Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication.

I have found that antineoplastic drugs which are known to be inhibitors of mammalian DNA topoisomerases have pronounced and selective effects on simian virus 40 DNA replication. Ellipticine, 4'-(9-acridinylamino)methanesulfon-m-aniside, and Adriamycin blocked decatenation of newly replicated simian virus 40 daughter chromosomes in vivo. The arrested decatenation intermediates produced by these drugs contained single-strand DNA breaks. Ellipticine in particular produced these catenated dimers rapidly and efficiently. Removal of the drug resulted in rapid reversal of the block and completion of decatenation. The demonstration that these drugs interfere with decatenation suggests that they may exert their cytotoxic and antineoplastic effects by preventing the separation of newly replicated cellular chromosomes. Camptothecin rapidly breaks replication forks in growing Cairns structures. It is likely that the target of camptothecin is the "swivel" topoisomerase required for DNA replication and that it is located at or very near the replication fork in vivo. Evidence is presented that many of the broken Cairns structures are in fact half-completed sister chromatid exchanges. One pathway for the resolution of these structures is completion of the sister chromatid exchange to produce a circular head-to-tail dimer.     

New insight for fluoroquinophenoxazine derivatives as possibly new potent topoisomerase I inhibitor

Fluoroquinolones, represented by ciproxacin and norfloxacin, are well-known clinical antimicrobial agents, and their phenyl ring expanded quinophenoxazines are reported as possible antitumor active compounds. These quinophenoxazines are known to inhibit DNA topoisomerase II essential for cell replication cycle. But there were no reports for topoisomerase I inhibition study for these compounds. In this report, we have prepared a few quinophenoxazine analogues and tested their topoisomerases I and II inhibitory activities and cytotoxicity. From the result, we found that quinophenoxazine analogues possessed strong topoisomerase I inhibitory capacity as well as topoisomerase II inhibition. Among the compounds prepared, A-62176 analogues showed strong topoisomerases I and II inhibitory activities. Interestingly, compound 8 missing the 3-aminopyrrolidine moiety at C2 position has similar potent inhibitory capacity against topoisomerases I and II at higher concentrations (20 and 10 microM, respectively). But compound 8 inhibited topoisomerase I function more selectively at lower concentration, 2 microM. Our observation might strongly implicate that fluoroquinophenoxazines can be developed as efficient topoisomerase I inhibitor with the elaborate modification.     

Molluscum Contagiosum Virus Topoisomerase: Purification, Activities, and Response to Inhibitors

Molluscum contagiosum virus (MCV), the only member of the Molluscipoxvirus genus, causes benign papules in healthy people but disfiguring lesions in immunocompromised patients. The sequence of MCV has been completed, revealing that MCV encodes a probable type I topoisomerase enzyme. All poxviruses sequenced to date also encode type I topoisomerases, and in the case of vaccinia virus the topoisomerase has been shown to be essential for replication. Thus, inhibitors of the MCV topoisomerase might be useful as antiviral agents. We have cloned the gene for MCV topoisomerase, overexpressed and purified the protein, and begun to characterize its activities in vitro. Like other eukaryotic type I topoisomerases, MCV topoisomerase can relax both positive and negative supercoils. An analysis of the cleavage of plasmid and oligonucleotide substrates indicates that cleavage by MCV topoisomerase is favored just 3′ of the sequence 5′ (T/C)CCTT 3′, resulting in formation of a covalent bond to the 3′ T residue, as with other poxvirus topoisomerases. We identified solution conditions favorable for activity and measured the rate of formation and decay of the covalent intermediate. MCV topoisomerase is sensitive to inhibition by coumermycin A1 (50% inhibitory concentration, 32 μM) but insensitive to five other previously reported topoisomerase inhibitors. This work provides the point of departure for studies of the mechanism of function of MCV topoisomerase and the development of medically useful inhibitors.     

Inhibition of Zn(II) Binding Type IA Topoisomerases by Organomercury Compounds and Hg(II)

Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3β may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.     

Topoisomerase inhibitors induce irreversible fragmentation of replicated DNA in concanavalin A stimulated splenocytes

Etoposide, a nonintercalative antitumor drug, is known to inhibit topoisomerase II. Its effects have been tested in concanavalin A stimulated splenocytes, a system of cell proliferation in which topoisomerase II is induced. The primary effect of etoposide was a strong inhibition of DNA synthesis and the production of reversible DNA breaks, presumably associated with topoisomerase II. However, prolonged (20 h) contact with the drug resulted in a secondary fragmentation by irreversible double-strand breaks that yielded unusually small DNA fragments. Surprisingly, the same effect was obtained with novobiocin, which does not produce topoisomerase II associated DNA breaks. Moreover, long-term treatment with camptothecin, a specific inhibitor of topoisomerase I which is known to induce single-strand breaks in vitro and in vivo, also produced double-strand breaks and DNA fragmentation into small pieces. These findings suggest that prolonged treatment of proliferating splenocytes by etoposide and other topoisomerase inhibitors induced DNA fragmentation by a mechanism that does not directly involve topoisomerases.     

Dual role of topoisomerase II in centromere resolution and aurora B activity

Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II–depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II–depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restores Aurora B kinase activity, and consequently, most syntelic attachments are released. Taken together, our results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule–kinetochore attachments by allowing proper activation of Aurora B kinase.     

Baseline sister chromatid exchange in human cell lines with different levels of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase is a chromatin enzyme which adds long chains of ADP-ribose to various acceptor proteins in response to DNA strand breaks. Its primary function is unknown; however, a role in DNA repair and radiation resistance has been postulated based largely on experiments with enzyme inhibitors. Recent reports of mutant cell lines, deficient in poly(ADP-ribose) polymerase activity, have supported previous studies with inhibitors, which suggests the involvement of poly(ADP-ribose) polymerase in maintaining baseline levels of sister chromatid exchanges. Mutant cells with even slightly depressed enzyme levels show large elevation of baseline sister chromatid exchanges. Since intracellular poly(ADP-ribose) polymerase levels can vary greatly between different nonmutant cell lines, we surveyed levels of baseline sister chromatid exchange in normal and tumor human cell lines and compared them with endogenous levels of poly(ADP-ribose) polymerase. Despite 10-fold differences in poly(ADP-ribose) polymerase, the baseline level of sister chromatid exchanges remained relatively constant in the different cell lines (0.13 +/- 0.03 SCE/chromosome), with no indication of a protective effect for cells with high levels of the enzyme.     

Synergistic interactions between tumor necrosis factor and inhibitors of DNA topoisomerase I and II

TNF is a pleiotropic cytokine that mediates diverse cellular responses, including cytotoxicity, cytostasis, proliferation, differentiation, and the expression of specific genes. Many of these processes require the activity of DNA topoisomerases I and II. We have investigated the interactions of TNF with inhibitors of both topoisomerases in 16-h assays using the murine L929 and human ME-180 cell lines, which undergo a cytotoxic TNF response. Camptothecin, a specific inhibitor of topoisomerase I, enhanced TNF cytotoxicity 150-fold against both cell lines. The topoisomerase II inhibitors VM-26 and VP-16, which stabilize covalent DNA-topoisomerase intermediates, greatly enhance TNF cytotoxicity against both cell lines. The most effective, VM-26, can lower the TNF LD50 to femtomolar levels. In contrast, the topoisomerase II inhibitors novobiocin and coumermycin, which bind to the enzyme ATPase site, protect L929 cells from TNF cytotoxicity but enhance TNF cytotoxicity in ME-180 cells. The large changes in TNF sensitivity induced by drug concentrations that by themselves show no effect, and the opposing synergistic effects of inhibitors with different inhibitory mechanisms (in L929 cells), suggest the active involvement of topoisomerases in TNF-mediated cytotoxicity. The correlation of cytotoxic synergy with the stabilization of DNA strand breaks indicates that DNA damage may play a significant role in TNF-mediated cytotoxicity.     

Evidence for a common mechanism of action for antitumor and antibacterial agents that inhibit type II DNA topoisomerases

Numerous antitumor and antibacterial agents inhibit type II DNA topoisomerases, yielding, in each case, a complex of enzyme covalently bound to cleaved DNA. We are investigating the mechanism of inhibitor action by using the type II DNA topoisomerase of bacteriophage T4 as a model. The T4 topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli. Two m-AMSA-resistant phage strains were previously isolated, one with a point mutation in topoisomerase subunit gene 39 and the other with a point mutation in topoisomerase subunit gene 52. We report here that the wild-type T4 topoisomerase is inhibited by six additional antitumor agents that also inhibit the mammalian type II topoisomerase: ellipticine, 9-OH-ellipticine, 2-me-9-OH-ellipticinium acetate, mitoxantrone diacetate, teniposide, and etoposide. Further, one or both of the m-AMSA-resistance mutations alters the enzyme sensitivity to each of these agents, conferring either cross-resistance or enhanced sensitivity. Finally, the gene 39 mutation confers on T4 topoisomerase a DNA gyrase-like sensitivity to the gyrase inhibitor oxolinic acid, thus establishing a direct link between the mechanism of action of the anti-bacterial quinolones and that of the antitumor agents. These results strongly suggest that diverse inhibitors of type II topoisomerases share a common binding site and a common mechanism of action, both of which are apparently conserved in the evolution of the type II DNA topoisomerases. Alterations in DNA cleavage site specificity caused by either the inhibitors or the m-AMSA-resistance mutations favor the proposal that the inhibitor binding site is composed of both protein and DNA.     

Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases.

In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.     

Sister and non-sister chromatid U-type exchange in rye meiosis

Aberrant meiotic chromosome configurations in an experimental population of rye lines are known to result from spontaneous U-type exchanges during meiotic prophase. Both sister chromatid and non-sister chromatid exchanges occur and this study is concerned with the relative frequencies of sister and non-sister exchanges. The anaphase I observations reveal a marked excess of non-sister U-type exchange configurations and it is argued that this reflects an original excess of prophase non-sister U-type exchanges. This conclusion is discussed with special reference to the origin of meiotic U-type exchanges and their relation to regular crossover exchanges.     

Characterization of sister chromatid exchange induction by 8-methoxypsoralen plus near UV light

The combination of 8-methoxypsoralen and near UV light is highly effective in inducing sister chromatid exchanges (SCEs) in Chinese hamster ovary cells. Appreciable increases in SCEs can be effected by treatments compatible with cell survival, and effects of a single dose of alkylation persist over multiple generations. Both the frequency and location of SCEs induced at different times within the DNA synthesis period varies in a manner indicating that exchange induction is restricted to regions which replicated during or after DNA damage.     

Gossypol Interferes with Both Type I and Type II Topoisomerase Activities Without Generating Strand Breaks

A considerable number of agents with chemotherapeutic potentials reported over the past years were shown to interfere with the reactions of DNA topoisomerases, the essential enzymes that regulate conformational changes in DNA topology. Gossypol, a naturally occurring bioactive phytochemical is a chemopreventive agent against various types of cancer cell growth with a reported activity on mammalian topoisomerase II. The compounds targeting topoisomerases vary in their mode of action; class I compounds act by stabilizing covalent topoisomerase-DNA complexes resulting in DNA strand breaks while class II compounds interfere with the catalytic function of topoisomerases without generating strand breaks. In this study, we report Gossypol as the interfering agent with type I topoisomerases as well. We also carried out an extensive set of assays to analyze the type of interference manifested by Gossypol on DNA topoisomerases. Our results strongly suggest that Gossypol is a potential class II inhibitor as it blocked DNA topoisomerase reactions with no consequently formed strand breaks.     

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.     

Swiveling and decatenation of replicating simian virus 40 genomes in vivo.

We have found that type II topoisomerase inhibitors have two effects on replicating simian virus 40 genomes in vivo: production of catenated dimers and slowed replication of the last 5% of the genome. This suggests that type II topoisomerase simultaneously decatenates and facilitates replication fork movement at this stage of DNA replication. On the basis of this observation, a detailed model is proposed for the roles of topoisomerases I and II in simian virus 40 DNA replication.     

Sister chromatid exchange induced by anti-herpes drugs.

The rate of sister chromatid exchange induced by several anti-herpes agents was measured to assess their potential mutagenicity. The agents--5-iodo-deoxyuridine (IDU), 5-trifluoromethyl-deoxyuridine (TFT), and [E]-5-(2-bromovinyl)-deoxyuridine (BVDU)--were incubated at various concentrations with human lymphocytes and fibroblasts, and that rate of sister chromatid exchanges was measured. In lymphocytes and fibroblasts BVDU and IDU did not induce exchange except at concentrations of 50 mg/l, while TFT increased the rate of exchange at a concentration of 0.5 mg/l. The rate of sister chromatid exchange is a sensitive index of chromosomal damage, and these findings provide information on the safety of some of the antiherpes agents tested. TFT increased the rate of exchange at a concentration that coincides with its minimal antiviral concentration, but BVDU did not induce exchange at therapeutic concentrations.     

Indenoindolone derivatives as topoisomerase II–inhibiting anticancer agents

Based on known heterocyclic topoisomerase II inhibitors and anticancer agents, various indenoindolone derivatives were predicted as potential topoisomerase II–inhibiting anticancer agents. They are hydrazones, (thio)semicarbazones, and oximes of indenoindolones, and indenoindolols. These derivatives with suitable substitutions exhibited potent specific inhibition of human DNA TopoIIα while not showing inhibition of topoisomerase I and DNA intercalation, despite the fact that parent indenoindolones are known poor/moderate inhibitors of topoisomerase II. The potent topoisomerase II inhibitor indenoindolone derivatives exhibited good anticancer activities compared to etoposide and 5-fluorouracil, and relatively low toxicity to normal cells. These derivatizations of indenoindolones were found to result in enhancement of anticancer activities.