Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.     

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.     

Effects of the DNA intercalators 4'-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage

DNA topoisomerase II is believed to be the enzyme that produces the protein-associated DNA strand breaks observed in mammalian cell nuclei treated with various intercalating agents. Two intercalators--4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA, amsacrine) and 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+)--differ in their effects on protein-associated double-strand breaks in isolated nuclei. m-AMSA stimulates their production at all concentrations, whereas 2-Me-9-OH-E+ stimulates at low concentrations and inhibits at high concentrations. We have reproduced these differential effects in experiments carried out in vitro with purified L1210 DNA topoisomerase II, and we have found that concentrations of 2-Me-9-OH-E+ above 5 microM prevent the trapping of DNA-topoisomerase II cleavable complexes irrespective of the presence of m-AMSA. It also stimulated topoisomerase II mediated DNA strand passage, again with or without inhibitory amounts of m-AMSA (this result suggests that extensive intercalation by 2-Me-9-OH-E+ destabilized the cleavable complexes). From these data, it is concluded that intercalator-induced protein-associated DNA strand breaks observed in intact eukaryotic cells and isolated nuclei are generated by DNA topoisomerase II and that intercalators can affect mammalian DNA topoisomerase II in more than one way. They can trap cleavable complexes and inhibit DNA topoisomerase II mediated DNA relaxation (m-AMSA and low concentrations of 2-Me-9-OH-E+) or destabilize cleavable complexes and stimulate DNA relaxation (high concentrations of 2-Me-9-OH-E+).     

Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis.

BACKGROUND: The ability of the comet assay to quantify DNA strand breaks and alkali labile sites has been widely demonstrated. In this study, this assay was tested for its ability to identify DNA fragmentation occurring during apoptosis in comparison with standard DNA flow cytometry analysis. METHODS: Staurosporine-induced apoptosis in CHO cells is an adequate model to study a rapid time- and dose-dependent appearance of this process. RESULTS: Nuclear staining with DAPI confirmed the induction of apoptosis with a typical chromatin condensation and fragmentation. Analysis of propidium-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G1 peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatment occurred earlier than the detection of apoptotic cells by flow cytometry. However, HDC were missed when the DNA fragmentation was too high, preventing accurate quantification of late apoptotic cells. CONCLUSIONS: The comet assay is more sensitive than standard DNA flow cytometry to detect early DNA fragmentation events occurring during apoptosis. However, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at later stages.     

In vivo bioassay to detect irinotecan-stabilized DNA/topoisomerase I complexes in rats

Irinotecan is an anticancer agent that stabilizes topoisomerase I/DNA complexes. So far, no test system has been reported for directly determining irinotecan-induced stabilization of topoisomerase I/DNA complexes in organs in vivo. We adapted an ‘in vivo complexes of enzyme to DNA’ (ICE) bioassay to assess irinotecan activity in the stomach, duodenum, colon and liver of male Wistar rats after a single treatment with irinotecan (100 mg/kg body weight, intraperitoneally). This was compared to the control group receiving 0.9% sodium chloride intraperitoneally. In addition, the DNA strand breaking properties of irinotecan were measured in mucosal cells from the distal colon by single-cell gel electrophoresis (comet assay) to investigate the association of topoisomerase poisoning and DNA damage in vivo. A single dose of irinotecan significantly increased amounts of topoisomerase I covalently bound to DNA in stomach, duodenum, colon and liver. Concomitantly, the irinotecan-treated group showed significantly higher amounts of DNA strand breaks in colon mucosa cells compared to the control group. The ICE bioassay and the comet assay represent two test systems for investigating the impact of topoisomerase I poisons on DNA integrity in colon tissues of Wistar rats.     

Apoptosis of unstimulated human lymphocytes and DNA strand breaks induced by the topoisomerase II inhibitor etoposide (VP-16)

Etoposide (VP-16)-induced DNA strand breaks and repair and apoptosis of unstimulated human lymphocytes have been studied using DNA comet assay, electrophoresis of low-molecular-weight DNA extracts, and fluorescence microscopy. Incubation of unstimulated human lymphocytes with VP-16 (50-200 microg/ml) for 3 or 24 h induced apoptosis. This conclusion is supported by results of morphological studies, evaluation of the proportion of hypodiploidy and internucleosomal degradation of DNA in lymphocytes. Etoposide-induced formation of DNA strand breaks preceded the appearance of these conventional apoptotic manifestations. The number of single-strand breaks depended on VP-16 concentration, and 2-3 h after its removal from the incubation medium they were repaired. The hydroxyl group at the C-4; position of the etoposide dimethoxyphenol ring may be responsible for the formation of single-strand breaks. Double-strand breaks were unrepaired 20 h after the change of the incubation medium. The number of double-strand breaks and a proportion of apoptotic cells did not exhibit any dependence on VP-16 concentration and/or duration of cell exposure to this agent. We suggest that the cytotoxic effect of VP-16 on unstimulated lymphocytes is mediated by a topoisomerase II isoform, topoisomerase II-beta, which is localized in the nucleolus and is not related to the cell cycle.     

Incomplete reversion of double stranded DNA cleavage mediated by Drosophila topoisomerase II: formation of single stranded DNA cleavage complex in the presence of an anti-tumor drug VM26.

Anti-tumor drug VM26 greatly stimulates topoisomerase II mediated DNA cleavage by stabilizing the cleavable complex. Addition of a strong detergent such as SDS to the cleavable complex induces the double stranded DNA cleavage. We demonstrate here that heat treatment can reverse the double stranded DNA cleavage; however, topoisomerase II remains bound to DNA even in the presence of SDS. This reversed complex has been shown to contain single strand DNA breaks with topoisomerase II covalently linked to the nicked DNA. Chelation of Mg++ by EDTA and the addition of salt to a high concentration also reverse the double strand DNA cleavage, and like heat reversion, topoisomerase II remains bound to DNA through single strand DNA break. The reversion complex can be analyzed and isolated by CsCl density gradient centrifugation. We have detected multiple discrete bands from such a gradient, corresponding to protein/DNA complexes with 1, 2, 3, ..... topoisomerase II molecules bound per DNA molecule. Analysis of topoisomerase II/DNA complexes isolated from the CsCl gradient indicates that there are single stranded DNA breaks associated with the CsCl stable complexes. Therefore, topoisomerase II/DNA complex formed in the presence of VM26 cannot be completely reversed to yield free DNA and enzyme. We discuss the possible significance of this finding to the mechanism of action of VM26 in the topoisomerase II reactions.     

DNA strand breaks induced by the anti-topoisomerase II bis-dioxopiperazine ICRF-193

The bis-dioxopiperazine ICRF-193 has long time been considered as a pure topoisomerase II catalytic inhibitor able to exert its inhibitory effect on the enzyme without stabilization of the so-called cleavable complex formed by DNA covalently bound to topoisomerase II. In recent years, however, this concept has been challenged, as a number of reports have shown that ICRF-193 really "poisons" the enzyme, most likely through a different mechanism from that shown by the classical topoisomerase II poisons used in cancer chemotherapy. In the present investigation, we have carried out a study of the capacity of ICRF-193 to induce DNA strand breaks, as classical poisons do, in cultured V79 and irs-2 Chinese hamster lung fibroblasts using the comet assay and pulsed-field gel electrophoresis (PFGE). Our results clearly show that ICRF-193 readily induces breakage in DNA through a mechanism as yet poorly understood.     

WRN protects against topo I but not topo II inhibitors by preventing DNA break formation

The Werner syndrome helicase/3′-exonuclease (WRN) is a major component of the DNA repair and replication machinery. To analyze whether WRN is involved in the repair of topoisomerase-induced DNA damage we utilized U2-OS cells, in which WRN is stably down-regulated (wrn-kd), and the corresponding wild-type cells (wrn-wt). We show that cells not expressing WRN are hypersensitive to the toxic effect of the topoisomerase I inhibitor topotecan, but not to the topoisomerase II inhibitor etoposide. This was shown by mass survival assays, colony formation and induction of apoptosis. Upon topotecan treatment WRN deficient cells showed enhanced DNA replication inhibition and S-phase arrest, whereas after treatment with etoposide they showed the same cell cycle response as the wild-type. A considerable difference between WRN and wild-type cells was observed for DNA single- and double-strand break formation in response to topotecan. Topotecan induced DNA single-strand breaks 6 h after treatment. In both wrn-wt and wrn-kd cells these breaks were repaired at similar kinetics. However, in wrn-kd but not wrn-wt cells they were converted into DNA double-strand breaks (DSBs) at high frequency, as shown by neutral comet assay and phosphorylation of H2AX. Our data provide evidence that WRN is involved in the repair of topoisomerase I, but not topoisomerase II-induced DNA damage, most likely via preventing the conversion of DNA single-strand breaks into DSBs during the resolution of stalled replication forks at topo I–DNA complexes. We suggest that the WRN status of tumor cells impacts anticancer therapy with topoisomerase I, but not topoisomerase II inhibitors.     

On the nature and origin of DNA strand breaks in elongating spermatids

Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by trichostatin A, whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished on incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity.     

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.     

Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals

The comet assay is a rapid, sensitive and inexpensive method for measuring DNA strand breaks. The comet assay has advantages over other DNA damage methods, such as sister chromatid exchange, alkali elution and micronucleus assay, because of its high sensitivity and that DNA strand breaks are determined in individual cells. This review describes a number of studies that used the comet assay to determine DNA strand breaks in aquatic animals exposed to genotoxicants both in vitro and in vivo, including assessment of DNA damage in aquatic animals collected from contaminated sites. One difficulty of using the comet assay in environmental work is that of comparing results from studies that used different methods, such as empirical scoring or comet tail lengths. There seems to be a consensus in more recent studies to use both the intensity of the tail and the length of the tail, i.e. DNA tail moment, percentage of DNA in the tail. The comet assay has been used to assess DNA repair and apoptosis in aquatic animals and modifications of the comet assay have allowed the detection of specific DNA lesions. There have been some recent studies to link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics. Further work is required before the comet assay can be used as a standard bio-indicator in aquatic environments, including standardization of methods (such as ASTM method E2186-02a) and measurements.     

Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I

Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug-induced cleavable complex.     

Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.     

Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II

Many intercalative antitumor drugs have been shown to cleave DNA indirectly through their specific effect on the stabilization of a cleavable complex formed between mammalian DNA topoisomerase II and DNA (Nelson, E.M., Tewey, K.M., and Liu, L.F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1361-1365). Antitumor epipodophyllotoxins (VP-16 and VM-26) which do not intercalate DNA can similarly induce protein-linked DNA breaks in cultured mammalian cells. In vitro studies using purified mammalian DNA topoisomerase II show that epipodophyllotoxins interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by stabilizing a cleavable complex. Treatment of this stabilized cleavable complex with protein denaturants results in DNA strand breaks and the covalent linking of a topoisomerase subunit to the 5'-end of the broken DNA. Furthermore, epipodophyllotoxins also inhibit the strand-passing activity of mammalian DNA topoisomerase II, presumably as a result of drug-enzyme interaction. The agreement between the in vivo and in vitro studies suggests that mammalian DNA topoisomerase II is a drug target in vivo. The similarity between the effect of epipodophyllotoxins on mammalian DNA topoisomerase II and the effect of nalidixic acid on Escherichia coli DNA gyrase suggests that the cytotoxic action of epipodophyllotoxins may be analogous to the bactericidal action of nalidixic acid.     

