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.     

Dysregulation of the DNA Damage Response and KMT2A Rearrangement in Fetal Liver Hematopoietic Cells

Etoposide, a topoisomerase 2 (TOP2) inhibitor, is associated with the development of KMT2A (MLL)-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL) rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL)-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP) assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq) identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll)-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective.     

The Epstein-Barr virus deubiquitinating enzyme BPLF1 regulates the activity of topoisomerase II during productive infection

Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 co-immunoprecipitated and deubiquitinated TOP2, and stabilized SUMOylated TOP2 trapped in cleavage complexes (TOP2ccs), which halted the DNA damage response to TOP2-induced double strand DNA breaks and promoted cell survival. Induction of the productive virus cycle in epithelial and lymphoid cell lines carrying recombinant EBV encoding the active enzyme was accompanied by TOP2 deubiquitination, accumulation of TOP2ccs and resistance to Etoposide toxicity. The protective effect of BPLF1 was dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair. These findings highlight a previously unrecognized function of BPLF1 in supporting a non-proteolytic pathway for TOP2ccs debulking that favors cell survival and virus production.     

Development of a multi-step leukemogenesis model of MLL-rearranged leukemia using humanized mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ(-/-) (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.     

Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.     

Stimulation of topoisomerase II-mediated DNA damage via a mechanism involving protein thiolation

The breakage/reunion reaction of DNA topoisomerase II (TOP2) can be interrupted by DNA intercalators (e.g., doxorubicin), enzyme binders (e.g., etoposide), or DNA lesions (e.g., abasic sites) to produce TOP2-mediated DNA damage. Here, we demonstrate that thiol alkylation of TOP2 can also produce TOP2-mediated DNA damage. This conclusion is supported by the following observations using purified TOP2: (1) Thiol-reactive quinones were shown to induce TOP2-mediated DNA cleavage. (2) Thiol-reactive compounds such as N-ethylmaleimide (NEM), disulfiram, and organic disulfides [e.g., 2,2'-dithiobis(5-nitropyridine)] were also shown to induce TOP2-mediated DNA cleavage with similar reaction characteristics as thiol-reactive quinones. (3) TOP2-mediated DNA cleavage induced by thiol-reactive quinones was completely abolished using mutant yeast TOP2 with all cysteine residues replaced with alanine (cysteineless TOP2). These results suggest the possibility that cellular DNA damage could occur indirectly through thiolation of a nuclear protein, TOP2. The implications of this reaction in carcinogenesis and apoptotic cell death are discussed.     

Topoisomerase II - drug interaction domains: identification of substituents on etoposide that interact with the enzyme

Etoposide is one of the most successful chemotherapeutic agents used for the treatment of human cancers. The drug kills cells by inhibiting the ability of topoisomerase II to ligate nucleic acids that it cleaves during the double-stranded DNA passage reaction. Etoposide is composed of a polycyclic ring system (rings A-D), a glycosidic moiety at the C4 position, and a pendent ring (E-ring) at the C1 position. Although drug-enzyme contacts, as opposed to drug-DNA interactions, mediate the entry of etoposide into the topoisomerase II-drug-DNA complex, the substituents on etoposide that interact with the enzyme have not been identified. Therefore, saturation transfer difference [1H]-nuclear magnetic resonance spectroscopy and protein-drug competition binding assays were employed to define the groups on etoposide that associate with yeast topoisomerase II and human topoisomerase IIalpha. Results indicate that the geminal protons of the A-ring, the H5 and H8 protons of the B-ring, and the H2' and H6' protons and the 3'- and 5'-methoxyl protons of the pendent E-ring interact with both enzymes in the binary protein-ligand complexes. In contrast, no significant nuclear Overhauser enhancement signals arising from the C-ring, the D-ring, or the C4 glycosidic moiety were observed with either enzyme, suggesting that there is limited or no contact between these portions of etoposide and topoisomerase II in the binary complex. The functional importance of E-ring substituents was confirmed by topoisomerase II-mediated DNA cleavage assays.     

Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes: Gas chromatographic–mass spectrometric determination of metabolites

Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17α-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17α-methyltestosterone, produced 6β-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography–mass spectrometry (GC–MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6β-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6β-hydroxyl metabolites with testosterone and 17α-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6β-hydroxylation.     

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.     

Bioconversion of deoxypodophyllotoxin into epipodophyllotoxin in E. coli using human cytochrome P450 3A4

Biotransformation of deoxypodophyllotoxin to epipodophyllotoxin by three major human hepatic enzymes, CYP1A2, CYP2C9 and CYP3A4, heterologously expressed in E. coli DH5alpha, was investigated. It was shown that CYP3A4 catalysed the hydroxylation of deoxypodophyllotoxin into epipodophyllotoxin in yields up to 90%. The structure of the metabolite was determined using HPLC-MS and HPLC-SPE-NMR techniques. There was no detectable production of epipodophyllotoxin or podophyllotoxin by CYP1A2 and CYP2C9 enzymes. The CYP3A4 enzyme shows a distinctly different reactivity to deoxypodophyllotoxin compared to the semi-synthetic derivatives etoposide and teniposide, which are degraded by 3-O-demethylation. These findings demonstrate a novel system for the production of 2,7'-cyclolignans, starting from the easily accessible deoxypodophyllotoxin.     

Eugenol inhibit 7,12-dimethylbenz[a]anthracene-induced genotoxicity in MCF-7 cells: Bifunctional effects on CYP1 and NAD(P)H:quinone oxidoreductase

Typically, chemopreventive agents either inhibit the cytochrome P450s (CYPs) that are essential for the metabolism of carcinogens or induce phase II detoxifying enzymes. This study examined the chemopreventive effect of eugenol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells. Eugenol inhibited the formation of the DMBA-DNA adduct in a dose dependent manner. CYP1A1 and CYP1B1 activity, which catalyze the biotransformation of DMBA, were strongly inhibited by eugenol. Eugenol also suppressed the CYP1A induction by DMBA through decreased aryl hydrocarbon receptor activation and subsequent DNA binding. Furthermore, eugenol increased the expression and activity of NAD(P)H:quinone oxidoreductase (QR), a major detoxifying enzyme for DMBA, through NF-E2 related factor2 binding to antioxidant response element in QR gene. Therefore, eugenol has a potent protective effect against DMBA-induced genotoxicity, presumably through the suppression of the DMBA activation and the induction of its detoxification. These results suggest that eugenol has potential as a chemopreventive.     

Nucleolytic cleavage of the mixed lineage leukemia breakpoint cluster region during apoptosis.

VP-16 (etoposide) has recently been shown to induce topoisomerase II (TOP2)-mediated DNA cleavage within the mixed lineage leukemia (MLL) breakpoint cluster region (bcr), suggesting a role of TOP2 in MLL gene rearrangement. In our current studies, we have compared the induction of DNA cleavage within the MLL bcr in different cell lines after treatment with various anticancer drugs. All anticancer drugs tested including VP-16 (a TOP2-directed drug), camptothecin (a topoisomerase I-directed drug), 5-fluorouracil and methotrexate (antimetabolites), and vinblastine (a microtubule inhibitor) induced the same site-specific cleavage within the MLL bcr. This cleavage was shown to be nuclease-mediated but not TOP2-mediated by the following observations: 1) drug-induced cleavage within the MLL bcr was not protein-linked; 2) unlike TOP2-mediated cleavage, drug-induced DNA cleavage within the MLL bcr was kinetically slow and coincided with the formation of the apoptotic nucleosomal DNA ladder; 3) drug-induced cleavage within the MLL bcr was unaffected in cells with reduced nuclear TOP2; and 4) drug-induced cleavage within the MLL bcr was abolished by the caspase inhibitor, Z-Asp(OCH(3))-Glu(OCH(3))-Val-Asp(OCH(3))-FMK. The possibility that an apoptotic nuclease may be involved in cleavage of the MLL bcr and MLL gene translocation is discussed.     

Effects of etoposide on the proliferation of hexaploid H1 (ES) cells

Etoposide is a specific inhibitor of topoisomerase II, which is an enzyme that enables double-stranded DNA to pass through another double-stranded DNA. Topoisomerase II is a major constituent of chromosome scaffold, existing at appreciable amounts in cells. To examine the effects of etoposide on the cell cycle, hexaploid H1 (ES) cells (6H1 cells) were used with diploid H1 (ES) cells (2H1 cells) as a control. Exponentially growing 2H1 and 6H1 cells were exposed to etoposide at various concentrations, and cultured for about 60 days in L15F10 medium with leukemia inhibitory factor. With a high concentration of etoposide (1 μM), the DNA histograms showed G(2)/M accumulation, suggesting that etoposide arrested the cell cycle at the G(2)/M phase. With a low concentration of etoposide (50 nM), the cell proliferation was suppressed with a doubling time of 98.4 h for 2H1 cells and 51.6 h for 6H1 cells, and without significant alteration in DNA histograms. Time-lapse videography revealed that 6H1 cells survived in the medium containing 50 nM etoposide had a cell cycle time of 18.8 h, which was equivalent to 19.2 h of the doubling time for the 6H1 cell population in drug-free medium, suggesting that a part of the cell population died and was excluded from the cell system. It was concluded that etoposide affected the cell cycle at a wide range of concentrations.     

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.     

Expression of human cytochromes P450 1A1 and P450 1A2 as fused enzymes with yeast NADPH-cytochrome P450 oxidoreductase in transgenic tobacco plants

Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1. The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv. Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did. The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.     

Etoposide-induced activation of c-jun N-terminal kinase (JNK) correlates with drug-induced apoptosis in salivary gland acinar cells.

We have examined the ability of etoposide to induce apoptosis in two recently established rat salivary acinar cell lines. Etoposide induced apoptosis in the parotid C5 cell line as evidenced by the appearance of cytoplasmic blebbing and nuclear condensation, DNA fragmentation and cleavage of PARP. Etoposide also induced activation of c-jun N-terminal kinase (JNK) in parotid C5 cells by 4 h after treatment, with maximal activation at 8 - 10 h. Coincident with activation of JNK, the amount of activated ERK1 and ERK2 decreased in etoposide-treated parotid C5 cells. In contrast to the parotid C5 cells, the vast majority of submandibular C6 cells appeared to be resistant to etoposide-induced apoptosis. Likewise, activation of JNKs was not observed in etoposide-treated submandibular C6 cells, and the amount of activated ERK1 and ERK2 decreased only slightly. Etoposide treatment of either cell line had no effect upon the activation of p38. Treatment of the parotid C5 cells with Z-VAD-FMK, a caspase inhibitor, inhibited etoposide-induced activation of JNK and DNA fragmentation. These data suggest that etoposide may induce apoptosis in parotid C5 cells by activating JNKs and suppressing the activation of ERKs, thus creating an imbalance in these two signaling pathways.