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.     

Analysis of etoposide binding to subdomains of human DNA topoisomerase II alpha in the absence of DNA

Epipodophyllotoxins are effective anti-tumor drugs that inhibit eukaryotic DNA topoisomerase II by trapping the enzyme in a covalent complex with DNA. We show that both the recombinant N-terminal ATPase domain and the B'A' core domain of human topoisomerase IIalpha (htopoIIalpha) bind radiolabeled etoposide specifically, even in the absence of DNA. The addition of ATP impairs etoposide binding to the holoenzyme and the N-terminal domain, but not to the core domain. To see if this interference resembles that between novobiocin and ATP in the bacterial GyrB subunit, we modeled the structure of the N-terminal domain of htopoIIalpha and performed molecular docking analysis with etoposide. Mutagenesis of critical amino acids, predicted to stabilize the drug within the N-terminal domain, reveals a less efficient binding of etoposide to the mutated proteins as monitored by direct drug binding assays, although the binding of ATP is not affected.     

ATP-independent DNA topoisomerase II as potential drug target in trypanosomes

Two categories of trypanosomal type II topoisomerases have been isolated from trypanosomes: one is unique since it is able to realize DNA topoisomerization reactions in the absence of ATP, in contrast to the other enzyme and mammalian topoisomerase II. The biochemical properties of ATP-independent topoisomerase II from Trypanosoma cruzi are described in this report. The enzyme can decatenate trypanosome kinetoplast DNA networks, catenate supercoiled DNA molecules, unknot P4 phage DNA, and cleave double-stranded DNA. The enzyme is inhibited by various classes of drugs and is more sensitive than mammalian topoisomerase II. Therefore, trypanosome ATP-independent topoisomerase II provides a potential target for chemotherapy.     

Altered DNA topoisomerase II in multidrug resistance

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected. We propose that MDR associated with alterations in topo II may have clinical consequences, and our current efforts involve exploiting these biochemical and molecular observations in the development of probes that may be useful to identify such drug resistant cells in the tumors of patients.     

Curcumin as a DNA topoisomerase II poison

Curcumin, the major active component of the spice turmeric, is recognised as a safe compound with great potential for cancer chemoprevention and cancer therapy. It induces apoptosis, but its initiation mechanism remains poorly understood. Curcumin has been assessed on the human cancer cell lines, TK-10, MCF-7 and UACC-62, and their IC50 values were 12.16, 3.63, 4.28 microM respectively. The possibility of this compound being a topoisomerase II poison has also been studied and it was found that 50 microM of curcumin is active in a similar fashion to the antineoplastic agent etoposide. These results point to DNA damage induced by topoisomerase II poisoning as a possible mechanism by which curcumin initiates apoptosis, and increase the evidence suggesting its possible use in cancer therapy.     

DNA topoisomerase II mutations and resistance to anti-tumor drugs

Mutations in DNA topoisomerase II are often correlated with drug-resistance in tumor cell lines. Studies of topoisomerase II-mediated drug-resistance in various model systems, as well as the sequencing of such mutations from drug-resistant tumors, have shed light on the functional domains of topoisomerase II, on how it interacts with inhibitors, and on the different mechanisms by which cells avoid the toxic effects of many clinically important anti-tumor drugs.     

The role of topoisomerase II in drug resistance.

The conventional laboratory approach to study the mechanisms of drug resistance has been the selection of drug-resistant cell lines by continuous exposure to cytotoxic agents. Such lines, which are selected for resistance to a single agent, frequently display cross-resistance to a number of cytotoxic agents that are unrelated in both structure and proposed mechanism of action. Multidrug-resistant cells display reduced drug accumulation, which is the result of overexpression of a surface glycoprotein (P170). Although resistance to multiple antitumor agents is a common clinical problem in the treatment of cancer, the precise role of the P-glycoprotein-mediated mechanism in human tumors remains to be established. Many alterations in multidrug-resistant cells selected in vitro have been identified. The concomitant expression of multiple phenotypic differences, which appear to be favored by continued and prolonged drug exposure, makes analysis of critical individual resistance pathways more difficult. However, multiple factors may also be involved in the development of clinical resistance. Recent studies have identified alterations in DNA topoisomerase II activity and function as an alternative mechanism that contributes to the multidrug-resistance phenomenon or is responsible for a different type of drug resistance. The precise nature of these changes remains unclear. Available evidence supports the view that expression of the enzyme is an important determinant of cell sensitivity to DNA topoisomerase poisons, but that other changes involved in regulation of enzyme function and/or in the cellular processing of drug-induced DNA damage may be critical in determining the differential pattern of cell response to antitumor agents.     

