Role of topoisomerases in cytotoxicity induced by DNA ligand Hoechst-33342 and UV-C in a glioma cell line

DNA ligand Hoechst-33342 significantly enhances UV induced cytotoxicity in human glioma cell lines (BMG-1 & U-87) with supra additive increase in cell death, cytogenetic damage, cell cycle delay, apoptosis and inhibition of PLDR. Cytotoxicity of Hoechst-33342 arises due to its interference in the breakage-rejoining reaction of DNA topoisomerases by stabilization of cleavable complexes. Since topoisomerases have also been implicated in the generation of potentially lethal DNA breaks by interaction with various types of DNA damage including UV induced DNA lesions, we investigated in present studies the role of functional topoisomerases in the synergistic cytotoxicity of Hoechst-33342 and UV in a human glioma cell line (BMG-1). Topoisomerase I activity analyzed by the plasmid relaxation assay, was significantly enhanced upon UV irradiation, implying a possible role of this enzyme in the processing of UV induced lesions. However, this increase in the activity was reduced by >50% in cells incubated with Hoechst-33342 for 1 hr prior to irradiation. Imunoflowcytometric analysis of the chromatin bound topoisomerases I and II levels (cleavable complex) using topoisomerases I and II anti-antibodies showed a good correlation between the induction of apoptosis by Hoechst-33342 and UV and enhancement in the level of topoisomerase II mediated cleavable complexes. Induction of apoptosis was associated with a decline in the level of Bcl2. Taken together, these studies show that supra additive cytotoxic effects of UV-C and Hoechst-33342 in BMG-1 cells are consequences of enhanced stabilization of topo II mediated cleavable complexes and alterations in specific signal transduction pathways of apoptosis, besides the inhibition of topoisomerase mediated repair processes.     

Iridoids as DNA topoisomerase I poisons

The discovery of new topoisomerase I inhibitors is necessary since most of the antitumor drugs are targeted against type II and only a very few can specifically affect type I. Topoisomerase poisons generate toxic DNA damage by stabilization of the covalent DNA-topoisomerase cleavage complex and some have therapeutic efficacy in human cancer. Two iridoids, aucubin and geniposide, have shown antitumoral activities, but their activity against topoisomerase enzymes has not been tested. Here it was found that both compounds are able to stabilize covalent attachments of the topoisomerase I subunits to DNA at sites of DNA strand breaks, generating cleavage complexes intermediates so being active as poisons of topoisomerase I, but not topoisomerase II. This result points to DNA damage induced by topoisomerase I poisoning as one of the possible mechanisms by which these two iridoids have shown antitumoral activity, increasing interest in their possible use in cancer chemoprevention and therapy.     

Iridoids as DNA topoisomerase I poisons

The discovery of new topoisomerase I inhibitors is necessary since most of the antitumor drugs are targeted against type II and only a very few can specifically affect type I. Topoisomerase poisons generate toxic DNA damage by stabilization of the covalent DNA-topoisomerase cleavage complex and some have therapeutic efficacy in human cancer. Two iridoids, aucubin and geniposide, have shown antitumoral activities, but their activity against topoisomerase enzymes has not been tested. Here it was found that both compounds are able to stabilize covalent attachments of the topoisomerase I subunits to DNA at sites of DNA strand breaks, generating cleavage complexes intermediates so being active as poisons of topoisomerase I, but not topoisomerase II. This result points to DNA damage induced by topoisomerase I poisoning as one of the possible mechanisms by which these two iridoids have shown antitumoral activity, increasing interest in their possible use in cancer chemoprevention and therapy.     

