Analysis of bisdioxopiperazine dexrazoxane binding to human DNA topoisomerase II alpha: decreased binding as a mechanism of drug resistance

Topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double stranded DNA segment and transporting it through another DNA molecule. Despite the extensive use of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, much remains unknown concerning the interactions between these agents and topoisomerase II. To identify the interaction of the bisdioxopiperazine dexrazoxane (ICRF-187) with topoisomerase II, we developed a rapid gel-filtration assay and characterized the binding of ((3)H)-dexrazoxane to human topoisomerase II alpha. Dexrazoxane binds to human topoisomerase II alpha in the presence of DNA and ATP with an apparent K(d) of 23 microM and a stoichiometry of 1 drug molecule per enzyme dimer. Various N-terminal single amino acid substitutions in human topoisomerase II alpha that were previously shown to confer specific bisdioxopiperazine resistance either totally abolished drug binding or resulted in less efficient binding. The effect of the various mutations on drug binding correlated well with their effect on drug resistance in vivo and in vitro. Interestingly, an altered active site tyrosine mutant of human topoisomerase II alpha, which is incapable of carrying out DNA strand passage, was unable to bind dexrazoxane, which agrees with the drug's proposed mechanism of action late in the topoisomerase II catalytic cycle. The direct correlation between the level of drug binding and dexrazoxane resistance is consistent with a decreased drug binding mechanism of action for these dexrazoxane resistance conferring mutations.     

Interaction of human DNA topoisomerase II alpha with DNA: quantification by surface plasmon resonance

DNA topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double-stranded DNA segment and transporting it through another duplex. Surface plasmon resonance (SPR) was used to characterize interactions of human topoisomerase II alpha with different topological forms of DNA. Using a linear fragment of pUC18 DNA, the equilibrium binding constant of topoisomerase II alpha was determined to be 0.16 nM. The affinity was not affected by the absence of ATP or the presence of the bisdioxopiperazine catalytic inhibitor ICRF-187. Besides, similar affinities were found for several bisdioxopiperazine-resistant mutant enzymes. These results suggest that the mechanism of topoisomerase II alpha inhibition by ICRF-187 and its resistance does not directly involve the interaction of DNA with the enzyme. SPR was also adapted to measure levels of the closed clamp form of topoisomerase II present on DNA. As expected, a stable closed clamp form of the enzyme was detectable on circular DNA but not on linear DNA. Detection of the closed clamp required the presence of ATP and a bisdioxopiperazine, or a non-hydrolyzable analogue of ATP. In the presence of ATP and ICRF-187, several bisdioxopiperazine-resistant mutant enzymes failed to form detectable levels of stable closed clamp. Interestingly, a mutant of human topoisomerase II alpha with an altered active site tyrosine showed lower levels of closed clamp formation. In conclusion, SPR is able to (1) determine the kinetics of topoisomerase II with its DNA substrate and (2) quantify the enzyme's closed clamp formation under varying circumstances.     

Separation of bisdioxopiperazine- and vanadate resistance in topoisomerase II

Bisdioxopiperazines are inhibitors of topoisomerase II trapping this protein as a closed clamp on DNA with concomitant inhibition of its ATPase activity. Here, we analyse the effects of N-terminal mutations identified in bisdioxopiperazine-resistant cells on ATP hydrolysis by this enzyme. We present data consistent with bisdioxopiperazine resistance arising by two different mechanisms; one involving reduced stability of the N-terminal clamp (the N-gate) and one involving reduced affinity for bisdioxopiperazines. Vanadate is a general inhibitor of type P ATPases and has recently been demonstrated to lock topoisomerase II as a salt-stable closed clamp on DNA analogous to the bisdioxopiperazines. We show that a R162K mutation in human topoisomerase II alpha renders this enzyme highly resistant towards vanadate while having little effect on bisdioxopiperazine sensitivity. The implications of these findings for the mechanism of action of bisdioxopiperazines versus vanadate with topoisomerase II are discussed.     