Changes of 8-OH-dG levels in DNA and its base excision repair activity in rat lungs after inhalation exposure to hexavalent chromium

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl2 the MMS-induced DNA strand breaks accumulated during the first 2h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.     

Induction of ATM Activation,Histone H2AX Phosphorylation and Apoptosis by Etoposide: Relation to Cell Cycle Phase

Etoposide (VP-16) belongs to the family of DNA topoisomerase II (topo2) inhibitors, drugs widely used in cancer chemotherapy. Their presumed mode of action is stabilization of “cleavable complexes” between topo2 and DNA; collisions of DNA replication forks with these complexes convert them into DNA double-strand breaks (DSBs), potentially lethal lesions that may trigger apoptosis. Immunocytochemical detection of activation of ATM (ATM-S1981P) and histone H2AX phosphorylation (γH2AX) provides a sensitive probe of the induction of DSBs in individual cells. Using multiparameter cytometry we measured the expression of ATM-S1981P and γH2AX as well as initiation of apoptosis (caspase-3 activation) in relation to the cell cycle phase in etoposide-treated human lymphoblastoid TK6 cells. The induction of ATM-S1981P and γH2AX was seen in all phases of the cell cycle. The G1-phase cells, however, preferentially underwent apoptosis. The extent of etoposide-induced H2AX phosphorylation was partially reduced by N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS).The maximal reduction of H2AX phosphorylation by NAC, seen in G1-phase cells, was nearly 50%. NAC also protected a fraction of G1 cells from etoposide-induced apoptosis, but had no such effect on S or G2M cells. However, no significant rise in the intracellular level of ROS upon treatment with etoposide was detected. The effects of etoposide were compared with the previously investigated effects of another topo2 inhibitor, mitoxantrone. The latter was seen to induce a maximal level of ATM-S1981P and γH2AX (partially abrogated by NAC) in G1-phase cells, but unlike etoposide, triggered apoptosis exclusively of S-phase cells. The data suggest that in addition to the generally accepted mechanism involving collisions of replication forks with the “cleavable complexes”, other mechanisms which appear to be different for etoposide vs. mitoxantrone, may contribute to formation of DSBs and to triggering of apoptosis.     

Topological complexes between DNA and topoisomerase II and effects of polyamines

The polyamines spermine and spermidine were found to enhance the formation of a stable noncovalent complex between mammalian topoisomerase II and DNA. This complex is not associated with DNA strand breaks and forms to a greater extent with supercoiled than with relaxed circular or with linear DNA. Polyamine-induced complex formation is associated with a stimulation of the enzymatic relaxation of DNA supercoils. In these respects, the polyamine-enhanced complex differs from the covalent cleavable complexes stabilized by DNA intercalators such as amsacrine (m-AMSA) or epipodophylotoxins such as teniposide (VM-26). In the polyamine-enhanced complex, the topoisomerase II may be a donutlike structure topologically bound to the DNA and able to migrate and dissociate from the ends of linear DNA molecules. At relatively high concentrations, spermine (1 mM) enhances topoisomerase II induced cleavage at certain sites on the SV40 genome that could have regulatory significance.     

In vivo mapping of DNA topoisomerase II-specific cleavage sites on SV40 chromatin

The antitumor drug, m-AMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidide), is known to interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by blocking the enzyme-DNA complex in its putative cleavable state. Treatment of SV40 virus infected monkey cells with m-AMSA resulted in both single- and double-stranded breaks on SV40 viral chromatin. These strand breaks are unusual because they are covalently associated with protein. Immunoprecipitation results suggest that the covalently linked protein is DNA topoisomerase II. These results are consistent with the proposal that the drug action in vivo involves the stabilization of a cleavable complex between topoisomerase II and DNA in chromatin. Mapping of these double-stranded breaks on SV40 viral DNA revealed multiple topoisomerase II cleavage sites. A major topoisomerase II cleavage site was preferentially induced during late infection and was mapped in the DNAase I hypersensitive region of SV40 chromatin.