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Aim of this study was to investigate the anticancer properties of the new lanthanum compound [tris(1,10-phenanthroline)lanthanum(III)]trithiocyanate (KP772; FFC24). In vitro, growth inhibition by KP772 was comparable for >60 tumour cell models with IC50 values generally in the low microM range. KP772 induced tumour cell apoptosis indicated by chromatin condensation, caspase substrate cleavage and mitochondrial membrane depolarisation. DNA is unlikely to represent the primary molecular target of KP772, as no significant interaction or damage of DNA was detectable both in vitro and in living cells. Moreover, we found no evidence for induction of radical species. In contrast, KP772 potently inhibited DNA synthesis paralleled by a massive block of cell cycle in G0/G1 phase and a selective decrease of cyclin B1. Although treatment with KP772 induced expression of p53 and p21Waf1, transfection of wild-type p53 into knock-out cells only marginally enhanced the cytostatic activity of KP772. In vivo, the anticancer activity of KP772 against human DLD-1 colon carcinoma xenografts was comparable to that of cisplatin and methotrexate at doses not causing significant adverse effects. With regard to toxicity, the LD50 and no-observed-adverse-effect levels (NOAEL) of KP772 in Sprague-Dawley rats were 21.6 and 7.5 mg/kg, in outbred albino mice 62 and 10 mg/kg, respectively. In summary, KP772 exerts anticancer activity via potent induction of cell cycle arrest and/or apoptosis and has promising in vivo anticancer activity against a human colon cancer xenograft. Together, these data suggest further development of KP772 as a new anticancer metal-drug.

Imidazoacridinones (IA) are a class of antitumor agents which includes C-1311, an interesting drug in clinical trials. This study investigated the mechanism of IA binding to DNA for a series of 13 analogs that differ in their cytotoxic potency. Using C-1311 as a model compound, crystallographic, spectroscopic and biochemical techniques were employed to characterize drug–DNA interactions. X-ray crystallographic analysis revealed a planar structure of imidazoacridinone core that is capable of intercalative DNA binding. Accordingly, C-1311 binding to DNA followed ‘classical’ pattern observed for intercalation, as proved by the DNA topoisomerase I—unwinding experiments, with relatively weak binding affinity (Ki=1.2×105 M−1), and the binding site size of 2.4 bp. Other IA also bound to DNA with the binding affinity in the range of 105 M−1 and binding site size of 2–3 bp, suggesting a prevalence of the intercalative mechanism, similar to C-1311. Considerable DNA binding affinity was displayed by all the highly cytotoxic derivatives. However, none of the analyzed drug–DNA binding parameters was significantly correlated with IA biological activities such as cell growth, DNA and RNA synthesis inhibition, or tumor growth inhibition, which suggests that the IA ability to non-covalently bind to DNA is not crucial for their biological activity. These results show that the ability to intercalate into DNA is a prominent attribute of IA, although factors other than intercalative binding seem to be required for the biological activities of IA drugs.

WP631, a new DNA-binding drug that bisintercalates into DNA with high affinity, seems to be highly cytotoxic against Jurkat T lymphocytes. The purpose of this study was to gain new insights into the mechanisms by which WP631 halts proliferation in this cell type. Treating Jurkat cells with nanomolar concentrations of WP631 roduced G2/M arrest, inhibited the transcription of c-myc and p53 genes, and induced limited apoptosis during the duration of treatment. Suppression of c-myc and p53 expression, and time-dependent decline in c-Myc and p53 protein levels, was associated with growth arrest. A weak interdependence was also found between the potent antiproliferative activity and the apoptotic response; treatment with WP631 for 24-36hr produced arrest in G2/M and allowed for partial DNA repair. Longer treatments with WP631 allowed some repaired cells to re-enter the cell cycle, but produced aneuploidy or apoptosis in others. © 2002 Elsevier Science Inc. All rights reserved., This work was financed by grants from the Spanish DGESIC (PB96-0812 and PB98-0469), The Welch Foundation and the Commission for Scientific Exchange between the United States of America and Spain. The support of the Centre de Referencia en Biotecnologia is also acknowledged