Telomeric DNA damage by topoisomerase I. A possible mechanism for cell killing by camptothecin

Topoisomerase I adjusts torsional stress in the genome by breaking and resealing one strand of the helix through a transient covalent coupling between enzyme and DNA. Camptothecin, a specific topoisomerase I poison, traps this covalent intermediate, thereby damaging the genome. Here we examined the activity of topoisomerase I at telomeric repeats to determine whether telomere structures are targets for DNA damage. We show that topoisomerase I is catalytically active in cleaving the G-rich telomeric strand in vitro in the presence of camptothecin but not in cleaving the C-rich strand. The topoisomerase I cleavage site is 5'-TT (downward arrow) AGGG-3' (cleavage site marked by the downward arrow). We also show that endogenous topoisomerase I can access telomeric DNA in vivo and form camptothecin-dependent covalent complexes. Therefore, each telomeric repeat represents a potential topoisomerase I cleavage site in vivo. Because telomere structures are comprised of a large number of repeats, telomeres in fact represent a high concentration of nested topoisomerase I sites. Therefore, more telomeric DNA damage by camptothecin could occur in cells with longer telomeres when cells possess equivalent levels of topoisomerase I. The evidence presented here suggests that DNA damage at telomeric repeats by topoisomerase I is a prominent feature of cell killing by camptothecin and triggers camptothecin-induced apoptosis.     

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.     

Comparison of effects of fostriecin, novobiocin, and camptothecin, inhibitors of DNA topoisomerases, on DNA replication and repair in human cells.

Fostriecin causes a delayed inhibition of replicative DNA synthesis in human cells, consistent with a role for DNA topoisomerase II (its target enzyme) at a late stage in replication. Fostriecin does not inhibit UV-induced excision repair. The less specific inhibitor novobiocin blocks repair in permeabilised cells given a low dose of UV, presumably through a mechanism other than the inhibition of topoisomerase II. Its effect cannot be accounted for by a depletion of the ATP required for incision. Camptothecin, an inhibitor of DNA topoisomerase I, blocks replicative DNA synthesis immediately but incompletely, suggesting a participation of topoisomerase I at the replication fork, but it, too, has no influence on DNA repair. We thus find no evidence for involvement of either topoisomerase I or II in the response of cells to UV damage.     

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.     

Topoisomerase I-DNA complexes contribute to arsenic trioxide-induced apoptosis

Topoisomerase I is an essential enzyme that relaxes DNA supercoiling by forming covalent DNA cleavage complexes, which are normally transient. Topoisomerase I-DNA complexes can be trapped by anticancer drugs (camptothecins) as well as by endogenous and exogenous DNA lesions. We show here that arsenic trioxide (a potent inducer of apoptosis that induces the intracellular accumulation of reactive oxygen species and targets mitochondria) induces cellular topoisomerase I cleavage complexes. Bcl-2 overexpression and quenching of reactive oxygen species, which prevent arsenic trioxide-induced apoptosis, also prevent the formation of topoisomerase I-DNA complexes, whereas enhancement of reactive oxygen species accumulation promotes these complexes. The caspase inhibitor, benzyloxycarbonyl-VAD partially prevents arsenic trioxide-induced topoisomerase I-DNA complexes and apoptosis, suggesting that activated caspases further maintain intracellular levels of reactive oxygen species that induce the formation of topoisomerase I-DNA complexes. Down-regulation of topoisomerase I expression decreases arsenic trioxide-induced apoptotic DNA fragmentation. Thus, we propose that arsenic trioxide induces topoisomerase I-DNA complexes that participate in chromatin fragmentation and programmed cell death during apoptosis.     

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.     

The requirement of Artemis in double-strand break repair depends on the type of DNA damage

Artemis is a recently identified factor involved in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Here, we performed targeted disruption of the Artemis gene (ARTEMIS) in the human pre-B cell line Nalm-6. Unexpectedly, we found that cells lacking Artemis exhibit increased sensitivity to low doses, but not high doses, of ionizing radiation. We also show that ARTEMIS-deficient cells are hypersensitive to the topoisomerase II inhibitor etoposide, but to a much lesser extent than cells lacking DNA ligase IV, a critical component of NHEJ. Unlike DNA ligase IV-deficient cells, ARTEMIS-deficient cells were not hypersensitive to ICRF-193, a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA cleavable complexes. Collectively, our results suggest that Artemis only partially participates in the NHEJ pathway to repair DSBs in human somatic cells.     

