A diazirine-based photoaffinity etoposide probe for labeling topoisomerase II

Etoposide is a widely used anticancer drug that targets topoisomerase II, an essential nuclear enzyme. However, despite the fact that it has been in use and studied for more than 30 years the specific site on the enzyme to which it binds is unknown. In order to identify the etoposide binding site(s) on topoisomerase II, a diazirine-based photoaffinity etoposide analog probe has been synthesized and its photoreactivity and biological activities have been characterized. Upon UV irradiation, the diazirine probe rapidly produced a highly reactive carbene species that formed covalent adducts containing stable carbon-based bonds indicating that it should also be able to form stable covalent adducts with amino acid residues on topoisomerase II. The human leukemia K562 cell growth and topoisomerase II inhibitory properties of the diazirine probe suggest that it targets topoisomerase II in a manner similar to etoposide. The diazirine probe was also shown to act as a topoisomerase II poison through its ability to cause topoisomerase IIα-mediated double-strand cleavage of DNA. Additionally, the diazirine probe significantly increased protein–DNA covalent complex formation upon photoirradiation of diazirine probe-treated K562 cells, as compared to etoposide-treated cells. This result suggests that the photoactivated probe forms a covalent adduct with topoisomerase IIα. In conclusion, the present characterization of the chemical, biochemical, and biological properties of the newly synthesized diazirine-based photoaffinity etoposide analog indicates that use of a proteomics mass spectrometry approach will be a tractable strategy for future identification of the etoposide binding site(s) on topoisomerase II through covalent labeling of amino acid residues.     

Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II

HL-60/AMSA is a human leukemia cell line that is 100 times more resistant to the cytotoxic actions of the antineoplastic, topoisomerase II-reactive DNA intercalating acridine derivative amsacrine (m-AMSA) than is its parent HL-60 line. HL-60/AMSA cells are minimally resistant to etoposide, a topoisomerase II-reactive drug that does not intercalate. Previously we showed that HL-60 topoisomerase II activity in cells, nuclei, or nuclear extracts was sensitive to m-AMSA and etoposide, while HL-60/AMSA topoisomerase II was resistant to m-AMSA but sensitive to etoposide. Now we show that purified topoisomerase II from the two cell lines exhibits the same drug sensitivity or resistance as that in the nuclear extracts although the magnitude of the m-AMSA resistance of HL-60/AMSA topoisomerase II in vitro is not as great as the resistance of the intact HL-60/AMSA cells. In addition HL-60/AMSA cells are cross-resistant to topoisomerase II-reactive intercalators from the anthracycline and ellipticine families and the pattern of sensitivity or resistance to the cytotoxic actions of the various topoisomerase II-reactive drugs is paralleled by topoisomerase II-reactive drug-induced DNA cleavage and protein cross-link production in cells and the production of drug-induced, topoisomerase II-mediated DNA cleavage and protein cross-linking in isolated biochemical systems. In addition to its lowered sensitivity to intercalators, HL-60/AMSA differed from HL-60 in 1) the susceptibility of its topoisomerase II to stimulation of DNA topoisomerase II complex formation by ATP, 2) the catalytic activity of its topoisomerase II in an ionic environment chosen to reproduce the environment found within the living cell, and 3) the observed restriction enzyme pattern on a Southern blot probed with a cDNA for human topoisomerase II. These data indicate that an m-AMSA-resistant form of topoisomerase II contributes to the resistance of HL-60/AMSA to m-AMSA and to other topoisomerase II-reactive DNA intercalating agents. The drug resistance is associated with additional biochemical and molecular alterations that may be important determinants of cellular sensitivity or resistance to topoisomerase II-reactive drugs.     