Mechanisms of drug resistance of two cell lines of human chronic promyelocytic leukemia K562, resistant to DNA topoisomerase II inhibitors adriamycin and etoposide]

Mechanisms of drug-resistance in two K562 cell lines selected for adriamycin and etoposide resistance (K562-ADR and K562-VP16, respectively) were studied. In K562-ADR cells, overexpression of mdr 1 gene and two-fold reduction of topoisomerase II alpha mRNA content were found, while topoisomerase II beta expression remained unchanged, compared to the parental cell line. Antiapoptotic bcl-2 mRNA level was four-fold decreased in K562-ADR cells, while the expression of other members of bcl-2 family was unaffected. In K562-VP16 cells five-fold reduction of topoisomerase II alpha expression was found with the absence of mdr 1 gene overexpression. The expression of antiapoptotic bcl-2 and proapoptotic bax genes was reduced in K562-VP16 cell line, while the content of bcl-2 mRNA was increased. Cytogenetic analysis of K562-VP16 cells revealed morphological changes in their cell karyotype and susceptibility of these cells to spontaneous polyploidization. Possible effects of etoposite on mitotic control in K562-VP16 cells are discussed.     

DNA topoisomerase II as the target for the anticancer drug TOP-53: mechanistic basis for drug action

TOP-53 is a promising anticancer agent that displays high activity against non-small cell lung cancer in animal tumor models [Utsugi, T., et al. (1996) Cancer Res. 56, 2809-2814]. Compared to its parent compound, etoposide, TOP-53 is considerably more toxic to non-small cell lung cancer cells, is more active at generating chromosomal breaks, and displays improved cellular uptake and pharmacokinetics in animal lung tissues. Despite the preclinical success of TOP-53, several questions remain regarding its cytotoxic mechanism. Therefore, this study characterized the basis for drug action. Results indicate that topoisomerase II is the primary cytotoxic target for TOP-53. Furthermore, the drug kills cells by acting as a topoisomerase II poison. TOP-53 exhibits a DNA cleavage site specificity that is identical to that of etoposide. Like its parent compound, the drug increases the number of enzyme-mediated DNA breaks by interfering with the DNA religation activity of the enzyme. TOP-53 is considerably more efficient than etoposide at enhancing topoisomerase II-mediated DNA cleavage and exhibits high activity against human topoisomerase IIalpha and IIbeta in vitro and in cultured cells. Therefore, at least in part, the enhanced cytotoxic activity of TOP-53 can be attributed to an enhanced activity against topoisomerase II. Finally, TOP-53 displays nearly wild-type activity against a mutant yeast type II enzyme that is highly resistant to etoposide. This finding suggests that TOP-53 can retain activity against systems that have developed resistance to etoposide, and indicates that substituents on the etoposide C-ring are important for topoisomerase II-drug interactions.     

Enhanced DNA repair as a mechanism of resistance to cis-diamminedichloroplatinum(II)

Murine leukemia L1210 cells, either sensitive or resistant to the toxic action of the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II), have been studied for potential differences in the formation and repair of drug-induced DNA damage. The sensitivity for these experiments was obtained by using the radiolabeled analogue [3H]-cis-dichloro(ethylenediamine)platinum(II). The resistant cells demonstrated a 40% reduction in drug accumulation but a qualitatively similar profile of DNA-bound adducts. These adducts resembled those previously characterized in pure DNA and represented intrastrand cross-links at GG, AG, and GNG (N is any nucleotide) sequences in DNA. Repair of these cross-links occurred in a biphasic manner: rapid for the first 6 h and then much slower. The resistant cells removed up to 4 times as many adducts during the rapid phase of repair. The extent of this repair did not directly correlate with the degree of resistance in that cells with 100-fold resistance were only slightly more effective at repair than cells with 20-fold resistance. Therefore, although enhanced DNA repair is thought to contribute markedly to drug resistance, other mechanisms for tolerance of DNA damage may also occur in these cells.     