Sanguinarine, a benzophenanthrine alkaloid, is potentially antineoplastic through induction of cell death pathways. The development of multidrug resistance (MDR) is a major obstacle to the success of chemotherapeutic agents. The aim of this study was to investigate whether sanguinarine is effective against uterine cervical MDR and, if so, by which mechanism. The effects of treatment with sanguinarine on human papillomavirus (HPV) type 16-immortalized endocervical cells and their MDR counterpart cells were compared. Trypan blue exclusion assays and clonogenic survival assays demonstrated that MDR human cervical cells are as sensitive as their drug-sensitive parental cells to death induced by sanguinarine. Upon treatment of both types of cells with sanguinarine, two distinct concentration-dependent modes of cell death were observed. Treatment with 2.12 or 4.24 microM sanguinarine induced death in most cells that was characterized as apoptosis using the criteria of cell surface blebbing, as determined by light and scanning electron microscopy, and proteolytic activation of caspase-3 and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), as detected by Western blot analysis. However, 8.48 and 16.96 microM sanguinarine caused a second mode of cell death, oncosis, distinguished by cell surface blistering, and neither caspase-3 activation nor PARP cleavage. This study provides the first evidence that sanguinarine is effective against MDR in cervical cells via bimodal cell death, which displays alternative mechanisms involving different morphologies and caspase-3 activation status.

A DNA-targeted hypoxic cell radiosensitizer and cytotoxin, 5-[3-(2-nitro-1-imidazoyl)-propyl]-phenanthridinium bromide (2-NLP-3), has been shown previously to have increased efficacy over untargeted analogues in vitro. To further study the mechanism of action of this compound, a cell line, CHO-1000, derived from Chinese hamster ovary (CHO) AA8-4 cells was isolated. This cell line is capable of continuously growing in a concentration of 2-NLP-3 approximately 10-fold greater than that tolerated by wild-type CHO cells. The resistance of CHO-1000 to 2-NLP-3 was compared with that of the P-glycoprotein overexpressing, multidrug resistant Chinese hamster cell line CHR-C5 (C5). The resistance of CHO-1000 cells to the acute toxic effects of 2-NLP-3 under both hypoxic and aerobic exposure conditions was intermediate to that of the sensitive CHO wild-type cells and the resistant C5 cells. A similar pattern was seen for the hypoxic cell radiosensitizing ability of 2-NLP-3. 2-NLP-3 produced significant depletion of glutathione under both hypoxic and aerobic conditions in all three cell lines studied, and the degree of depletion was correlated with drug toxicity. CHO-1000 and C5 cells were significantly more resistant to colchicine and doxorubicin compared with wild-type cells. The toxicity pattern of 2-NLP-3 and its comparison phenanthridinium ion, P3, was not the same for CHO-1000 cells compared with C5 cells. Verapamil was an effective agent for reversing the hypoxic resistance to 2-NLP-3 in both CHO-1000 and C5 cells, but only a partial reversal of aerobic resistance was observed in CHO-1000 cells. These results indicate that the resistant phenotype of CHO-1000 is mediated to some degree by P-glycoprotein expression, but that other as yet unidentified factors are also involved.