Contributions of the D-Ring to the activity of etoposide against human topoisomerase IIα: potential interactions with DNA in the ternary enzyme--drug--DNA complex

Etoposide is a widely prescribed anticancer drug that stabilizes covalent topoisomerase II-cleaved DNA complexes. The drug contains a polycyclic ring system (rings A-D), a glycosidic moiety at C4, and a pendant ring (E-ring) at C1. Interactions between human topoisomerase IIα and etoposide in the binary enzyme--drug complex appear to be mediated by substituents on the A-, B-, and E-rings of etoposide. These protein--drug contacts in the binary complex have predictive value for the actions of etoposide within the ternary topoisomerase IIα--drug--DNA complex. Although the D-ring of etoposide does not appear to contact topoisomerase IIα in the binary complex, etoposide derivatives with modified D-rings display reduced cytotoxicity against murine leukemia cells [Meresse, P., et al. (2003) Bioorg. Med. Chem. Lett. 13, 4107]. This finding suggests that alterations in the D-ring may affect etoposide activity toward topoisomerase IIα in the ternary enzyme--drug--DNA complex. Therefore, to address the potential contributions of the D-ring to the activity of etoposide, we characterized drug derivatives in which the C13 carbonyl was moved to the C11 position (retroetoposide and retroDEPT) or the D-ring was opened (D-ring diol). All of the D-ring alterations decreased the ability of etoposide to enhance DNA cleavage mediated by human topoisomerase IIα in vitro and in cultured cells. They also weakened etoposide binding in the ternary enzyme--drug--DNA complex and altered sites of enzyme-mediated DNA cleavage. On the basis of these findings, we propose that the D-ring of etoposide has important interactions with DNA in the ternary topoisomerase II cleavage complex.     

Effects of antineoplastic drugs on the post-strand-passage DNA cleavage/religation equilibrium of topoisomerase II.

The post-strand-passage DNA cleavage/religation equilibrium of Drosophila melanogaster topoisomerase II was examined. This was accomplished by including adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analogue which supports strand passage but not enzyme turnover, in assays. Levels of post-strand-passage enzyme-mediated DNA breakage were 3-5 times higher than those generated by topoisomerase II prior to the strand-passage event. This finding correlated with a decrease in the apparent first-order rate of topoisomerase II mediated DNA religation in the post-strand-passage cleavage complex. Since previous studies demonstrated that antineoplastic drugs stabilize the pre-strand-passage cleavage complex of topoisomerase II by impairing the enzyme's ability to religate cleaved DNA [Osheroff, N. (1989) Biochemistry 28, 6157-6160; Robinson, M.J., & Osheroff, N. (1990) Biochemistry 29, 2511-2515], the effects of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide on the enzyme's post-strand-passage DNA cleavage complex were characterized. Both drugs stimulated the ability of topoisomerase II to break double-stranded DNA after strand passage. As determined by two independent assay systems, m-AMSA and etoposide stabilized the enzyme's post-strand-passage DNA cleavage complex primarily by inhibiting DNA religation. These results strongly suggest that both the pre- and post-strand-passage DNA cleavage complexes of topoisomerase II serve as physiological targets for these structurally disparate antineoplastic drugs.     

Topoisomerase-specific drug sensitivity in relation to cell cycle progression.