Rapid and prolonged stalling of human DNA topoisomerase I in UVA-irradiated genomic areas

DNA topoisomerase I appears to be involved in DNA damage and repair in a complex manner. The enzyme is required for DNA maintenance and repair, but it may also damage DNA through its covalently DNA-bound, catalytic intermediate. The latter mechanism plays a role in tumor cell killing by camptothecins, but seems also involved in oxidative cell killing and certain stages of apoptosis. Stalling and/or suicidal DNA cleavage of topoisomerase I adjacent to nicks and modified DNA bases has been demonstrated in vitro. Here, we investigate the enzyme's interactions with UVA-induced DNA lesions inside living cells. We irradiated cells expressing GFP-tagged topoisomerase I with an UVA laser focused through a confocal microscope at confined areas of the nuclei. At irradiated sites, topoisomerase I accumulated within seconds, and accumulation lasted for more than 90 min. This effect was apparently due to reduced mobility, although the enzyme was not immobilized at the irradiated nuclear sites. Similar observations were made with mutant versions of topoisomerase I lacking the active site tyrosine or the N-terminal domain, but not with the N-terminal domain alone. Thus, accumulation of topoisomerase I at UVA-modified DNA sites is most likely due to non-covalent binding to damaged DNA, and not suicidal cleavage of such lesions. The rapid onset of accumulation suggests that topoisomerase I functions in this context as a component of DNA damage recognition and/or a cofactor of fast DNA-repair processes. However, the prolonged duration of accumulation suggests that it is also involved in more long-termed processes.     

DNA damage-mediated apoptosis induced by selenium compounds

Selenium (Se) compounds, which are the most extensively studied cancer chemopreventive agents, induce apoptotic death of tumor cells. In the current study, we show that selenite-induced apoptosis involves DNA damage. We showed that selenite-induced apoptosis as evidenced by cleavage of poly(ADP-ribose) polymerase was reduced in NIH 3T3 cells treated with ATM small interfering RNA, suggesting the involvement of the DNA damage regulator ATM. Consistent with ATM/ATR involvement, selenite was also shown to stimulate Ser-139 phosphorylation of the ATM/ATR substrate H2AX. Selenite-induced apoptosis was shown to involve DNA topoisomerase II (Top II) as selenite-induced apoptosis was reduced in Top II-deficient HL-60/MX2 cells and in HL-60 cells co-treated with the Top II catalytic inhibitor ICRF-193. Using purified human recombinant Top II, selenite was shown to induce reversible Top II cleavage complexes in vitro. In the aggregate, these results suggest that selenite-induced apoptosis, which involves ATM/ATR and Top II, is likely to be because of DNA damage.     

Recombinogenic flap ligation mediated by human topoisomerase I

Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3'-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks. Here, we characterize flap ligation mediated by human topoisomerase I. We demonstrate that cleavage complexes containing the enzyme at a blunt end allow invasion of a 3'-acceptor tail matching the scissile strand of the donor, which facilitates ligation of the recessed 5'-hydroxyl end. However, the reaction was strictly dependent on the length of double-stranded DNA of the donor complexes, and longer stretches of base-pairing inhibited strand invasion. The stabilization of the DNA helix was most probably provided by the covalently bound enzyme itself, since deleting the N-terminal domain of human topoisomerase I stimulated flap ligation. We suggest that stabilization of the DNA duplex upon enzyme binding may play an important role during normal topoisomerase I catalysis by preventing undesired strand transfer reactions. For flap ligation to function in a repair pathway, factors other than topoisomerase I, such as helicases, would be necessary to unwind the DNA duplex and allow strand invasion.     

Green tea constituent (--)-epigallocatechin-3-gallate inhibits topoisomerase I activity in human colon carcinoma cells

DNA topoisomerases I and II are essential for cell survival and play critical roles in DNA metabolism and structure. Inhibitors of topoisomerase constitute a novel family of antitumor agents with demonstrated clinical activity in human malignancies. The clinical use of these agents is limited due to severe toxic effects on normal cells. Therefore, there is a need to develop novel, nontoxic topoisomerase inhibitors that have the ability to spare normal cells. Recent studies have shown that green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG), impart growth inhibitory responses to cancer cells but not to normal cells. Based on the knowledge that EGCG induces DNA damage, cell cycle arrest, and apoptosis, we considered the possibility of the involvement of topoisomerase in the antiproliferative response of EGCG. Here, for the first time, we show that EGCG inhibits topoisomerase I, but not topoisomerase II in several human colon carcinoma cell lines. Based on this study it is tempting to suggest that combination of EGCG with other conventional topoisomerase inhibitors could be an improved strategy for treatment of colon cancer. The possible role of EGCG as a chemotherapeutic agent needs to be investigated.     

Human topoisomerase I cleavage complexes are repaired by a p53-stimulated recombination-like reaction in vitro

Several studies have shown that human topoisomerase I (htopoI) cleaves in the vicinity of various DNA lesions and thereby forms covalent intermediates known as ‘cleavage complexes’. Such complexes are detrimental to cells if they are not repaired. Therefore, it is generally accepted that repair pathways must exist for such lesions. We have demonstrated that a htopoI cleavage complex can be recognized by a second topoisomerase I molecule and thereby perform a so-called htopoI ‘double cleavage’ in vitro. In addition, we found that the double cleavage reaction was stimulated by p53. Here we show that the double cleavage reaction results in the removal of the original htopoI cleavage complex and the generation of a single-stranded gap of ~13 nt. This gap supports a sequence-dependent DNA recombination reaction mediated by the second htopoI molecule. Furthermore, we show that p53 strongly stimulates the recombination reaction. We suggest that this reaction may represent a novel p53-dependent topoisomerase I-induced recombination repair (TIRR) pathway for htopoI cleavage complexes.     

The nucleotide excision repair pathway is required for UV-C-induced apoptosis in Caenorhabditis elegans

Ultraviolet (UV) radiation is a mutagen of major clinical importance in humans. UV-induced damage activates multiple signaling pathways, which initiate DNA repair, cell cycle arrest and apoptosis. To better understand these pathways, we studied the responses to UV-C light (254 nm) of germ cells in Caenorhabditis elegans. We found that UV activates the same cellular responses in worms as in mammalian cells. Both UV-induced apoptosis and cell cycle arrest were completely dependent on the p53 homolog CEP-1, the checkpoint proteins HUS-1 and CLK-2, and the checkpoint kinases CHK-2 and ATL-1 (the C. elegans homolog of ataxia telangiectasia and Rad3-related); ATM-1 (ataxia telangiectasia mutated-1) was also required, but only at low irradiation doses. Importantly, mutation of genes encoding nucleotide excision repair pathway components severely disrupted both apoptosis and cell cycle arrest, suggesting that these genes not only participate in repair, but also signal the presence of damage to downstream components of the UV response pathway that we delineate here. Our study suggests that whereas DNA damage response pathways are conserved in metazoans in their general outline, there is significant evolution in the relative importance of individual checkpoint genes in the response to specific types of DNA damage.     

A novel approach for organelle-specific DNA damage targeting reveals different susceptibility of mitochondrial DNA to the anticancer drugs camptothecin and topotecan

DNA is susceptible of being damaged by chemicals, UV light or gamma irradiation. Nuclear DNA damage invokes both a checkpoint and a repair response. By contrast, little is known about the cellular response to mitochondrial DNA damage. We designed an experimental system that allows organelle-specific DNA damage targeting in Saccharomyces cerevisiae. DNA damage is mediated by a toxic topoisomerase I allele which leads to the formation of persistent DNA single-strand breaks. We show that organelle-specific targeting of a toxic topoisomerase I to either the nucleus or mitochondria leads to nuclear DNA damage and cell death or to loss of mitochondrial DNA and formation of respiration-deficient ‘petite’ cells, respectively. In wild-type cells, toxic topoisomerase I–DNA intermediates are formed as a consequence of topoisomerase I interaction with camptothecin-based anticancer drugs. We reasoned that targeting of topoisomerase I to the mitochondria of top1Δ cells should lead to petite formation in the presence of camptothecin. Interestingly, camptothecin failed to generate petite; however, its derivative topotecan accumulates in mitochondria and induces petite formation. Our findings demonstrate that drug modifications can lead to organelle-specific DNA damage and thus opens new perspectives on the role of mitochondrial DNA-damage in cancer treatment.     

Reverse gyrase recruitment to DNA after UV light irradiation in Sulfolobus solfataricus

Induction of DNA damage triggers a complex biological response concerning not only repair systems but also virtually every cell function. DNA topoisomerases regulate the level of DNA supercoiling in all DNA transactions. Reverse gyrase is a peculiar DNA topoisomerase, specific to hyperthermophilic microorganisms, which contains a helicase and a topoisomerase IA domain that has the unique ability to introduce positive supercoiling into DNA molecules. We show here that reverse gyrase of the archaean Sulfolobus solfataricus is mobilized to DNA in vivo after UV irradiation. The enzyme, either purified or in cell extracts, forms stable covalent complexes with UV-damaged DNA in vitro. We also show that the reverse gyrase translocation to DNA in vivo and the stabilization of covalent complexes in vitro are specific effects of UV light irradiation and do not occur with the intercalating agent actinomycin D. Our results suggest that reverse gyrase might participate, directly or indirectly, in the cell response to UV light-induced DNA damage. This is the first direct evidence of the recruitment of a topoisomerase IA enzyme to DNA after the induction of DNA damage. The interaction between helicase and topoisomerase activities has been previously proposed to facilitate aspects of DNA replication or recombination in both Bacteria and Eukarya. Our results suggest a general role of the association of such activities in maintaining genome integrity and a mutual effect of DNA topology and repair.     

Homocamptothecin, an E-ring-modified camptothecin analogue, generates new topoisomerase I-mediated DNA breaks

Homocamptothecin (hCPT) contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone ring found in camptothecin and its tumor active analogues, including topotecan and irinotecan. The homologation of the lactone E-ring reinforces the stability of the lactone, thus reducing considerably its conversion into a carboxylate form which is inactive. We have recently shown that hCPT is much more active than the parent compound against a variety of tumor cells in vitro and in xenograft models, suggesting that a highly reactive lactone is not essential for topoisomerase I-mediated anticancer activity [Lesueur-Ginot et al. (1999) Cancer Res. 59, 2939-2943]. In the present study, we provide further evidence that hCPT has superior topoisomerase I inhibition capacities to CPT. In particular, we show that replacement of the camptothecin lactone E-ring with a homologous seven-membered lactone ring changes the sequence-specificity of the drug-induced DNA cleavage by topoisomerase I. Both CPT and hCPT stimulate the cleavage by topoisomerase I at T( downward arrow)G sites, but in addition, hCPT stabilizes cleavage at specific sites containing the sequence AAC( downward arrow)G. At low drug concentrations, the cleavage at the T( downward arrow)G sites and at the hCPT-specific C( downward arrow)G sites is more pronounced and more stable with hCPT than with CPT. The in vitro data were confirmed in cells. Higher levels of protein-DNA complexes were detected in P388 leukemia cells treated with hCPT than those treated with CPT. Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped onto DNA by hCPT in cells. Finally, the use of a leukemia cell line resistant to CPT provided evidence that topoisomerase I is involved in the cytotoxicity of hCPT. Altogether, the results show that the beta-hydroxylactone ring of hCPT plays an important and positive role in the poisoning of topoisomerase I. An explanation is proposed to account for such remarkable changes in the sequence specificity of topoisomerase I cleavage consequent to the modification of the lactone. The study sheds new light on the importance of the lactone ring of camptothecins for the stabilization of topoisomerase I-DNA complexes.