The action of two structurally related DNA intercalating agents has been studied and compared, namely 4'-(9-acridinylamino) methanesulphon-m-anisidide (amsacrine, mAMSA) and 1,4-bis(butylamino)benzo[g]phthalazine (ABP) on the cell cycle and differentiation of U-937 human promonocytic leukemia cells. mAMSA (0.1 microM) and ABP (4 microM) reduced the proliferation activity to a similar extent and caused little cell mortality. At these subcytotoxic concentrations mAMSA induced the cells to accumulate at the G2 phase of the cycle, while cycle inhibition provoked by ABP was not phase specific. In addition, mAMSA caused an increase in the cell mass while ABP provoked cell shrinkage. This was consistent with the fact that ABP considerably inhibited protein synthesis, while mAMSA did not significantly affect this activity. SDS/K+DNA precipitation assays indicated that mAMSA, but not ABP, stimulated protein-DNA covalent complex formation. Finally, it was found that mAMSA, but not ABP, elicited the expression of differentiation markers, namely nitroblue tetrazolium reduction, activation of vimentin and leukocyte integrin (CD11b/CD18 and CD11c/CD18) expression, and downregulation of c-myc expression. The DNA intercalators doxorubicin and mitoxantrone, which like mAMSA induced the cells to accumulate at the G2 phase and increased the cell mass, induced the expression of differentiation markers. In contrast, the intercalators aclarubicin and caffeine and the non-intercalator novobiocin, which produced minor alterations on cell-cycle distribution and caused cell shrinkage, did not significantly elicit differentiation. These results support the conclusion that differentiation of myeloid leukemia cells by cytostatic drugs depends on the perturbations of the cell cycle, leading to disproportionate increases in cell mass.

In a search for new antiretroviral agents acting at the nucleic acid level, two hybrid molecules composed of a bispyrrolecarboxamide chain related to netropsin, linked to the intercalating chromophore oxazolopyridocarbazole, were tested on the cycle of a defective Moloney murine leukemia virus (M.MuLV) derived from the SVX shuttle and expressing resistance to the G418 antibiotic. The drug netropsin-oxazolopyridocarbazole (Net-OPC), which displays a binding preference to duplex DNA containing A + T bases, inhibits the retroviral replicative cycle ( ic 50 = 4.8 {rmmM ). In contrast, the related molecule (bis)pyrollecarboxamide-oxazolopyridocarbazole (Bpc-OPC) devoid of sequence preference as well as the elemental components of Net-OPC, namely OPC, pentyl-OPC and netropsin, displays no significant action on the viral cycle. The estimation of cytosolic viral DNA in infected cells using quantitative polymerase chain reaction suggests that Net-OPC impairs a post retrotranscriptional step of the viral cycle.

The DNA intercalating, ellipticine analog drug, 5,11-dimethyl-5 H -indolo[2,3- b ]quinoline, is able to stabilize in vitro the topoisomerase II-DNA cleavable complex and to induce DNA breaks in BPV I episome in rat fibroblasts. Cytotoxicity studies with DC3F cells resistant to ellipticine strongly suggest that topoisomerase II is a cellular target involved in the mechanism of cytotoxic action of this carboline derivative.

Two isoflavones, genistein (4',5,7-trihydroxyisoflavone) (1) and orobol (5,7,3',4'-tetrahydroxyisoflavone) (2) induced mammalian topoisomerase II dependent DNA cleavage in vitro. The cleavage activities of 1 and 2 were comparable to those of known antitumor agents with topoisomerase II dependent DNA cleavage activity such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP-16). Two flavones, fisetin (3,7,3',4'-tetrahydroxyflavone) (3) and quercetin (3,5,7,3',4'-pentahydroxyflavone) (4) showed topoisomerase II dependent DNA cleavage activity with similar potentials to that of Adriamycin. Addition of salt (0.5 M NaCl) to the reaction mixture containing genistein and topoisomerase II resulted in a great reduction of DNA cleavage, suggesting that the mechanism of the topoisomerase II dependent DNA cleavage induced by flavonoids is through the cleavable complex formation as seen with m-AMSA and VP-16. DNA unwinding assay using mammalian topoisomerase I showed that both 1 and 2 did not intercalate into DNA but both 3 and 4 intercalated like m-AMSA. Other structurally related flavonoids could not induce topoisomerase II dependent DNA cleavage, indicating that the restricted structures of flavonoids were required for the cleavage activity.