The nuclear enzyme DNA topoisomerase II catalyzes the breakage and resealing of duplex DNA and plays an important role in several genetic processes. It also mediates the DNA cleavage activity and cytotoxicity of clinically important anticancer agents such as etoposide. We have examined the activity of topoisomerase II during the first cell cycle of quiescent BALB/c 3T3 cells following serum stimulation. Etoposide-mediated DNA break frequency in vivo was used as a parameter of topoisomerase II activity, and enzyme content was assayed by immunoblotting. Density-arrested A31 cells exhibited a much lower sensitivity to the effects of etoposide than did actively proliferating cells. Upon serum stimulation of the quiescent cells, however, there was a marked increase in drug sensitivity which began during S phase and reached its peak just before mitosis. Maximal drug sensitivity during this period was 2.5 times greater than that of log-phase cells. This increase in drug sensitivity was associated with an increase in intracellular topoisomerase II content as determined by immunoblotting. The induction of topoisomerase II-mediated drug sensitivity was aborted within 1 h of exposure of cells to the protein synthesis inhibitor cycloheximide, but the DNA synthesis inhibitor aphidicolin had no effect. In contrast to the sensitivity of cells to drug-induced DNA cleavage, maximal cytotoxicity occurred during S phase. A 3-h exposure to cycloheximide before etoposide treatment resulted in nearly complete loss of cytotoxicity. Our findings indicate that topoisomerase II activity fluctuates with cell cycle progression, with peak activity occurring during the G2 phase. This increase in topoisomerase II is protein synthesis dependent and may reflect a high rate of enzyme turnover. The dissociation between maximal drug-induced DNA cleavage and cytotoxicity indicates that the topoisomerase-mediated DNA breaks may be necessary but are not sufficient for cytotoxicity and that the other factors which are particularly expressed during S phase may be important as well.     

Numerical Analysis of Etoposide Induced DNA Breaks

Background

Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs.

Methodology/Principal Findings

To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity.

Conclusions/Significance

These results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity.     

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.     

Etoposide stimulates 1,25-dihydroxyvitamin D3 differentiation activity,hormone binding and hormone receptor expression in HL-60 human promyelocytic cells

The simultaneous administration of the DNA topoisomerase II inhibitor etoposide (0.15 mM) and 1,25-dihydroxyvitamin D₃ (VD₃) (10 nM) synergistically induced the differentiation of HL-60 human promyelocytic leukemia cells. Similar results were obtained using U-937 human promonocytic cells, or the topoisomerase II inhibitors doxorubicin (15 nM) and mitoxantrone (2.5 nM). When sequential treatments were used, pre-incubation with VD₃ had little effect on the subsequent action of etoposide, while pre-incubation with etoposide greatly potentiated the subsequent action of VD₃. In addition, etoposide treatment stimulated VD₃ binding activity and increased VD₃ receptor mRNA and protein levels. The increase in hormone receptor expression may explain, at least in part, the capacity of topoisomerase inhibitors to potentiate the differentiation inducing activity of VD₃.     

Ciprofloxacin and etoposide (VP16) produce a similar pattern of DNA cleavage in a plasmid of an archaebacterium

The fluoroquinolone ciprofloxacin, an inhibitor of eubacterial DNA gyrase, induces single- and double-stranded DNA breaks in the plasmid pGRB-1 from the halophilic archaebacterium Halobacterium GRB when the cells are treated by this drug in a magnesium-depleted medium. This reaction is prevented by a dose of novobiocin known to specifically inhibit DNA gyrase. Cleavage of pGRB-1 DNA induced by either ciprofloxacin or the antitumoral drug etoposide (VP16) produces DNA fragments of identical lengths. These results indicate that ciprofloxacin, novobiocin, and etoposide have a common target in Halobacterium GRB: an archaebacterial type II DNA topoisomerase. The similarity of DNA cleavage patterns induced by ciprofloxacin and etoposide is a new and strong argument that quinolone and epipodophyllotoxins have the same mode of interaction with the DNA-DNA topoisomerase II complexes. The plasmid pGRB-1 could be used to prescreen in the same system both antibiotics that inhibit bacterial gyrase and antitumoral drugs that inhibit eukaryotic DNA topoisomerase II.     