Random mutagenesis of the B'A' core domain of yeast DNA topoisomerase II and large-scale screens of mutants resistant to the anticancer drug etoposide

Mutagenic PCR method was applied to introduce point mutations to the B'A' core domain of yeast DNA topoisomerase II. Screens for mutants resistant to the anticancer drug etoposide were carried out in a yeast ts system in the presence of high concentrations of the drug or in a drug-hypersensitive genetic background. 129 mutants were obtained from a total of 47,000 transformants. Nucleotide sequencing of 40 selected mutants showed that a large number of the mutations map to regions encoding the linker that joins the ATPase domain to the B' module and the B'A' linker. Significant reduction in catalytic activity was evident for a large fraction of mutant enzymes and all mutants were also resistant to amsacrine, another topoisomerase II drug with a different chemical structure, suggesting that few of the mutations reflect simple changes of specific amino acid side chains that are directly involved in enzyme-drug interactions.     

The cytoplasmic trafficking of DNA topoisomerase IIalpha correlates with etoposide resistance in human myeloma cells

In this study we have investigated the role of topoisomerase (topo) IIalpha trafficking in cellular drug resistance. To accomplish this, it was necessary to separate the influence of cell cycle, drug uptake, topo protein levels, and enzyme trafficking on drug sensitivity. Thus, we developed a cell model (called accelerated plateau) using human myeloma H929 cells that reproducibly translocates topo IIalpha to the cytoplasm. Compared to log-phase cells, the cytoplasmic redistribution of topo IIalpha in plateau-phase cells correlated with a 10-fold resistance to VP-16 and a 40-60% reduction in the number of drug-induced double-strand DNA breaks. In addition, 7-fold more VP-16 was necessary to achieve 50% topo IIalpha band depletion, suggesting that there are fewer drug-induced topo-DNA complexes formed in quiescent cells than in log-phase cells. The total cellular amount of topo IIalpha and topo IIbeta protein in log- and plateau-phase cells was similar as determined by Western blot analysis. There was a 25% reduction in S-phase cell number in plateau cells (determined by bromodeoxyuridine (BrdU) incorporation), while there was no significant difference in the equilibrium concentrations of [(3)H]-VP-16 when log cells were compared with plateau cells. Furthermore, the nuclear/cytoplasmic ratio of topo IIalpha is increased 58-fold in accelerated-plateau H929 cells treated with leptomycin B (LMB) when compared to untreated cells. It appears that the nuclear-cytoplasmic shuttling of topo IIalpha, which decreases the amount of nuclear target enzyme, is a major mechanism of drug resistance to topo II inhibitors in plateau-phase myeloma cells.     

Binding mode and affinity studies of DNA-binding agents using topoisomerase I DNA unwinding assay

A topoisomerase I DNA unwinding assay has been used to determine the relative DNA-binding affinities of a model pair of homologous naphthalene diimides. Binding affinity data were corroborated using calorimetric (ITC) and spectrophotometric (titration and T(m)) studies, with substituent size playing a significant role in binding. The assay was also used to investigate the mode of binding adopted by several known DNA-binding agents, including SYBR Green and PicoGreen. Some of the compounds exhibited unexpected binding modes.     

Antitumor agents. Part 235: Novel 4'-ester etoposide analogues as potent DNA topoisomerase II inhibitors with improved therapeutic potential

Eight 4'-ester epipodophyllotoxin derivatives (9-16) were designed and synthesized with the aim to overcome drug-resistance and improve water-solubility simultaneously. These compounds were superior to etoposide (1) in causing cellular protein-linked DNA breaks and inhibiting KB and 1-resistant KB-7d cell replication. Compounds 9 and 10 showed significant inhibitory activity against DNA topoisomerase II in vitro. Compound 10 also exhibited an in vitro DNA cleavage pattern similar to that of GL-331 (5). A hypothetical model on the action mode of 1-analogues is proposed based on the results.     

Sitamaquine as a putative antileishmanial drug candidate: from the mechanism of action to the risk of drug resistance

Sitamaquine is a 8-aminoquinoline in development for the treatment of visceral leishmaniasis by oral route, no activity being observed on the experimental cutaneous leishmaniasis experimental models. Recent data explain how sitamaquine accumulate in Leishmania parasites, however its molecular targets remain to be identified. An advantage of sitamaquine is its short elimination half-life, preventing a rapid resistance emergence. The antileishmanial action of its metabolites is not known. The selection of a sitamaquine-resistant clone of L. donovani in laboratory and the phase II clinical trials pointing out some adverse effects such as methemoglobinemia and nephrotoxicity are considered for a further development decision.     

Defective DNA repair as a potential mechanism for the rapid development of drug resistance in Plasmodium falciparum

The development and spread of highly drug-resistant parasites pose a central problem in the control of malaria.Understanding mechanisms that regulate genomic stability, such as DNA repair, in drug-resistant parasites and during drug treatment may help determine whether this rapid onset of resistance is due to an increase in the rate at which resistance-causing mutations are generated. This is the first report to demonstrate DNA repair activities from the malaria-causing parasite Plasmodium falciparum that are specific for ultraviolet light-induced DNA damage. The efficiency of DNA repair differs dramatically among P. falciparum strains with varying drug sensitivities. Most notable is the markedly reduced level of repair in the highly drug-resistant W2 isolate, which has been shown to develop resistance to novel drugs at an increased rate when compared to drug-sensitive strains. Additionally, the antimalarial drug chloroquine and other quinoline-like compounds interfered with the DNA synthesis step of the repair process, most likely a result of direct binding to repair substrates. We propose that altered DNA repair, either through defective repair mechanisms or drug-mediated inhibition, may contribute to the accelerated development of drug resistance in the parasite.     

Effect of antineoplastic agents on the DNA cleavage/religation reaction of eukaryotic topoisomerase II: inhibition of DNA religation by etoposide

Beyond its essential physiological functions, topoisomerase II is the primary cellular target for a number of clinically relevant antineoplastic drugs. Although the chemotherapeutic efficacies of these drugs correlate with their abilities to stabilize the covalent topoisomerase II-DNA cleavage complex, their molecular mechanism of action has yet to be described. In order to characterize the drug-induced stabilization of this enzyme-DNA complex, the effect of etoposide on the DNA cleavage/religation reaction of Drosophila melanogaster topoisomerase II was studied. Under the conditions employed, etoposide increased levels of enzyme-mediated double-stranded DNA cleavage 5-6-fold and single-stranded cleavage approximately 4-fold. Maximal stimulation was observed at 80-100 microM etoposide with 50% of the maximal effect at approximately 15 microM drug. By employing a topoisomerase II mediated DNA religation assay [Osheroff, N. & Zechiedrich, E.L. (1987) Biochemistry 26, 4303-4309], etoposide was found to stabilize the enzyme-DNA cleavage complex (at least in part) by inhibiting the enzyme's ability to religate cleaved DNA. Moreover, in order for the drug to affect religation, it has to be present at the time of DNA cleavage.     

Synthetic lanostane-type triterpenoids as inhibitors of DNA topoisomerase II

DNA topoisomerase (Topo) II is one of the target enzymes for chemotherapeutic drug development. Lanostane-type triterpenoids with various functional groups (-Cl, -Br, -OMe, -CHO, -CN, -COOH, and -COOMe) at C-2 were synthesized from 3-oxolanost-9(11)-en-24S,25-diol (9) isolated from Pinus luchuensis and their inhibitory effects on Topo II activity and cytotoxic activities against A549 cells were examined. All the derivatives showed Topo II inhibitory effects with IC50 values ranging from 1.86 to 149.97 microM and cytotoxic activities with ED50 values ranging from 3.96 to 38.15 microM.     

Sequence-selective topoisomerase II inhibition by anthracycline derivatives in SV40 DNA: relationship with DNA binding affinity and cytotoxicity

Topoisomerase II mediated double-strand breaks produced by anthracycline analogues were studied in SV40 DNA. The compounds included doxorubicin, daunorubicin, two doxorubicin stereoisomers (4'-epimer and beta-anomer), and five chromophore-modified derivatives, with a wide range of cytotoxic activity and DNA binding affinity. Cleavage of 32P-end-labeled DNA fragments was visualized by autoradiography of agarose and polyacrylamide gels. Structure-activity relationships indicated that alterations in the chromophore structure greatly affected drug action on topoisomerase II. In particular, removal of substituents on position 4 of the D ring resulted in more active inducers of cleavage with lower DNA binding affinity. The stereochemistry between the sugar and the chromophore was also essential for activity. All the active anthracyclines induced a single region of prominent cleavage in the entire SV40 DNA, which resulted from a cluster of sites between nucleotides 4237 and 4294. DNA cleavage intensity patterns exhibited differences among analogues and were also dependent upon drug concentration. Intensity at a given site depended on both stimulatory and suppressive effects depending upon drug concentration and DNA sequence. A good correlation was found between cytotoxicity and intensity of topoisomerase II mediated DNA breakage.