The bifunctional intercalator Ditercalinium (NSC 335153) demonstrates an anti-tumoral cytotoxicity markedly different from other intercalating agents. A delayed toxicity is observed in eucaryotic cells, both in vitro and in vivo , at drug concentrations far below those required to observe immediate toxic effects. Fluorescence microscopy demonstrates that Ditercalinium and the mit-ochondrial-staining fluorophore DiOC 2 (5) are concentrated in the same cellular organelles of L1210 cells. Electron microscopy of Ditercalinium-treated cells reveals extensive and progressive swelling of mitochondria, with no other ultrastructural changes observed. Ditercalinium uptake and toxicity are in part related to mitochondrial membrane potential. However, drug accumulation itself does not immediately alter the mitochondrial membrane potential. Cellular ATP pool levels and the rate of respiration fall progressively after drug treatment. Nucleotide pools in DC3F cells, measured between drug treatment and death, show marked drops in pyrimidine levels while purine nucleotide levels decline more slowly. Addition of uridine or cytidine partially rescues Ditercalinium-treated cells, while toxicity is increased in the presence of 2-deoxyglucose. The combined evidence indicates that the toxicity of Ditercalinium to murine leukemia cells (L1210) and Chinese Hamster lung cells (DC3F) is due to disruption of mitochondrial function.

Aza-bioisosteres of anthrapyrazoles (Aza-APs) bearing the C–N substitution at position 9 are powerful anticancer agents now in clinical trials. In contrast, their 8-substituted regioisomers are practically devoid of chemotherapeutic effects. To understand the molecular basis for a dramatically different response by otherwise very similar compounds, we performed a detailed investigation on the physico-chemical properties of several aza-APs belonging to the two families, on their DNA-binding affinity and specificity as well as on their capacity to impair the activity of the two isoforms of human Topoisomerase II (top2α and top2β). Our results indicate that molecular size and shape, electronic distribution, redox properties, lipophilicity and protonation equilibria are essentially the same when comparing 9- with 8-substituted congeners. Although no major difference could be picked up when comparing the DNA binding properties of corresponding members of the 8- and 9-aza families, interestingly the affinity and specificity for the nucleic acid is modulated by the nature of the side-arms linked to the aza-AP scaffold, suggesting structural motifs that may determine DNA sequence recognition by the studied drug. Topoisomerase II poisoning activity was much higher for 9-aza derivatives than 8-aza analogues as shown by a cleavage assay with purified recombinant top2 isoforms. The difference appears to account for the divergent anticancer potential exhibited by different aza-AP regioisomers and suggests a specific molecular recognition of the cleavage complex by the studied drugs.

The aporphine alkaloids (+)-dicentrine and (+)-bulbocapnine are non-planar molecules lacking features normally associated with DNA binding by intercalation or minor groove binding. Surprisingly, dicentrine showed significant activity as a topoisomerase II (EC 5.99.1.3) inhibitor and also was active in a DNA unwinding assay. The DNA unwinding suggests DNA intercalation, which could explain the inhibition of topoisomerase II. Bulbocapnine, which differs from dicentrine only by the presence of a hydroxyl group at position 11 and the absence of a methoxyl group at position 9, was inactive in all assays. Molecular modeling showed that dicentrine can attain a relatively planar conformation, whereas bulbocapnine cannot, due to steric interaction between the 11-hydroxyl group and an oxygen of the methylenedioxy ring. These observations suggest that dicentrine is an “adaptive” DNA intercalator, which can bind DNA only by adopting a somewhat strained planar conformation. The requirement of a suboptimal conformation to achieve DNA binding appears to make dicentrine a weaker topoisomerase II inhibitor than the very planar oxoaporphine alkaloid liriodenine. These results suggest that it may be possible to modulate DNA binding and biologic activity of drugs by modifications affecting their ability to adopt planar conformations.

The antileukemic alkaloid, fagaronine, is a potent differentiation inducer of various hematopoietic cell lines. We show here that fagaronine is a DNA base-pair intercalator with a K(app) of 2.1 x 10(5) M-1 for calf thymus DNA. Fagaronine inhibits the catalytic activity of purified calf thymus topoisomerase I as shown by relaxation of supercoiled plasmid DNA followed by electrophoresis in neutral as well as in chloroquine-containing gels. The catalytic activity of topoisomerase I is inhibited at concentrations above 30 microM. Fagaronine also inhibits the catalytic activity of purified calf thymus topoisomerase II at concentrations above 25 microM as shown by decatenation of kinetoplast DNA. Fagaronine stabilizes the covalent DNA-enzyme reaction intermediate (the cleavable complex) between topoisomerase I and linear pBR322 DNA at concentrations up to 1 microM. Further increase of the fagaronine concentration leads to a progressive decrease in the cleavable complex formation, which is totally inhibited at 100 microM. In contrast, up to 1 microM fagaronine has no effect on cleavable complex formation between purified calf thymus topoisomerase II and linear pBR322 DNA, whereas cleavable complex formation is inhibited at higher concentrations. Exposure to fagaronine results in an increase in DNA-protein complex formation in intact P388 murine leukemia cells. P388CPT5 cells, which have an altered topoisomerase I activity, are 4-fold resistant to the growth inhibitory effects of fagaronine compared to the parental cell line. Similarly, DC-3F/9-OH-E Chinese hamster fibrosarcoma cells, which have an altered topoisomerase II activity, are about 5-fold resistant to the growth inhibitory effects of fagaronine. We conclude that fagaronine is an inhibitor of both DNA topoisomerase I and II and propose that this might play a role in the cytotoxic activity.

We have examined the activities of two novel azaanthracene-9,10-diones (aza), 1-aza and 2-aza, in HL-60 human leukemia cell lines containing type II topoisomerases with different sensitivities to inhibition by other intercalating agents. The sensitive line, HL-60, was sensitive to 2-aza but not to 1-aza, whereas the resistant HL-60/AMSA was sensitive to neither agent. Measurements of 1- and 2-aza-induced, topoisomerase II-mediated DNA cross-linking in the cells revealed patterns of resistance and sensitivity that paralleled the results in the cytotoxicity assays. However, measurements of drug-induced topoisomerase II-mediated DNA cross-linking using purified HL-60 and HL-60/AMSA topoisomerase II indicated that both agents could stabilize a covalent complex between DNA and the HL-60 enzyme. HL-60/AMSA topoisomerase II resisted stabilization by either agent. This suggests that the resistance of HL-60 cells to 1-aza is not due to the inability of this drug to inhibit topoisomerase II but rather to another, undefined mechanism.

The novel, topoisomerase II-reactive anthracycline intercalator idarubicin (IDA) was demonstrated to produce protein-associated DNA cleavage in HL-60 human leukemia cells. Like a host of other antineoplastic intercalating agents, IDA produced this effect to a much lesser extent in HL-60/AMSA cells, a line that is primarily resistant to the intercalator amsacrine, but is cross-resistant to a variety of topoisomerase II-reactive DNA intercalating agents including IDA. This resistance is thought to be secondary to the resistance of the topoisomerase II within HL-60/AMSA cells. Surprisingly, HL-60/AMSA cells were minimally resistant to the cytotoxic and DNA cleaving actions of another anthracycline, doxorubicin (ADR). Comparing other effects of the two anthracyclines revealed that IDA, but not ADR, produced endonucleolytic cleavage, a marker of apoptosis. These results suggest that DNA intercalating anthracyclines can have different effects in human leukemia cells. In the case of IDA, drug actions were similar to those produced by the majority of intercalating agents examined in this cellular system. In the case of ADR, the ability to circumvent the resistance of HL-60/AMSA suggests additional, non-topoisomerase II-mediated mechanisms of cytolysis that may also explain the broad spectrum of clinical activity of ADR.

Malaria constitutes one of the major health threats in the tropical and sub-tropical areas of the world. Yet, few advances were made in recent years in revealing the mode of action of the common and most economically affordable antimalarial drugs, the schizontocidal 4-aminoquinolines. Data presented indubitably repudiate the previous notions that these drugs act by either halting the feeding of the parasite on its host erythrocyte cytosol or repressing nucleic acid synthesis due to intercalation into the parasite's DNA. A novel target for drugs is outlined, i.e. they are shown to inhibit in vitro the release of iron from acidified host cell cytosol, consisting mostly of hemoglobin, a process that could provide this trace element to the parasite. Resistance to quinoline-containing drugs is the principal reason for the present resurgence of malaria. Drug-resistant parasites accumulate less of these weak base-like drugs in the acidic digestive vacuoles. A kinetic model is presented, indicating that diminishing drug accumulation is due to decreased vacuolar proton pump activity and is not a result of a putative multidrug resistance (MDR) efflux pump. Findings to date on the molecular biology of parasite mdr genes are reviewed. These indicate no correlation between gene expression or mutations and phenotypic drug resistance. Reversal of parasite drug resistance by relevant compounds in MDR cancer cells seems to involve mechanism(s) different from the inhibition of the MDR pump in cancer cells.

The cytotoxicity anti-tumour intercalating agents such as the anthraquinone mitoxantrone is thought to relate to DNA binding and the trapping of DNA topoisomerase II complexes on cellular DNA. We have studied the uptake, nuclear location, DNA binding mode and DNA damaging capacity of mitoxantrone in a small cell lung carcinoma cell line (NCI-H69) compared with an in vitro-derived variant subline (NCI-H69/LX4) that exhibits "classical" multi-drug resistance (MDR). Variant cells maintained under doxorubicin selection showed reduced RNA levels that returned to control values within 7 days of growth under non-selective conditions. Variant cells released from selection stress showed resistance to DNA cleavage by doxorubicin, mitoxantrone, 4'-epidoxorubicin, 4'-deoxy-doxorubicin but reduced resistance to aclacinomycin A and a 9-alkyl substituted anthracycline in broad agreement with the cross-resistance patterns for cytotoxicity. Mitoxantrone treated NCI-H69 cells were found to accumulate DNA-protein crosslinks during a 4 hr post-treatment incubation period whereas variant cells maintained depressed levels of crosslinking. There was no apparent abnormality in the availability or drug sensitivity of topoisomerase II assayed in crude nuclear extracts of NCI-H69/LX4 cells. Whole cell uptake of radiolabelled mitoxantrone was depressed (50%) in NCI-H69/LX4 compared with NCI-H69, whereas assessment of nuclear-bound drug in individual cells by a fluorescence quenching technique showed at least a 10-fold greater level of target protection. The quenching results provide evidence of a high affinity, saturable mode of drug binding, favoured at low drug concentrations, that correlated with DNA cleavage capacity. We propose that the cytotoxic action of mitoxantrone is dependent upon a restricted and persistent form of binding to DNA that favours the long-term or progressive trapping of topoisomerase II complexes.

The interaction of aristololactam-beta-D-glucoside with calf thymus DNA has been studied by measuring the changes in the absorbance of the alkaloid over a wide range of temperatures and sodium chloride concentrations. The binding parameters obtained are best fit by the neighbour exclusion model. The salt and temperature dependence of the binding constants are used to estimate the thermodynamic parameters involved in the interaction of the alkaloid with DNA. It is observed that aristololactam-beta-D-glucoside binding to DNA is an exothermic process over the entire range of salt and temperature, and the estimated values of enthalpy and entropy change are strongly dependent on the ionic strength of the solution. The enthalpy and entropy changes compensate one another to produce a relatively small Gibbs' free energy change. The possibility that aristololactam-beta-D-glucoside exists as a monovalent cation at neutral pH and the possible molecular contribution to the enthalpy and entropy changes of the aristololactam-beta-D-glucoside-DNA complex are discussed.

The anticancer drug 4′-(9-acridinylamino)-methanesulfon- m -anisidide (m-AMSA) is known to bind to DNA by intercalation and to produce protein-associated DNA strand breaks in cells. Previous work [Zwelling et al., Biochemistry 20 , 6553 (1981)] had shown that m-AMSA is in rapid equilibrium between extracellular and intracellular compartments, and that the DNA strand breaks exist in a steady state of rapid formation and resealing. The current work reports an unusual uptake phenomenon of m-AMSA by mouse leukemia L1210 cells that occurs at higher drug concentrations than previously studied. The new uptake phenomenon was characterized by cooperativity, hysteresis, irreversibility, saturability, slowness and temperature dependence. It is concluded that m-AMSA concentrations above a critical value can initiate the irreversible sequestration of m-AMSA into a new phase, probably in an extranuclear compartment of the cell, from which the drug has no access to the nuclear DNA and probably does not contribute to cytotoxicity.

The results reported in this paper show the changes in chromatin structure caused by the binding of adriamycin (ADR), daunorubicin (DR) and ethidium bromide (EtdBr) to DNA in chromatin, either isolated or in nuclei or whole cells. Micrococcal nuclease was used as the structural probe of chromatin. The binding of the drugs to chromatin DNA induced two structural changes. First, it produced an unfolding of the overall chromatin structure as evidenced by the increased production of acid-soluble oligonucleotides for the drug-treated samples above the level of the control sample. Second, it caused a disruption of the core particle structure with increased production of DNA of subnucleosomal size and smearing of the nucleosome pattern. The effects were greatest for daunorubicin, followed by adriamycin and ethidium bromide.

Computer graphics and non-bonded energy calculations have been used to model the intercalative binding of the anti-tumour antibiotic nogalamycin, to double-stranded DNA. The drug is predicted to bind preferentially to A-T rich sites. Specific hydrogen bonds to the exocyclic amino group of adenine bases on the 3' side of an intercalative site have been found by the modelling procedure.

An attempt was made to analyze the mechanism by which 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) inhibits mammalian DNA topoisomerase II. The effects of various 9-aminoacridine derivatives differing by their DNA affinities and DNA sequence selectivity of binding were compared in the presence of purified mouse leukemia L1210 DNA topoisomerase II. No correlation was found between DNA unwinding and topoisomerase II inhibition. 9-Aminoacridine was inactive as a topoisomerase II inhibitor and o-AMSA was only weakly active. The location of L1210 topoisomerase II mediated DNA breaks produced in the absence or presence of 9-aminoacridines were studied in [32P]-end-labeled pBR 322 DNA. All 9-aminoacridines, even those differing by their DNA sequence selectivity of binding, produced similar DNA cleavage patterns. Most drug-induced topoisomerase II mediated DNA breaks appeared at sites that were already cleaved by the enzyme in the absence of drug. The present results suggest that 9-aminoacridines inhibit L1210 DNA topoisomerase II by interacting at or near enzyme-DNA complexes by some unknown DNA effect or by direct protein interaction.

Cultured human fibroblasts were exposed to various concentrations of actinomycin D, a DNA intercalating agent, and studied by various alkaline elution techniques for the presence of DNA lesions. DNA-protein crosslinks increased proportionately with increasing actinomycin D, with 1.53 crosslinks/10(6) nucleotides after 5 micrograms/ml exposure. However, in the single-strand DNA break assay, elution of DNA initially increased as expected with increasing actinomycin D but thereafter decreased, with only 0.09 breaks/10(6) nucleotides detected after 25 micrograms/ml exposure, suggesting the presence of DNA crosslinking. A standard alkaline elution assay for DNA-DNA crosslinking was performed, and lesions which mimicked such crosslinks were detected, with a relative crosslink frequency of 2.30 after 5 micrograms/ml exposure. These actinomycin D-associated lesions disappeared when the alkaline elution procedure was modified to include additional proteinase digestion and use of the detergent sodium dodecyl sulfate (SDS) in the elution buffer, suggesting that they represented undigested DNA-protein crosslinks or nonspecific protein on the filters inhibiting DNA elution. Greater than ten times as many DNA-protein crosslinks were detected in fibroblasts than the number of single-strand DNA breaks after cellular exposure to actinomycin, even after determining breaks using the modified methodology for decreasing cellular protein interference. The data suggest that the actinomycin-DNA complex is associated with the formation of DNA-protein crosslinks which represent lesions other than endonuclease-associated DNA strand scission.