The efficacy of topoisomerase II-targeted anticancer agents reflects the persistence of drug-induced cleavage complexes in cells

Genistein, a widely consumed bioflavonoid with chemopreventative properties in adults, and etoposide, a commonly prescribed anticancer drug, are well-characterized topoisomerase II poisons. Although both compounds display similar potencies against human topoisomerase IIalpha and IIbeta in vitro and induce comparable levels of DNA cleavage complexes in cultured human cells, their cytotoxic and genotoxic effects differ significantly. As determined by assays that monitored cell viability or the phosphorylation of histone H2AX, etoposide was much more toxic in CEM cells than genistein. Further studies that characterized the simultaneous treatment of cells with genistein and etoposide indicate that the differential actions of the two compounds are not related to the effects of genistein on cellular processes outside of its activity against topoisomerase II. Rather, they appear to result from a longer persistence of cleavage complexes induced by etoposide as compared to genistein. Parallel in vitro studies with purified type II enzymes led to similar conclusions regarding cleavage complex persistence. Isoform-specific differences were observed in vitro and in cells treated with etoposide. To this point, the t 1/2 of etoposide-induced DNA cleavage complexes formed with topoisomerase IIalpha in CEM cells was approximately 5 times longer than those formed with topoisomerase IIbeta. The cytotoxicity of etoposide following four treatment-recovery cycles was similar to that induced by continuous exposure to the drug over an equivalent time period. Taken together, these findings suggest that it may be possible to preferentially target topoisomerase IIalpha with etoposide by employing a schedule that utilizes pulsed drug treatment-recovery cycles.     

Etoposide quinone is a redox-dependent topoisomerase II poison

Etoposide is a topoisomerase II poison that is used to treat a variety of human cancers. Unfortunately, 2-3% of patients treated with etoposide develop treatment-related leukemias characterized by 11q23 chromosomal rearrangements. The molecular basis for etoposide-induced leukemogenesis is not understood but is associated with enzyme-mediated DNA cleavage. Etoposide is metabolized by CYP3A4 to etoposide catechol, which can be further oxidized to etoposide quinone. A CYP3A4 variant is associated with a lower risk of etoposide-related leukemias, suggesting that etoposide metabolites may be involved in leukemogenesis. Although etoposide acts at the enzyme-DNA interface, several quinones poison topoisomerase II via redox-dependent protein adduction. The effects of etoposide quinone on topoisomerase IIα-mediated DNA cleavage have been examined previously. Although findings suggest that the activity of the quinone is slightly greater than that of etoposide, these studies were carried out in the presence of significant levels of reducing agents (which should reduce etoposide quinone to the catechol). Therefore, we examined the ability of etoposide quinone to poison human topoisomerase IIα in the absence of reducing agents. Under these conditions, etoposide quinone was ～5-fold more active than etoposide at inducing enzyme-mediated DNA cleavage. Consistent with other redox-dependent poisons, etoposide quinone inactivated topoisomerase IIα when incubated with the protein prior to DNA and lost activity in the presence of dithiothreitol. Unlike etoposide, the quinone metabolite did not require ATP for maximal activity and induced a high ratio of double-stranded DNA breaks. Our results support the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis.     

Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.     

Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks

Topoisomerase II is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating transient double-strand DNA breaks. While the enzyme cannot perform its essential cellular functions without cleaving DNA, this scission activity is inherently dangerous to chromosomal integrity. In fact, etoposide and other clinically important anticancer drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. Cells rely heavily on recombination to repair double-strand DNA breaks, but the specific pathways used to repair topoisomerase II-generated DNA damage have not been defined. Therefore, Saccharomyces cerevisiae was used as a model system to delineate the recombination pathways that repair DNA breaks generated by topoisomerase II. Yeast cells that expressed wild-type or a drug-hypersensitive mutant topoisomerase II or overexpressed the wild-type enzyme were examined. Based on cytotoxicity and recombination induced by etoposide in different repair-deficient genetic backgrounds, double-strand DNA breaks generated by topoisomerase II appear to be repaired primarily by the single-strand invasion pathway of homologous recombination. Non-homologous end joining also was triggered by etoposide treatment, but this pathway was considerably less active than single-strand invasion and did not contribute significantly to cell survival in S.cerevisiae.     

A truncated form of DNA topoisomerase IIbeta associates with the mtDNA genome in mammalian mitochondria.

