Induction of topoisomerase II-mediated DNA cleavage by a protoberberine alkaloid, berberrubine

Topoisomerase II is the cytotoxic target for a number of clinically relevant antitumor drugs. Berberrubine, a protoberberine alkaloid which exhibits antitumor activity in animal models, has been identified as a specific poison of topoisomerase II in vitro. Topoisomerase II-mediated DNA cleavage assays showed that berberrubine poisons the enzyme by stabilizing topoisomerase II-DNA cleavable complexes. Subsequent proteinase K treatments revealed that berberrubine-induced DNA cleavage was generated solely by topoisomerase II. Topoisomerase II-mediated DNA religation with elevated temperature revealed a substantial reduction in DNA cleavage induced by berberrubine, to the extent comparable to that of other prototypical topoisomerase II poison, etoposide, suggesting that DNA cleavage involves stabilization of the reversible enzyme-DNA cleavable complex. However, the step at which berberrubine induces cleavable complex may differ from that of etoposide as revealed by the difference in the formation of the intermediate product, nicked DNA. This suggests that berberrubine's primary mode of linear formation may involve trapping nicked molecules, formed at transition from linear to covalently closed circular DNA. Unwinding of the duplex DNA by berberrubine is consistent with an intercalative binding mode for this compound. In addition to the ability to induce the cleavable complex mediated with topoisomerase II, berberrubine at high concentrations was shown to specifically inhibit topoisomerase II catalytic activity. Berberrubine, however, did not inhibit topoisomerase I at concentrations up to 240 microM. Cleavage sites induced by topoisomerase II in the presence of berberrubine and etoposide were mapped in DNA. Berberrubine induces DNA cleavage in a site-specific and concentration-dependent manner. Comparison of the cleavage pattern of berberrubine with that of etoposide revealed that they share many common sites of cleavage. Taken together, these results indicate that berberrubine represents a new class of antitumor agent which exhibits the topoisomerase II poison activity as well as catalytic inhibition activity and may have a potential clinical value in cancer treatment.     

Human DNA topoisomerase I-mediated cleavages stimulated by ultraviolet light-induced DNA damage

DNA topoisomerases have been proposed as the proteins involved in the formation of the DNA-protein cross-links detected after ultraviolet light (UV) irradiation of cellular DNA. This possibility has been investigated by studying the effects of UV-induced DNA damage on human DNA topoisomerase I action. UV lesions impaired the enzyme's ability to relax negatively supercoiled DNA. Decreased relaxation activity correlated with the stimulation of cleavable complexes. Accumulation of cleavable complexes resulted from blockage of the rejoining step of the cleavage-religation reaction. Mapping of cleavage sites on the pAT153 genome indicated UV-induced cleavage at discrete positions corresponding to sites stimulated also by the topoisomerase I inhibitor camptothecin, except for one. Subsequent analysis at nucleotide level within the sequence encompassing the UV-specific cleavage site revealed the precise positions of sites stimulated by camptothecin with respect to those specific for UV irradiation. Interestingly, one of the UV-stimulated cleavage sites was formed within a sequence that did not contain dimerized pyrimidines, suggesting transmission of the distortion, caused by photodamage to DNA, into the neighboring sequences. These results support the proposal that DNA structural alterations induced by UV lesions can be sufficient stimulus to induce cross-linking of topoisomerase I to cellular DNA.     

Induction of mammalian DNA topoisomerase I-mediated DNA cleavage and DNA winding by bulgarein

Bulgarein, a fungal metabolite, induced mammalian topoisomerase I-mediated DNA cleavage in vitro. The cleavage activity of bulgarein was comparable to that of camptothecin at a drug concentration range of 0.025-approximately 5 microM. The DNA cleavage induced by bulgarein was suppressed at concentrations above 12.5 microM. Treatment of a reaction mixture containing bulgarein and topoisomerase I with elevated temperature (65 degrees C) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I-mediated DNA cleavage induced by bulgarein is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex." Intensity of cleaved DNA fragments induced by bulgarein with topoisomerase I was different from that induced by camptothecin. Bulgarein inhibited catalytic activities of both topoisomerase I and topoisomerase II. The changes in absorption spectra of bulgarein in the visible region observed upon addition of increasing amounts of calf thymus DNA indicate that bulgarein interacts with DNA. DNA (un)winding assay by two-dimensional gel electrophoresis showed that bulgarein induced the winding of DNA in the opposite direction to that of an intercalator so that positively supercoiled molecules are produced. Thus, bulgarein represents a new class of drugs which stabilizes the cleavable complex of topoisomerase I and alters the structure of DNA in a manner leading to a tightening of the helical twist.     