A comparative study of genotoxic effects of anti-topoisomerase II drugs ICRF-193 and bufalin in Chinese hamster ovary cells

With the ultimate purpose of testing the existence of possible differences in the effectiveness of the topoisomerase II catalytic inhibitor ICRF-193 (a bisdioxopiperazine) and the enzyme suppressor bufalin (a bufadienolide from toad venom) we have carried out a series of experiments aimed at inducing cytotoxicity as well as DNA and chromosome damage in transformed CHO cells. In order to assess any possible influence of DNA repair capacity of the treated cells on the final outcome, we have made use of the repair-defective CHO mutant EM9, which shows a defect in DNA single- and double-strand breaks repair for comparison with its repair-proficient parental line AA8.Our results seem to indicate that, while both ICRF-193 and bufalin suppress cell growth and result in a clear inhibition of topoisomerase II catalytic activity, only ICRF-193 has been shown as able to induce both chromosome and DNA damage, with a more pronounced effect in the CHO mutant EM9 than in the repair-proficient line AA8.     

Enhanced processing of UVA-irradiated DNA by human topoisomerase II in living cells

Solar UV light induces a variety of DNA lesions in the genome. Enhanced cleavage of such base modifications by topoisomerase II has been demonstrated in vitro, but it is unclear what will arise from an interplay of these mechanisms in the genome of a living cell exposed to UV light. To address this question, we have subjected cells expressing biofluorescent topoisomerase IIalpha or IIbeta to DNA base modifications inflicted by a UVA laser at 364 nm through a confocal microscope in a locally confined manner. At DNA sites thus irradiated, we observed rapid, long term (>90 min) accumulation of topoisomerase IIalpha and IIbeta, which was accompanied by a decrease in mobility but not immobilization of the enzyme. The catalytic topoisomerase II inhibitor ICRF-187 prevented the effect when added to the cell culture before the UVA pulse but promoted it when added thereafter. Self-primed in situ extension with rhodamine-dUTP revealed massive DNA breakage at the UVA-exposed spot. Culturing the cells with ICRF-187 before UVA-exposure prevented such breaks. In conclusion, we show in a living cell nucleus that UVA-modified DNA is preferentially targeted and processed by topoisomerase IIalpha and IIbeta. This results in increased levels of topoisomerase II-mediated DNA breaks, but formation of immobile, stable topoisomerase II.DNA intermediates is not notably promoted. Inhibition of topoisomerase II activity by ICRF-187 greatly diminishes UVA-induced DNA breakage, implying topoisomerase IIalpha and IIbeta as endogenous co-factors modulating and possibly aggravating the impact of UVA light on the genome.     

Mitotic Bypass Via An Occult Cell Cycle Phase Following DNA Topoisomerase II Inhibition In p53 Functional Human Tumor Cells

Cell cycle checkpoints guard against the inappropriate commitment to critical cell events such as mitosis. The bisdioxopiperazine ICRF-193, a catalytic inhibitor of DNA topoisomerase II, causes a reversible stalling of the exit of cells from G2 at the decatenation checkpoint (DC) and can generate tetraploidy via the compromising of chromosome segregation and mitotic failure. We have addressed an alternative origin – endocycle entry - for the tetraploidisation step in ICRF-193 exposed cells. Here we show that DC-proficient p53-functional tumour cells can undergo a transition to tetraploidy and subsequent aneuploidy via an initial bypass of mitosis and the mitotic spindle checkpoint. DC-deficient SV40-tranformed cells move exclusively through mitosis to tetraploidy. In p53-functional tumour cells, escape through mitosis is enhanced by dominant negative p53 co-expression. The mitotic bypass transition phase (termed G2endo) disconnects cyclin B1 degradation from nuclear envelope breakdown and allows cells to evade the action of Taxol. G2endo constitutes a novel and alternative cell cycle phase - lasting some 8 h - with distinct molecular motifs at its boundaries for G2 exit and subsequent entry into a delayed G1 tetraploid state. The results challenge the paradigm that checkpoint breaching leads directly to abnormal ploidy states via mitosis alone. We further propose that the induction of bypass could: facilitate the covert development of tetraploidy in p53 functional cancers, lead to a misinterpretation of phase allocation during cell cycle arrest and contribute to tumour cell drug resistance.     

A Topoisomerase II-Dependent Checkpoint in G2-Phase Plant Cells Can Be Bypassed by Ectopic Expression of Mitotic Cyclin B2

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclin-dependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.