Despite the likely requirement for a DNA topoisomerase II activity during synthesis of mitochondrial DNA in mammals, this activity has been very difficult to identify convincingly. The only DNA topoisomerase II activity conclusively demonstrated to be mitochondrial in origin is that of a type II activity found associated with the mitochondrial, kinetoplast DNA network in trypanosomatid protozoa [Melendy, T., Sheline, C., and Ray, D.S. (1988) Cell 55, 1083-1088; Shapiro, T.A., Klein, V.A., and Englund, P.A. (1989) J. Biol. Chem.264, 4173-4178]. In the present study, we report the discovery of a type DNA topoisomerase II activity in bovine mitochondria. Identified among mtDNA replicative proteins recovered from complexes of mtDNA and protein, the DNA topoisomerase relaxes a negatively, supercoiled DNA template in vitro, in a reaction that requires Mg2+ and ATP. The relaxation activity is inhibited by etoposide and other inhibitors of eucaryotic type II enzymes. The DNA topoisomerase II copurifies with mitochondria and directly associates with mtDNA, as indicated by sensitivity of some mtDNA circles in the isolated complex of mtDNA and protein to cleavage by etoposide. The purified activity can be assigned to a approximately 150-kDa protein, which is recognized by a polyclonal antibody made against the trypanosomal mitochondrial topo II enzyme. Mass spectrometry performed on peptides prepared from the approximately 150-kDa protein demonstrate that this bovine mitochondrial activity is a truncated version of DNA topoisomerase IIbeta, one of two DNA topoisomerase II activities known to exist in mammalian nuclei.     

Cross-resistance of an amsacrine-resistant human leukemia line to topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions.

HL-60/AMSA is a human leukemia cell line that is 50-100-fold more resistant than its drug-sensitive HL-60 parent line to the cytotoxic actions of the DNA intercalator amsacrine (m-AMSA). HL-60/AMSA topoisomerase II is also resistant to the inhibitory actions of m-AMSA. HL-60/AMSA cells and topoisomerase II are cross-resistant to anthracycline and ellipticine intercalators but relatively sensitive to the nonintercalating topoisomerase II reactive epipodophyllotoxin etoposide. We now demonstrate that HL-60/AMSA and its topoisomerase II are cross-resistant to the DNA intercalators mitoxantrone and amonafide, thus strongly indicating that HL-60/AMSA and its topoisomerase II are resistant to topoisomerase II reactive intercalators but not to nonintercalators. At high concentrations, mitoxantrone and amonafide were also found to inhibit their own, m-AMSA's, and etoposide's abilities to stabilize topoisomerase II-DNA complexes. This appears to be due to the ability of these concentrations of mitoxantrone and amonafide to inhibit topoisomerase II mediated DNA strand passage at a point in the topoisomerization cycle prior to the acquisition of the enzyme-DNA configuration that yields DNA cleavage and topoisomerase II-DNA cross-links. In addition, amonafide can inhibit the cytotoxic actions of m-AMSA and etoposide. Taken together, these results suggest that the cytotoxicity of m-AMSA and etoposide is initiated primarily by the stabilization of the topoisomerase II-DNA complex. Other topoisomerase II reactive drugs may inhibit the enzyme at other steps in the topoisomerization cycle, particularly at elevated concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between the etoposide derivative F14512 and human type II topoisomerases: implications for the C4 spermine moiety in promoting enzyme-mediated DNA cleavage

