoisomerase II that is dominant and specific with regard to bisdioxopiperazine resistance (Wessel et al., 2002).
Crosses between heterozygous TOP2AY165S/+ animals failed to produce any homozygous TOP2AY165S/Y165S mice. Instead, only TOP2A+/+ and TOP2AY165S/+ animals were born at the ratio of 1:2, suggesting that the homozygous TOP2AY165S/Y165S embryos die in utero. The mechanistic basis for this is not clear, but the simplest explanation would be that the level of topoisomerase II catalytic activity in TOP2AY165S/Y165S embryos is insufficient to meet the requirements during development. Another possibility would be that the embryonic lethality of the homozygous TOP2AY165S/Y165S mutation is related to the decreased levels of DNA-stimulated ATP hydrolysis seen with the Y165S protein (Sorensen et al., 2005). The DNA transport mechanism of eukaryotic topoisomerase II is directly coupled to ATP hydrolysis (Baird et al., 1999). Consequently, the reduced ATP hydrolysis of the Y165S protein could be associated with subtle alterations in its catalytic DNA strand passage reaction that could be lethal in the absence of wt topoisomerase II. The phenotype of the homozygous mutant embryos will be described elsewhere.
As described above, we assumed that the heterozygous TOP2AY165S/+ animals would show an increased resistance to treatment with bisdioxopiperazines. To test this hypothesis, we conducted toxicity experiments with groups of wt and mutant mice (Figs. 4 and 5). When the survival data for ICRF-187, as presented in Fig. 4, A to D, are pooled, it is evident that the mutant mice are resistant to the general toxicity of ICRF-187 (Fig. 4E). In experiments in which the more topoisomerase II-potent analog ICRF-193 was used, topoisomerase II mutant status was clearly a highly significant determinant for toxicity even in small experiments where groups of six mice were used (Fig. 5, B and C). When pooled, these ICRF-193 experiments demonstrate that the mutant mice are highly resistant toward the general toxicity of ICRF-193 (Fig. 5D).
To identify a biological mechanism underlying the bisdioxopiperazine resistance conferred by the TOP2AY165S/+ allele, we looked at a well known effect of bisdioxopiperazines, namely myelosuppression (Bakowski et al., 1979; Von Hoff et al., 1981). Bisdioxopiperazines, including ICRF-187 and ICRF-193, trap eukaryotic topoisomerase II as a closed protein clamp on DNA, which is toxic to cells (Ishida et al., 1995; van Hille and Hill, 1998; Jensen et al., 2000a; Xiao et al., 2003; Oestergaard et al., 2004). This configuration of topoisomerase II on DNA has furthermore been associated with increased levels of DNA strand breaks in some reports (Huang et al., 2001; Hajji et al., 2003). Because bone marrow stem cells are rapidly proliferating and contain high levels of topoisomerase II, we hypothesized that topoisomerase II would be involved in this effect and that the heterozygous TOP2AY165S/+ allele might abrogate or reduce this effect in TOP2AY165S/+ animals (Wessel et al., 2002). This turned out to be the case. The TOP2AY165S/+ allele had a significant impact on the ability of both ICRF-187 and ICRF-193 to cause depression of bone marrow cell counts (Figs. 6A and 7A). The same picture was seen for peripheral white blood cell counts, which were also significantly less depressed in TOP2AY165S/+ animals (Figs. 6B and 7B). These results for the first time demonstrate directly that topoisomerase II is playing an important and significant role in the myelosuppressive effect of bisdioxopiperazines. Furthermore, our finding that intestinal toxicity was not related to the selective toxicity of ICRF-187 and ICRF-193 toward the wt mice points toward hemotoxicity being responsible for this.
We found the RBC and PLT counts to be decreased after ICRF-187 treatment (p < 0.0001 for both RBC and PLT), regardless of genotype (p = 0.48 and 0.60, respectively; see Fig. 6, C and D). This suggests that the acute toxicity of ICRF-187 on RBC and platelets is caused by a mechanism independent of topoisomerase II, which also is in concordance with the fact that the time point for blood sampling (day 3 after start of treatment) is too early to reveal an effect on the erythropoietic precursor cells in the bone marrow/spleen, because erythrocytes and platelets have a longer transit time in the blood than white blood cells. As seen for ICRF-187, the red blood cells and platelet counts also tended to decrease with increasing ICRF-193 dose (Fig. 7, C and D; p < 0.0001 in both cell types), and in the case of platelets, there was no effect of genotype, whereas the TOP2AY165S/+ mice had lower RBC counts than TOP2A+/+ mice (p = 0.0003). This difference in RBC counts is probably caused by the dehydration and consequent hemoconcentration in the TOP2A+/+ mice after treatment with ICRF-193, because these mice were much less tolerant to the general toxicity of ICRF-193. The short-term effects observed on RBC and platelets after bisdioxopiperazine treatments are not associated with effects depending on topoisomerase II but possibly connected to the compound's iron chelating properties.
In this article, we present a model for assessing the role for topoisomerase II in bisdioxopiperazine action in the mouse. We found this enzyme to be associated with resistance to the general toxicity of both ICRF-187 and ICRF-193, and we used this model to demonstrate directly that topoisomerase II is involved in mediating bisdioxopiperazine-induced myelosuppression. Experiments aimed at clarifying the role of the TOP2AY165S/+ allele on the ability of ICRF-187 to antagonize the effects of topoisomerase II poisons in vivo are under way. We also aim at using this model to investigate the role of topoisomerase II in ICRF-187-mediated protection against anthracycline-induced cardiomyopathy and to assess whether topoisomerase II might also be involved in ICRF-187-mediated protection against anthracycline-mediated extravasation necrosis.
Acknowledgements
The technical assistance of Annette Nielsen, Sanne Christiansen, and Mette Frandsen is highly appreciated. The expertise of Anita, Lillian, and Jannie in animal care is appreciated. Support from the Danish Biotechnology Instrument Center is also highly appreciated.
ABBREVIATIONS: CNS, central nervous system; CFU-GM, colony forming unit-granulocyte/macrophage; ES, embryonic stem cell; ICRF, Imperial Cancer Research Foundation; kDNA, kinetoplast DNA; PLT, platelet; PCR, polymerase chain reaction; RBC, red blood cell; WBC, white blood cell; bp, base pair(s); kbp, kilobase pair(s); kb, kilobase(s); wt, wild type with regard to the topoisomerase II gene; ICRF-187, (+)-(S)-4,4'-propylenedi-2,6-piperazinedione; ICRF-154, 1,2-bis(3,5-dioxopiperazin-1-yl)ethane; ICRF-193, 4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione; ADR-925, the iron chelating hydrolysis product of ICRF-187.
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作者单位：Experimental Pathology Unit, Department of Pathology, Copenhagen University Hospital, Copenhagen, Denmark (M.G., M.S., L.H.J.); TopoTarget A/S, Copenhagen, Denmark (M.G., A.V.T., K.F.H., P.B.J., M.S., L.H.J.); Department of Molecular Biology, University of Aarhus, Aarhus, Denmark (A.F., P.H.H., E.-M