Antitumor agents--CLXXIII. Synthesis and evaluation of camptothecin-4 beta-amino-4'-O-demethyl epipodophyllotoxin conjugates as inhibitors of mammalian DNA topoisomerases and as cytotoxic agents

Two conjugates composed of a camptothecin and a 4'-O-demethyl epipodophyllotoxin derivative joined by an imine linkage were prepared and evaluated as inhibitors of mammalian DNA topoisomerases I and II. Target compounds stimulated cleavable complex formation with both types of enzyme in vitro although activities were reduced at least twofold relative to the activity of unconjugated constituents. The behavior of the most active conjugate as an inhibitor of cell growth closely resembled both topoisomerase I- and II- inhibitory components in that the compound displayed a combined spectrum of activity against various drug-resistant KB sublines. Cytotoxic activity and selectivity were largely retained through conjugation, the exception being a lower than expected activity against a pleiotrophic multidrug-resistant subline. The induced levels and the properties of cellular protein-associated DNA complexes were consistent with topoisomerase involvement and with the in vitro cleavage assay results. Based on the present findings, conjugation afforded cleavable complex-forming topoisomerase inhibitors which display dual target specificity and a broad spectrum of cytotoxic activity against drug-resistant cells.     

Differential effects of camptothecin derivatives on topoisomerase I-mediated DNA structure modification

The effects of eleven camptothecin derivatives on calf thymus topoisomerase I-mediated cleavage of synthetic DNA duplex have revealed that the A ring of camptothecin is very important for its biochemical activity. Depending on the type, number, and location of substituents, highly active or inactive analogues were obtained. The persistence of CPT-induced topoisomerase I-DNA covalent binary complexes was investigated by using as substrates DNA containing several good topoisomerase I cleavage sites, or else a synthetic DNA duplex of defined structure with a single high-efficiency cleavage site. The ligation kinetics at a given topoisomerase I cleavage site were sometimes quite different in the presence of CPT derivatives whose structures were closely related. Even in the presence of a single CPT analogue, topoisomerase I-DNA covalent binary complexes underwent ligation with different kinetics, presumably reflecting a dependence on DNA sequences flanking the individual topoisomerase I cleavage sites. Individual camptothecin derivatives also exhibited a spectrum of inhibitory potentials in blocking the topoisomerase I-mediated rearrangement of branched, nicked, and gapped DNA duplex substrates; in some cases the potencies of inhibition observed in these assays for individual camptothecin analogues were quite different than those determined for stabilization of the unmodified DNA-topoisomerase I binary complex.     

Mutations of vaccinia virus DNA topoisomerase I that stabilize the cleavage complex

Two mutations in vaccinia virus topoisomerase I, K167D and G226N, have been characterized. SOS induction was observed in Escherichia coli expressing vaccinia topoisomerase I with either one of these mutations. The mutant enzymes were purified to homogeneity and compared with the wild type enzyme for relaxation activity and the partial activities of substrate binding, site-specific DNA cleavage and DNA religation to determine the mechanism of SOS induction. The K167D mutant enzyme had reduced binding affinity for the DNA substrate with a Kapp that was 10-fold higher than wild type. Nevertheless, in reactions with high enzyme concentration, its substrate cleavage activity was 90% that of wild type. The G226N mutant enzyme had virtually wild type binding and cleavage activities. However, intermolecular religation by these two mutants were observed to be significantly reduced. The cleavage complexes formed with the K167D and G226N mutants were more stable to high salt than the wild type cleavable complex. We propose that these mutants in vivo induce the SOS response in E. coli due to the shift of topoisomerase cleavage-religation equilibrium towards cleavage and increased stability of the cleavage complex. The mutation thus has a similar effect as the topoisomerase-targeting inhibitors that turn topoisomerases into DNA damaging agents.     

Diospyrin, a bisnaphthoquinone: a novel inhibitor of type I DNA topoisomerase of Leishmania donovani

Diospyrin is a plant product that has significant inhibitory effect on the growth of Leishmania donovani promastigotes. This compound inhibits the catalytic activity of DNA topoisomerase I of the parasite. Like camptothecin, it induces topoisomerase I mediated DNA cleavage in vitro. Treatment of DNA with diospyrin before addition of topoisomerase I has no effect. Preincubation of topoisomerase I with diospyrin before the addition of DNA in the relaxation reaction increases this inhibition. Our results suggest that this bis-naphthoquinone compound exerts its inhibitory effect by binding with the enzyme and stabilizing the topoisomerase I-DNA "cleavable complex." Diospyrin is a specific inhibitor of the parasitic topoisomerase I. It does not inhibit type II topoisomerase of L. donovani and requires much higher concentrations to inhibit type I topoisomerase of calf thymus. The potent inhibitory effect of diospyrin on type I DNA topoisomerase from L. donovani can be exploited for rational drug design in human leishmaniasis.     

Drug-specific sites of topoisomerase II DNA cleavage in Drosophila chromatin: heterogeneous localization and reversibility

DNA cleavage stimulated by different topoisomerase II inhibitors shows in vitro a characteristic sequence specificity. Since chromatin structure and genome organization are expected to influence drug-enzyme interactions and repair of drug-induced DNA lesions, we investigated topoisomerase II DNA cleavage sites stimulated by teniposide (VM-26), 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (dh-EPI, a doxorubicin derivative), 4'-(9-acridinylamino)-methanesulfon-m-anisidide, and amonafide in the histone gene locus and satellite III DNA of Drosophila cells with Southern blottings and genomic sequencing by primer extension. VM-26 stimulated cleavage in the satellite III DNA, whereas the other studied drugs did not. All four drugs stimulated cleavage in the histone gene cluster, but they yielded drug-specific cleavage intensity patterns. Cleavage sites by dh-EPI and VM-26 were sequenced in the histone H2A gene promoter and were shown to be distinct. DNA cleavage analysis in cloned DNA fragments with Drosophila topoisomerase II showed that drugs stimulated the same sites in vivo and in vitro. Strand cuts were in vivo staggered by 4 bases, and base sequences at major dh-EPI and VM-26 sites completely agreed with known in vitro drug sequence specificities. Moreover, DNA cleavage reverted faster in the satellite III than in the histone repeats. While stimulating similar levels of DNA breakage in bulk genomic DNA, dh-EPI and VM-26 markedly differed for cleavage extent and reversibility in specific chromatin loci. The results demonstrate a high heterogeneity in the localization, extent, and reversibility of drug-stimulated DNA cleavage in the chromatin of living cells.     

Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity

Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.     

Potentiation of CD95L-induced apoptosis of human malignant glioma cells by topotecan involves inhibition of RNA synthesis but not changes in CD95 or CD95L protein expression

Topotecan is a novel topoisomerase I inhibitor that may have a role in the adjuvant chemotherapy of several solid tumors, including malignant glioma. Here, we have characterized the time- and concentration-dependent toxicity of topotecan in four human malignant glioma cell lines, LN-18, LN-229, LN-308 and T98G. High micromolar concentrations of topotecan, which are unlikely to be achieved in plasma in human patients in vivo, were cytotoxic within 48 hr, induced DNA fragmentation, did not induce major cell cycle changes, failed to consistently alter BCL-2 or BAX protein levels but inhibited RNA synthesis and induced cleavable DNA/topoisomerase I complex formation. Prolonged exposure for 72 hr to high nanomolar to low micromolar concentrations of topotecan augmented p21 protein levels and induced G2/M arrest but failed to consistently alter BCL-2 and BAX protein levels, did not induce significant DNA/topoisomerase I complex formation and did not inhibit RNA synthesis. Neither short-term nor long-term topotecan toxicity was blocked by ectopic expression of bcl-2 or wild-type p53. Transfer of a mutant p53 gene enhanced topotecan sensitivity in wild-type p53 LN-229 but not mutant p53 LN-18 cells. CD95 ligand (CD95L)-induced apoptosis was synergistically enhanced by short-term/high concentration but not long-term/low concentration exposure to topotecan, suggesting that topotecan sensitizes human malignant glioma cells to CD95L-induced apoptosis via inhibition of RNA synthesis. These data suggest that topotecan needs to be administered in high concentrations, such as an intratumoral polymer, to limit glioma cell growth in synergy with CD95L in vivo.     

The responses of secondary endings of cat soleus muscle spindles to succinyl choline

In this report we examine biochemical and genetic alterations in DNA topoisomerase II (topoisomerase II) in K562 cells selected for resistance in the presence of etoposide (VP-16). Previously, we have demonstrated that the 30-fold VP-16-resistant K/VP.5 cell line exhibits decreased stability of drug-induced topoisomerase II/DNA covalent complexes, requires greater ATP concentrations to stimulate VP-16-induced topoisomerase II/DNA complex formation, and contains reduced mRNA and protein levels of the M(r) 170,000 isoform of topoisomerase II, compared with parental K562 cells. K/VP.5 cells grown in the absence of VP-16 for 2 years maintained resistance to VP-16, decreased levels of topoisomerase II, and attenuated ATP stimulation of VP-16-induced topoisomerase II/DNA binding, compared with K562 cells. Sequencing of cDNA coding for two consensus ATP binding sites and the active site tyrosine in the K/VP.5 topoisomerase II gene indicated that no mutations were present in these domains. In addition, single-strand conformational polymorphism analysis of restriction fragments encompassing the entire topoisomerase II cDNA revealed no evidence of mutations in the gene for this enzyme in K/VP.5 cells. Nuclear extracts from K562 (but not K/VP.5) cells contained a heat-labile factor that potentiated VP-16-induced topoisomerase II/DNA covalent complex formation in isolated nuclei from K/VP.5 cells. Immunoprecipitated topoisomerase II from K/VP.5 cells was 2.5-fold less phosphorylated, compared with enzyme from K562 cells. Collectively, our data suggest that acquired VP-16 resistance is mediated, at least in part, by altered levels or activity of a kinase that regulates topoisomerase II phosphorylation and hence drug-induced topoisomerase II/DNA covalent complex formation and stability.     

Intermittent exposure of medulloblastoma cells to topotecan produces growth inhibition equivalent to continuous exposure

Camptothecin analogues such as topotecan increase the number of covalent topoisomerase I-DNA complexes, which, in turn, have been proposed to initiate apoptosis. If induction of apoptosis by the camptothecins is, in fact, dependent on the formation of topoisomerase I-DNA complexes, then it would be of clinical relevance to identify schedules of exposure to the camptothecins that maximize the formation of these complexes but minimize the total amount of the drug administered. The time and dose dependence of topoisomerase I-DNA complex formation was determined by incubating Daoy pediatric medulloblastoma cells in vitro with topotecan at concentrations equivalent to those achievable in the plasma clinically (10, 50, or 200 nM) and measuring the number of complexes present in cells incubated for 15 min to 48 h with the drug. Regardless of the concentration of topotecan used, covalent topoisomerase I-DNA complexes were maximal within 15 min following addition of the lactone form of topotecan to the tissue culture medium. After 2 h of exposure to topotecan, complexes had decreased from maximum to approximately half of that value. Few, if any, complexes were detectable with topotecan incubations of 24-48 h. Growth inhibition studies showed that the IC50s of topotecan for the Daoy cell line (2.2 x 10(-9) M) and also for a second pediatric medulloblastoma cell line, SJ-Med3 (3.6 x 10(-9) M), exposed to topotecan 8 h daily for 5 days or continuous exposure were equivalent. The decrease in topoisomerase I-DNA complexes between 15 min and 1 h was consistent with a pH-dependent re-equilibration of topotecan to the less active hydroxyacid form of the drug. The decrease in complexes after a 2-48-h incubation with the drug was attributable neither to biological inactivation of topotecan as shown by sequential growth inhibition studies nor to a decrease in amount of topoisomerase I in the drug-treated cells. Indirect immunofluorescence labeling of topoisomerase I in Daoy cells incubated for 48 h with 10 nM topotecan showed a redistribution of nucleolar topoisomerase I. We are currently evaluating the antitumor effect of intermittent repetitive exposures to topotecan in mice bearing Daoy cells as a xenograft. The clinical utility of each effective schedule of exposure will depend on whether the therapeutic index of repetitive intermittent exposure to the drug is more or less favorable than the therapeutic index of continuous exposure.     

Camptothecins inhibit the utilization of hydrogen peroxide in the ligation step of topoisomerase I catalysis

The antitumor compounds camptothecin and its derivatives topotecan and irinotecan stabilize topoisomerase I cleavage complexes by inhibiting the religation reaction of the enzyme. Previous studies, using radiolabeled camptothecin or affinity labeling reagents structurally related to camptothecin, suggest that the agent binds at the topoisomerase I-DNA interface of the cleavage complexes, interacting with both the covalently bound enzyme and with the +1 base. In this study, we have investigated the molecular mechanism of camptothecin action further by taking advantage of the ability of topoisomerase I to couple non-DNA nucleophiles to the cleaved strand of the covalent enzyme-DNA complexes. This reaction of topoisomerase I was originally observed at moderate basic pH where active cleavage complexes mediate hydrolysis or alcoholysis by accepting water or polyhydric alcohol compounds as substitutes for a 5'-OH DNA end in the ligation step. Here, we report that a H2O2-derived nucleophile, presumably, the peroxide anion, facilitates the release of topoisomerase I from the cleavage complexes at neutral pH, and we present evidence showing that this reaction is mechanistically analogous to DNA ligation. We find that camptothecin, topotecan, and SN-38 (the active metabolite of irinotecan) inhibit H2O2 ligation mediated by cleavage complexes not containing DNA downstream of the cleavage site, indicating that drug interaction with DNA 3' to the covalently bound enzyme is not strictly required for the inhibition, although the presence of double-stranded DNA in this region enhances the drug effect. The results suggest that camptothecins prevent ligation by blocking the active site of the covalently bound enzyme.     

Low-level resistance to camptothecin in a human small-cell lung cancer cell line without reduction in DNA topoisomerase I or drug-induced cleavable complex formation

To study the evolution of camptothecin (CPT) resistance, we have established two small-cell lung cancer cell lines with low (3.2-fold, NYH/CAM15) and high (18-fold, NYH/CAM50) resistance to CPT by stepwise drug exposure. NYH/CAM50 cells had reduced topoisomerase I (topo I) content and activity, and consequently CPT-induced DNA single strand breaks (SSBs) were reduced, as measured by alkaline elution. In contrast, NYH/CAM15 cells had identical topo I content and activity as compared with wild-type (wt) cells. CPT-mediated SSBs and the rate of their reversal after drug removal were also equal in wt and NYH/CAM15 cells, as were doubling time, the fraction of cells in S-phase and DNA synthesis rate in response to CPT. As the conversion of DNA SSBs to DNA double strand breaks (DSBs) is thought to represent a critical event leading to cell death, we measured DNA DSBs by neutral elution. In contrast to DNA SSBs, CPT induced fewer DNA DSBs in NYH/CAM15 than in wt cells. DNA flow cytometry showed that, in CPT-treated cells, the G1 phase was emptied as cells accumulated in late S- and G2M phase. A Spearman rank correlation showed that depletion of G1 and accumulation in late S and G2M correlated to CPT sensitivity in these three cell lines. In conclusion, acquired resistance to CPT can occur without a reduction in either topo I enzyme or CPT-induced cleavable complex formation, while a decrease in the level of CPT-induced DNA DSBs may be of major importance in the early stages of CPT resistance.     

Differences in the topoisomerase I cleavage complexes formed by camptothecin and wakayin, a DNA-intercalating marine natural product

Wakayin is bispyrroloiminoquinone isolated from a Clavelina sp. ascidian by cytotoxicity directed fractionation. Like camptothecin, it has been found to inhibit the topoisomerase I catalyzed relaxation of supercoiled DNA. Wakayin enhanced cleavage complex formation at the same DNA sequences as camptothecin. Both compounds showed dose-related increases in cleavage complex formation, though wakayin's effect is attenuated at high concentrations. Wakayin is a string DNA binder. Wakayin also differed from camptothecin in that its cleavage complexes were much less stable than those of camptothecin in 0.5 M NaCl. Again in contrast to camptothecin, wakayin stabilized cleavage complexes poorly, if at all, at 0 degree C.     

Transforming growth factor-beta, endothelin-1, and c-fos expression in necrotizing/crescentic IgA glomerulonephritis

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA. The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.     

Mechanism of quinolone action. A drug-induced structural perturbation of the DNA precedes strand cleavage by topoisomerase IV

Quinolones are potent broad spectrum antibacterial drugs that target the bacterial type II DNA topoisomerases. Their cytotoxicity derives from their ability to shift the cleavage-religation equilibrium required for topoisomerase action toward cleavage, thereby effectively trapping the enzyme on the DNA. It has been proposed that these drugs act by binding to the enzyme-DNA complex. Using catalytically inactive and quinolone-resistant mutant topoisomerase IV proteins, nitrocellulose filter DNA binding assays, and KMnO4 probing of drug-DNA and drug-DNA-enzyme complexes, we show: (i) that norfloxacin binding to DNA induces a structural alteration, which probably corresponds to an unwinding of the helix, that is exacerbated by binding of the topoisomerase and by binding of the drug to the enzyme and (ii) that formation of this structural perturbation in the DNA precedes DNA cleavage by the topoisomerase in the ternary complex. We conclude that cleavage of the DNA and the resultant opening of the DNA gate during topoisomerization requires the induction of strain in the DNA that is bound to the enzyme. We suggest that quinolones may act to accelerate the rate of DNA cleavage by stimulating acquisition of this structural perturbation in the ternary complex.     

Formation of topoisomerase II alpha complexes with nascent DNA is related to VM-26-induced cytotoxicity

Several clinically active anticancer drugs are known to interfere with DNA topoisomerase II activity. However, the importance of the individual alpha (170 kDa) and beta (180 kDa) isozymes as targets of topoisomerase II-active drugs is not clear. To address this question, human CCRF-CEM leukemia cells were incubated with bromodeoxyuridine, and either the nascent DNA or bulk DNA not undergoing replication was purified by immunoprecipitation with an anti-bromodeoxyuridine antibody. The topoisomerase II isozymes that coprecipitated with either the nascent DNA or bulk DNA were analyzed by Western blotting. The alpha isozyme formed complexes with nascent DNA in cells pretreated with either VM-26 or mitoxantrone, while the beta isozyme was only bound to bulk DNA. At moderately cytotoxic concentrations, VM-26 enhanced the binding of topoisomerase II alpha to nascent DNA at least 5.2-fold compared to bulk DNA. However, in VM-26 resistant CEM/VM-1 cells incubated with equitoxic concentrations of VM-26, topoisomerase II alpha complex formation with nascent DNA was decreased at least 5.5-fold compared to bulk DNA. Drug-induced binding of topoisomerase II beta with bulk DNA in CEM/VM-1 cells did not correlate with cytotoxicity. Collectively, these results indicate that the formation of VM-26 stabilized complexes of topoisomerase II alpha with nascent DNA are critical to the development of cytotoxicity, and that resistance of CEM/VM-1 cells to VM-26 is related to impaired formation of these complexes. The results also provide indirect evidence that topoisomerase II alpha is involved in DNA, replication.