Key Words:

Plant cyclin B2, Topoisomerase II, ICRF-193, G2 checkpoint, Microtubules     

Topoisomerase II poisoning by ICRF-193

Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerase-targeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very significant topoisomerase II poison. Detection of topoisomerase II poisoning by ICRF-193 required the use of a chaotropic protein denaturant in the topoisomerase poisoning assays. ICRF-193 caused dose-dependent cross-linking of human topoisomerase IIbeta to DNA and stimulated topoisomerase IIbeta-mediated DNA cleavage at specific sites on (32)P-end-labeled DNA. Human topoisomerase IIalpha-mediated DNA cleavage was stimulated to a lesser extent by ICRF-193. In vivo experiments with MCF-7 cells also showed the requirement of a chaotropic protein denaturant in the assays and selectivity for the beta-isozyme of human topoisomerase II. Studies with two topoisomerase IIbeta-negative cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with beta-isozyme selectivity. Common use of only the detergent, SDS, in assays may have led to failure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.     

ATR enforces the topoisomerase II-dependent G2 checkpoint through inhibition of Plk1 kinase

An ATR-dependent G(2) checkpoint responds to inhibition of topoisomerase II and delays entry into mitosis by sustaining nuclear exclusion of cyclin B1-Cdk1 complexes. Here we report that induction of this checkpoint with ICRF-193, a topoisomerase II catalytic inhibitor that does not cause DNA damage, was associated with an ATR-dependent inhibition of polo-like kinase 1 (Plk1) kinase activity and a decrease in cyclin B1 phosphorylation. Expression of constitutively active Plk1 but not wild type Plk1 reversed ICRF-193-induced mitotic delay in HeLa cells, suggesting that Plk1 kinase activity is important for the checkpoint response to ICRF-193. G(2)/M synchronized normal human fibroblasts, when treated with ICRF-193, showed a decrease in cyclin B1 phosphorylation and Plk1 kinase activity despite high cyclin B1-Cdk1 kinase activity. G(2) fibroblasts that were treated with caffeine to override the checkpoint response to ICRF-193 displayed a high incidence of chromosomal aberrations. Taken together, these results suggest that ATR-dependent inhibition of Plk1 kinase activity may be one mechanism to regulate cyclin B1 phosphorylation and sustain nuclear exclusion during the G(2) checkpoint response to topoisomerase II inhibition. Moreover, the results demonstrate an important role for the topoisomerase II-dependent G(2) checkpoint in the preservation of human genomic stability.     

A topoisomerase II-dependent checkpoint in G2-phase plant cells can be bypassed by ectopic expression of mitotic cyclin B2

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.     

Overall Prevalence and Distribution of Knockdown Resistance (kdr) Mutations in Aedes aegypti from Mandalay Region,Myanmar

Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) of mosquitoes confer resistance to insecticides. Although insecticide resistance has been suspected to be widespread in the natural population of Aedes aegypti in Myanmar, only limited information is currently available. The overall prevalence and distribution of kdr mutations was analyzed in Ae. aegypti from Mandalay areas, Myanmar. Sequence analysis of the VGSC in Ae. aegypti from Myanmar revealed amino acid mutations at 13 and 11 positions in domains II and III of VGSC, respectively. High frequencies of S989P (68.6%), V1016G (73.5%), and F1534C (40.1%) were found in domains II and III. T1520I was also found, but the frequency was low (8.1%). The frequency of S989P/V1016G was high (55.0%), and the frequencies of V1016G/F1534C and S989P/V1016G/F1534C were also high at 30.1% and 23.5%, respectively. Novel mutations in domain II (L963Q, M976I, V977A, M994T, L995F, V996M/A, D998N, V999A, N1013D, and F1020S) and domain III (K1514R, Y1523H, V1529A, F1534L, F1537S, V1546A, F1551S, G1581D, and K1584R) were also identified. These results collectively suggest that high frequencies of kdr mutations were identified in Myanmar Ae. aegypti, indicating a high level of insecticide resistance.     

Cell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1

Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving gamma-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation.     

The p53 tumor suppressor stimulates the catalytic activity of human topoisomerase IIalpha by enhancing the rate of ATP hydrolysis

DNA topoisomerase II is an essential nuclear enzyme for proliferation of eukaryotic cells and plays important roles in many aspects of DNA processes. In this report, we have demonstrated that the catalytic activity of topoisomerase IIalpha, as measured by decatenation of kinetoplast DNA and by relaxation of negatively supercoiled DNA, was stimulated approximately 2-3-fold by the tumor suppressor p53 protein. In order to determine the mechanism by which p53 activates the enzyme, the effects of p53 on the topoisomerase IIalpha-mediated DNA cleavage/religation equilibrium were assessed using the prototypical topoisomerase II poison, etoposide. p53 had no effect on the ability of the enzyme to make double-stranded DNA break and religate linear DNA, indicating that the stimulation of the enzyme catalytic activity by p53 was not due to alteration in the formation of covalent cleavable complexes formed between topoisomerase IIalpha and DNA. The effects of p53 on the catalytic inhibition of topoisomerase IIalpha were examined using a specific catalytic inhibitor, ICRF-193, which blocks the ATP hydrolysis step of the enzyme catalytic cycle. Clearly manifested in decatenation and relaxation assays, p53 reduced the catalytic inhibition of topoisomerase IIalpha by ICRF-193. ATP hydrolysis assays revealed that the ATPase activity of topoisomerase IIalpha was specifically enhanced by p53. Immunoprecipitation experiments revealed that p53 physically interacts with topoisomerase IIalpha to form molecular complexes without a double-stranded DNA intermediary in vitro. To investigate whether p53 stimulates the catalytic activity of topoisomerase II in vivo, we expressed wild-type and mutant p53 in Saos-2 osteosarcoma cells lacking functional p53. Wild-type, but not mutant, p53 stimulated topoisomerase II activity in nuclear extract from these transfected cells. Our data propose a new role for p53 to modulate the catalytic activity of topoisomerase IIalpha. Taken together, we suggest that the p53-mediated response of the cell cycle to DNA damage may involve activation of topoisomerase IIalpha.     

Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II.

Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II was studied in the absence and presence of DNA, and in the absence and presence of inhibitor ICRF-193. The results indicate that purified Top2-(1-409), a fragment containing the NH(2)-terminal 409 amino acids of the yeast enzyme, is predominantly monomeric, with a low level of ATPase owing to weak association of two monomers to form a catalytically active dimer. The ATPase activity of Top2-(1-409) is independent of DNA in a buffer containing 100 mM NaCl, in which intact yeast DNA topoisomerase II exhibits robust DNA-dependent ATPase and DNA transport activities. Purified Top2-(1-660), a fragment containing the NH(2)-terminal 660 amino acid of the yeast enzyme, appears to be dimeric in the absence or presence of DNA, and the ATPase activity of the protein is significantly stimulated by DNA. These results are consistent with a model in which binding of an intact DNA topoisomerase II to DNA places the various subfragments of the enzyme in a way that makes the intramolecular dimerization of the ATPase domains more favorable. We believe that this alignment of subfragments is mainly achieved through the binding of the enzyme to the DNA segment within which the enzyme makes transient breaks. The ATPase activity of Top2-(1-409) is inhibited by ICRF-193, suggesting that the bisdioxopiperazine class of DNA topoisomerase II inhibitors directly interacts with the paired ATPase domains of the enzyme.     

Topoisomerase IIα mediates E2F-1-induced chemosensitivity and is a target for p53-mediated transcriptional repression

Mutations of the retinoblastoma tumor suppressor, pRb, or its cyclin-cyclin-dependent kinase (CDK) regulatory kinases or CDK inhibitors, allows unrestrained E2F activity, leading to unregulated cell cycle progression. However, overexpression of E2F-1 also sensitizes cells to apoptosis, suggesting that targeting this pathway may be of therapeutic benefit. Enforced expression of E2F-1 in interleukin-3-dependent myeloid cells led to preferential sensitivity to the topoisomerase II inhibitor, etoposide, which was independent of p53 accumulation. Pretreatment of the E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not cause DNA damage, protected these cells against etoposide-induced apoptosis. However, ICRF-193 cooperated with other DNA-damaging agents to induce apoptosis. Enforced expression of E2F-1 led to accumulation of p53 protein. An E2F-1 mutant that is defective in inducing cell cycle progression also induced p53, suggesting that p53 was responding directly to E2F, and not to secondary events caused by inappropriate cell cycle progression (i.e., DNA damage). Thus, topoisomerase II inhibition and DNA damage cooperate to selectively induce apoptosis in cells that have mutations in the pRb pathway.     

Revised genetic requirements for the decatenation G2 checkpoint: The role of ATM

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.     

Dissecting the cell-killing mechanism of the topoisomerase II-targeting drug ICRF-193

Topoisomerase II is an essential enzyme that is targeted by a number of clinically valuable anticancer drugs. One class referred to as topoisomerase II poisons works by increasing the cellular level of topoisomerase II-mediated DNA breaks, resulting in apoptosis. Another class of topoisomerase II-directed drugs, the bis-dioxopiperazines, stabilizes the conformation of the enzyme where it attains an inactive salt-stable closed clamp structure. Bis-dioxopiperazines, similar to topoisomerase II poisons, induce cell killing, but the underlying mechanism is presently unclear. In this study, we use three different biochemically well characterized human topoisomerase IIalpha mutant enzymes to dissect the catalytic requirements needed for the enzyme to cause dominant sensitivity in yeast to the bis-dioxopirazine ICRF-193 and the topoisomerase II poison m-AMSA. We find that the clamp-closing activity, the DNA cleavage activity, and even both activities together are insufficient for topoisomerase II to cause dominant sensitivity to ICRF-193 in yeast. Rather, the strand passage event per se is an absolute requirement, most probably because this involves a simultaneous interaction of the enzyme with two DNA segments. Furthermore, we show that the ability of human topoisomerase IIalpha to cause dominant sensitivity to m-AMSA in yeast does not depend on clamp closure or strand passage but is directly related to the capability of the enzyme to respond to m-AMSA with increased DNA cleavage complex formation.     

DNA strand breaks induced by the anti-topoisomerase II bis-dioxopiperazine ICRF-193

The bis-dioxopiperazine ICRF-193 has long time been considered as a pure topoisomerase II catalytic inhibitor able to exert its inhibitory effect on the enzyme without stabilization of the so-called cleavable complex formed by DNA covalently bound to topoisomerase II. In recent years, however, this concept has been challenged, as a number of reports have shown that ICRF-193 really "poisons" the enzyme, most likely through a different mechanism from that shown by the classical topoisomerase II poisons used in cancer chemotherapy. In the present investigation, we have carried out a study of the capacity of ICRF-193 to induce DNA strand breaks, as classical poisons do, in cultured V79 and irs-2 Chinese hamster lung fibroblasts using the comet assay and pulsed-field gel electrophoresis (PFGE). Our results clearly show that ICRF-193 readily induces breakage in DNA through a mechanism as yet poorly understood.     

ATPase domain of eukaryotic DNA topoisomerase II. Inhibition of ATPase activity by the anti-cancer drug bisdioxopiperazine and ATP/ADP-induced dimerization

We have prepared full-length Drosophila and human topoisomerase II and truncation constructs containing the amino-terminal ATPase domain, and we have analyzed their biochemical properties. The ATPase activity of the truncation proteins, similar to that of the full-length proteins, is greatly stimulated by the presence of DNA. This activity of the truncation proteins is also sensitive to the inhibition by the drug bisdioxopiperazine, ICRF-193, albeit at a much lower level than the full-length protein. Therefore, bisdioxopiperazine can directly interact with the NH(2)-terminal ATPase domain, but the drug-enzyme interaction may involve other domains as well. The ATPase activity of the ATPase domain protein showed a quadratic dependence on enzyme concentration, suggesting that dimerization of the NH(2)-terminal domain is a rate-limiting step. Using both protein cross-linking and sedimentation equilibrium analysis, we showed that the ATPase domain exists as a monomer in the absence of cofactors but can readily dimerize in the presence of a nonhydrolyzable analog of ATP, 5'-adenylyl-beta,gamma-imidodiphosphate. More interestingly, both ATP and ADP can also promote protein dimerization. This result thus suggests that the protein clamp, mediated through the dimerization of ATPase domain, remains closed after ATP hydrolysis and opens upon the dissociation of ADP.