F14512 is a novel etoposide derivative that contains a spermine in place of the C4 glycosidic moiety. The drug was designed to exploit the polyamine transport system that is upregulated in some cancers. However, a preliminary study suggests that it is also a more efficacious topoisomerase II poison than etoposide [Barret et al. (2008) Cancer Res. 68, 9845-9853]. Therefore, we undertook a more complete study of the actions of F14512 against human type II topoisomerases. As determined by saturation transfer difference (1)H NMR spectroscopy, contacts between F14512 and human topoisomerase IIα in the binary enzyme-drug complex are similar to those of etoposide. Although the spermine of F14512 does not interact with the enzyme, it converts the drug to a DNA binder [Barret et al. (2008)]. Consequently, the influence of the C4 spermine on drug activity was assessed. F14512 is a highly active topoisomerase II poison and stimulates DNA cleavage mediated by human topoisomerase IIα or topoisomerase IIβ. The drug is more potent and efficacious than etoposide or TOP-53, an etoposide derivative that contains a C4 aminoalkyl group that strengthens drug-enzyme binding. Unlike the other drugs, F14512 maintains robust activity in the absence of ATP. The enhanced activity of F14512 correlates with a tighter binding and an increased stability of the ternary topoisomerase II-drug-DNA complex. The spermine-drug core linkage is critical for these attributes. These findings demonstrate the utility of a C4 DNA binding group and provide a rational basis for the development of novel and more active etoposide-based topoisomerase II poisons.     

The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.     

DNA topoisomerases as targets for the anticancer drug TAS-103: primary cellular target and DNA cleavage enhancement

TAS-103 is a novel antineoplastic agent that is active against in vivo tumor models [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. This drug is believed to be a dual topoisomerase I/II-targeted agent, because it enhances both topoisomerase I- and topoisomerase II-mediated DNA cleavage in treated cells. However, the relative importance of these two enzymes for the cytotoxic actions of TAS-103 is not known. Therefore, the primary cellular target of the drug and its mode of action were determined. TAS-103 stimulated DNA cleavage mediated by mammalian topoisomerase I and human topoisomerase IIalpha and beta in vitro. The drug was less active than camptothecin against the type I enzyme but was equipotent to etoposide against topoisomerase IIalpha. A yeast genetic system that allowed manipulation of topoisomerase activity and drug sensitivity was used to determine the contributions of topoisomerase I and II to drug cytotoxicity. Results indicate that topoisomerase II is the primary cellular target of TAS-103. In addition, TAS-103 binds to human topoisomerase IIalpha in the absence of DNA, suggesting that enzyme-drug interactions play a role in formation of the ternary topoisomerase II.drug.DNA complex. TAS-103 induced topoisomerase II-mediated DNA cleavage at sites similar to those observed in the presence of etoposide. Like etoposide, it enhanced cleavage primarily by inhibiting the religation reaction of the enzyme. Based on these findings, it is suggested that TAS-103 be classified as a topoisomerase II-targeted drug.     

Polyamine-containing etoposide derivatives as poisons of human type II topoisomerases: Differential effects on topoisomerase IIα and IIβ

Etoposide is an anticancer drug that acts by inducing topoisomerase II-mediated DNA cleavage. Despite its wide use, etoposide is associated with some very serious side-effects including the development of treatment-related acute myelogenous leukemias. Etoposide targets both human topoisomerase IIα and IIβ. However, the contributions of the two enzyme isoforms to the therapeutic vs. leukemogenic properties of the drug are unclear. In order to develop an etoposide-based drug with specificity for cancer cells that express an active polyamine transport system, the sugar moiety of the drug has been replaced with a polyamine tail. To analyze the effects of this substitution on the specificity of hybrid molecules toward the two enzyme isoforms, we analyzed the activity of a series of etoposide-polyamine hybrids toward human topoisomerase IIα and IIβ. All of the compounds displayed an ability to induce enzyme-mediated DNA cleavage that was comparable to or higher than that of etoposide. Relative to the parent drug, the hybrid compounds displayed substantially higher activity toward topoisomerase IIβ than IIα. Modeling studies suggest that the enhanced specificity may result from interactions with Gln778 in topoisomerase IIβ. The corresponding residue in the α isoform is a methionine.     

S phase and G2 arrests induced by topoisomerase I poisons are dependent on ATR kinase function.

ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as well as prolonged exposure to the topoisomerase II poison etoposide, resulted in subsequent G(2) arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10-hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints.