Atp-bound topoisomerase ii as a target for antitumor drugs

Topoisomerase II (TOP2) poisons interfere with the breakage/reunion reaction of TOP2 resulting in DNA cleavage. In the current studies, we show that two different classes (ATP-sensitive and -insensitive) of TOP2 poisons can be identified based on their differential sensitivity to the ATP-bound conformation of TOP2. First, in the presence of 1 mm ATP or the nonhydrolyzable analog adenosine 5'-(beta,gamma-imino)triphosphate, TOP2-mediated DNA cleavage induced by ATP-sensitive TOP2 poisons (e.g. doxorubicin, etoposide, mitoxantrone, and 4'-(9-acridinylamino)methanesulfon-m-anisidide) was 30-100-fold stimulated, whereas DNA cleavage induced by ATP-insensitive TOP2 poisons (e.g. amonafide, batracylin, and menadione) was only slightly (less than 3-fold) affected. In addition, ADP was shown to strongly antagonize TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive TOP2 poisons. Second, C427A mutant human TOP2alpha, which exhibits reduced ATPase activity, was shown to exhibit cross-resistance to all ATP-sensitive but not ATP-insensitive TOP2 poisons. Third, using ciprofloxacin competition assay, TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive poisons was shown to be antagonized by ciprofloxacin. These results suggest that ATP-bound TOP2 may be the specific target of ATP-sensitive TOP2 poisons. Using Lac repressor-operator complexes as roadblocks, we show that ATP-bound TOP2 acts as a circular clamp capable of entering DNA ends and sliding on unobstructed duplex DNA.     

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.     

Topoisomerase I-mediated DNA cleavage induced by the minor groove-directed binding of bibenzimidazoles to a distal site

Many agents (e.g. camptothecins, indolocarbazoles, indenoisoquinolines, and dibenzonaphthyridines) stimulate topoisomerase I (TOP1)-mediated DNA cleavage (a behavior termed topoisomerase I poisoning) by interacting with both the DNA and the enzyme at the site of cleavage (typically by intercalation between the -1 and +1 base-pairs). The bibenzimidazoles, which include Hoechst 33258 and 33342, are a family of DNA minor groove-directed agents that also stimulate topoisomerase I-mediated DNA cleavage. However, the molecular mechanism by which these ligands poison TOP1 is poorly understood. Toward this goal, we have used a combination of mutational, footprinting, and DNA binding affinity analyses to define the DNA binding site for Hoechst 33258 and a related derivative that results in optimal induction of TOP1-mediated DNA cleavage. We show that this DNA binding site is located downstream from the site of DNA cleavage, encompassing the base-pairs from position +4 to +8. The distal nature of this binding site relative to the site of DNA cleavage suggests that minor groove-directed agents like the bibenzimidazoles poison TOP1 via a mechanism distinct from compounds like the camptothecins, which interact at the site of cleavage.     

Critical roles of tyrosyl‐DNA phosphodiesterases in cell tolerance to carnosol‐induced DNA damage

Carnosol is a natural compound with pharmacological action due to its anti‐cancer properties. However, the precise mechanism for its anti‐carcinogenic effect remains elusive. In this study, we used lymphoblastoid TK6 cell lines to identify the DNA damage and repair mechanisms of carnosol. Our results showed that carnosol induced DNA double‐strand breaks (DSBs). We also found that cells lacking tyrosyl‐DNA phosphodiesterase 1 (TDP1), an enzyme related to topoisomerase 1 (TOP1), and tyrosyl‐DNA phosphodiesterase 2 (TDP2), an enzyme related to topoisomerase 2 (TOP2), were supersensitive to carnosol. Carnosol was found to induce the formation of the TOP1‐DNA cleavage complex (TOP1cc) and TOP2‐DNA cleavage complex (TOP2cc). When comparing the accumulation of γ‐H2AX foci and the number of chromosomal aberrations (CAs) with wild‐type (WT) cells, the susceptivity of the TDP1^?/? and TDP2^?/? cells were associated with an increased DNA damage. Our results provided evidence of carnosol inducing DNA lesions in TK6 cells and demonstrated that the damage induced by carnosol was associated with abnormal topoisomerase activity. We conclude that TDP1 and TDP2 play important roles in the anti‐cancer effect of carnosol.     

Nucleotide-resolution mapping of topoisomerase-mediated and apoptotic DNA strand scissions at or near an MLL translocation hotspot

The emergence of therapy-related acute myeloid leukemia (t-AML) has been associated with DNA topoisomerase II (TOP2)-targeted drug treatments and chromosomal translocations frequently involving the MLL, or ALL-1, gene. Two distinct mechanisms have been implicated as potential triggers of t-AML translocations: TOP2-mediated DNA cleavage and apoptotic higher-order chromatin fragmentation. Assessment of the role of TOP2 in this process has been hampered by a lack of techniques allowing in vivo mapping of TOP2-mediated DNA cleavage at nucleotide resolution in single-copy genes. A novel method, extension ligation-mediated polymerase chain reaction (ELMPCR), was used here for mapping topoisomerase-mediated DNA strand breaks and apoptotic DNA cleavage across a translocation-prone region of MLL in human cells. We report the first genomic map integrating translocation breakpoints and topoisomerase I, TOP2, and apoptotic DNA cleavage sites at nucleotide resolution across an MLL region harboring a t-AML translocation hotspot. This hotspot is flanked by a TOP2 cleavage site and is localized at one extremity of a minor apoptotic cleavage region, where multiple single- and double-strand breaks were induced by caspase-activated apoptotic nucleases. This cleavage pattern was in sharp contrast to that observed approximately 200 bp downstream in the exon 12 region, which displayed much stronger apoptotic cleavage but where no double-strand breaks were detected and no t-AML-associated breakpoints were reported. The localization and remarkable clustering of the t-AML breakpoints cannot be explained simply by the DNA cleavage patterns but might result from potential interactions between TOP2 poisoning, apoptotic DNA cleavage, and DNA repair attempts at specific sites of higher-order chromatin structure in apoptosis-evading cells. ELMPCR provides a new tool for investigating the role of DNA topoisomerases in fundamental genetic processes and translocations associated with cancer treatments involving topoisomerase-targeted drugs.     

Plasmid linking number change induced by topoisomerase I-mediated DNA damage.

The state of cellular chromatin in response to DNA damage has been examined by monitoring the change in the linking number of circular episomes. COS cells transfected with an SV40-based vector were treated with camptothecin (CPT), a eukaryotic DNA topoisomerase I (TOP1) poison which induces TOP1-mediated DNA damage. Within minutes, a large increase in the linking number (over 10 linking number) of a small fraction (5-15%) of the episomal DNA was observed. A similar CPT-induced increase in plasmid DNA linking number was observed in Saccharomyces cerevisae expressing human DNA TOP1. In this case, the majority of the plasmid DNA can undergo rapid relaxation. The large increase in the plasmid linking number suggests major chromatin structural reorganization in response to TOP I-mediated DNA damage.     

Hydrogen peroxide induces topoisomerase I-mediated DNA damage and cell death

Reactive oxygen species modify DNA, generating various DNA lesions including modified bases such as 8-oxoguanine (8-oxoG). These base-modified DNA lesions have been shown to trap DNA topoisomerase I (TOP1) into covalent cleavage complexes. In this study, we have investigated the role of TOP1 in hydrogen peroxide toxicity. We showed that ectopic expression of TOP1 in Saccharomyces cerevisiae conferred sensitivity to hydrogen peroxide, and this sensitivity was dependent on RAD9 checkpoint function. Moreover, in the mammalian cell culture system, hydrogen peroxide-induced growth inhibition and apoptosis were shown to be partly TOP1-dependent as evidenced by a specific increase in resistance to hydrogen peroxide in TOP1-deficient P388/CPT45 murine leukemia cells as compared with their TOP1-proficient parental cell line P388. In addition, hydrogen peroxide was shown to induce TOP1-DNA cross-links. These results support a model in which hydrogen peroxide promotes the trapping of TOP1 on oxidative DNA lesions to form TOP1-DNA cleavage complexes that contribute to hydrogen peroxide toxicity.     

Dietary isothiocyanate-induced apoptosis via thiol modification of DNA topoisomerase IIα

Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.     

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.     

Topoisomerase II-mediated breaks in spermatozoa cause the specific degradation of paternal DNA in fertilized oocytes

We have demonstrated that mouse spermatozoa can cleave their DNA into 50-kb fragments when treated with Triton X-100, MnCl(2), and CaCl(2). This cleavage, which is termed sperm chromatin fragmentation (SCF), is mediated by topoisomerase IIB (TOP2B) following stimulation by a factor in the epididymal fluid, most likely a nuclease, and can be at least partially religated by EDTA. When the protamines are removed, this DNA breakage is followed by digestion of the DNA by a nuclease(s). We tested whether the oocyte could repair TOP2B-induced sperm DNA breaks and whether partial religation by EDTA would allow spermatozoa to fertilize the oocytes normally. Oocytes injected with untreated spermatozoa developed normally. However, oocytes injected with spermatozoa treated with MnCl(2) and CaCl(2) to induce SCF, with or without subsequent EDTA treatment, failed to develop. In both of these treatment groups, the maternal pronuclei developed normally and replicated their DNA. However the paternal pronuclei did not replicate their DNA and this DNA began to disappear 6 h postinjection, which corresponded approximately to the time at which maternal DNA replication was initiated. These data suggest that when TOP2B is induced to cleave sperm DNA before fertilization, the paternal DNA is subsequently degraded by a highly regulated mechanism that does not affect the maternal chromatin. Furthermore, partial religation by EDTA of TOP2B-induced breaks prevents neither the inhibition of DNA synthesis nor DNA degradation.     

Site-specific topoisomerase I-mediated DNA cleavage induced by nogalamycin: a potential role of ligand-induced DNA bending at a distal site

Many DNA binding ligands (e.g., nogalamycin, actinomycin D, terbenzimidazoles, indolocarbazoles, nitidine, and coralyne) and various types of DNA lesions (e.g., UV dimers, DNA mismatches, and abasic sites) are known to stimulate topoisomerase I-mediated DNA cleavage. However, the mechanism(s) by which these covalent and noncovalent DNA interactions stimulate topoisomerase I-mediated DNA cleavage remains unclear. Using nogalamycin as a model, we have studied the mechanism of ligand-induced topoisomerase I-mediated DNA cleavage. We show by both mutational and DNA footprinting analyses that the binding of nogalamycin to an upstream site (from position -6 to -3) can induce highly specific topoisomerase I-mediated DNA cleavage. Substitution of this nogalamycin binding site with a DNA bending sequence (A(5)) stimulated topoisomerase I-mediated DNA at the same site in the absence of nogalamycin. Replacement of the A(5) sequence with a disrupted DNA bending sequence (A(2)TA(2)) significantly reduced the level of topoisomerase I-mediated DNA cleavage. These results, together with the known DNA bending property of nogalamycin, suggest that the nogalamycin-DNA complex may provide a DNA structural bend to stimulate topoisomerase I-mediated DNA cleavage.     

Induction of reversible complexes between eukaryotic DNA topoisomerase I and DNA-containing oxidative base damages. 7, 8-dihydro-8-oxoguanine and 5-hydroxycytosine

We recently showed that abasic sites, uracil mismatches, nicks, and gaps can trap DNA topoisomerase I (top1) when these lesions are introduced in the vicinity of a top1 cleavage site (Pourquier, P., Ueng, L.-M., Kohlhagen, G., Mazumder, A., Gupta, M., Kohn, K. W., and Pommier, Y. (1997) J. Biol. Chem. 272, 7792-7796; Pourquier, P., Pilon, A. A., Kohlhagen, G., Mazumder, A., Sharma, A., and Pommier, Y. (1997) J. Biol. Chem. 26441-26447). In this study, we investigated the effects on top1 of an abundant base damage generated by various oxidative stresses: 7,8-dihydro-8-oxoguanine (8-oxoG). Using purified eukaryotic top1 and oligonucleotides containing the 8-oxoG modification, we found a 3-7-fold increase in top1-mediated DNA cleavage when 8-oxoG was present at the +1 or +2 position relative to the cleavage site. Another oxidative lesion, 5-hydroxycytosine, also enhanced top1 cleavage by 2-fold when incorporated at the +1 position of the scissile strand. 8-oxoG at the +1 position enhanced noncovalent top1 DNA binding and had no detectable effect on DNA religation or on the incision step. top1 trapping by 8-oxoG was markedly enhanced when asparagine adjacent to the catalytic tyrosine was mutated to histidine, suggesting a direct interaction between this residue and the DNA major groove immediately downstream from the top1 cleavage site. Altogether, these results demonstrate that oxidative base lesions can increase top1 binding to DNA and induce top1 cleavage complexes.     

Overexpression of type I topoisomerases sensitizes yeast cells to DNA damage

DNA topoisomerases play essential roles in many DNA metabolic processes. It has been suggested that topoisomerases play an essential role in DNA repair. Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent protein-DNA intermediate of the topoisomerase reaction. Lesions in DNA are also able to trap topoisomerase-DNA intermediates, suggesting that topoisomerases have the potential to either assist in DNA repair by locating sites of damage or exacerbating DNA damage by generation of additional damage at the site of a lesion. We have shown that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not. Overexpression of topoisomerase II did not change the sensitivity of cells to these DNA-damaging agents. Yeast cells lacking TOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme. Yeast topoisomerase I covalent complexes can be trapped efficiently on UV-damaged DNA. We suggest that TOP1 does not participate in the repair of DNA damage in yeast cells. However, the enzyme has the potential of exacerbating DNA damage by forming covalent DNA-protein complexes at sites of DNA damage.     

TDP2–Dependent Non-Homologous End-Joining Protects against Topoisomerase II–Induced DNA Breaks and Genome Instability in Cells and In Vivo

Anticancer topoisomerase “poisons” exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5′-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.     

Inhibition of DNA alkylation damage with inorganic salts

Human exposure to alkylating agents metabolized from tobacco- and food-borne carcinogens occurs regularly. Dietary inorganic compounds such as selenium and vanadium have been shown previously to provide chemoprotective benefits in rat and human trials. Here, we present biochemical data on the ability of inorganic compounds to protect DNA from alkylation damage. An enzyme cleavage assay is used to observe alkylated DNA. Simple salts (e.g., NaCl or NiCl₂) did not prevent DNA alkylation, whereas anionic oxo species (e.g., Na₂SeO₄ or Na₃VO₄) did inhibit alkylation. We propose that these oxo species behave as nucleophilic targets for the electrophilic alkylating agents, thereby preventing DNA damage.