The effects of two 7 H -pyridocarbazole dimers, PyDi1 and PyDi2, on Chinese hamster lung cells in culture in vitro , were compared to those of the corresponding monomers, PyMo1 and PyMo2, by measuring the rates of macromolecule syntheses, the growth kinetics of the drug-treated cells, and the cell cycle progression. The dimers, which are endowed with a very high DNA affinity, were about 10- and 40-fold more cytotoxic than the monomers from which they markedly differ in the following ways: in contrast to monomers, the dimers do not provoke the arrest of cell cycle progression in the G 2 + M phase; after a transitory exposure to either one of the dimers, the cell growth arrest was delayed for 6–8 generations. Therefore, the 7 H -pyridocarbazole dimers express their cytotoxicity through a mechanism of action different from that of their mono-intercalating counterparts. They might then constitute a new series of antitumour drugs.

The binding of aristololactam-beta-D-glucoside to DNA is characterized by hypochromism and bathochromism in the absorption band, quenching of the fluorescence intensity, increase in the positive and negative ellipticity of DNA, enhancement of thermal transition temperature, sign and magnitude of thermodynamic parameters, increase of the contour length of sonicated rod-like DNA and induction of the unwinding-rewinding process of covalently closed superhelical DNA. Binding parameters determined from absorbance and fluorescence titration by Scatchard analysis, according to an excluded-site model, indicate a very high affinity towards DNA. The binding of the alkaloid is an exothermic process with Gibbs free energy of -7.4 kcal/mol, van't Hoff enthalpy of -13.8 kcal/mol and entropy of -21.5 cal/degree/mol at 25 degrees. On the basis of these observations it is concluded that aristololactam-beta-D-glucoside binds to DNA by a mechanism of intercalation.

The interrelationship between affinity for DNA, cytotoxicity and induction of single-strand DNA breaks in cultured L1210 cells was studied for 21 compounds belonging to two series of tricyclic intercalators: 1-amino-substituted 4-methyl-5H-pyrido[4,3-b]indoles (gamma CARB) and 1-amino-substituted 4-methyl-5H-pyrido[3',4':4,5]pyrrolo[2,3-c]pyridines (PPP), which are simplified analogues of Ellipticine derivatives obtained by deletion of one cycle. Adriamycin, m-AMSA (4'-(9-acridinylamino) methanesulfon-m-anisidide), PZE (10-[diethylaminopropyl amino]-6-methyl-5H-pyrido[3',4':4,5]-pyrrolo[2,3-g] isoquinoline and RTE [( 1-(3-diethylaminopropylamino)-9-methoxy ellipticine, bimaleate) are used as reference compounds. The intercalation of these compounds into DNA was strongly suggested by three experimental observations: (i) the competitive inhibition of ethidium bromide intercalation, (ii) bathochromic and hypochromic effects on absorption spectra induced by DNA, and (iii) drug-induced increase of the DNA length, measured by viscosimetry. PPP derivatives are generally less cytotoxic and induce DNA breaks less efficiently than the gamma CARB ones, both in terms of maximum breakage frequencies and required drug concentrations. The most active compounds induced SSB in the DNA of L1210 cells, in a bell-shaped manner: the SSB frequency increased, rose to a maximum and then decreased as the drug concentrations increased. The maximum SSB frequencies induced by the most active compounds are of the same order as those of reference compounds Adriamycin and PZE. The structurally important requirements are essentially the same for both DNA breakage activity and cytotoxicity: (i) a N-CH3 in the 5-position, (ii) a CH3 in the 4-position, (iii) a hydroxy in the 8-position and (iv) the presence of an (aminoalkyl)amino side chain with preferentially a 3 carbon unit. There is no direct relationship between DNA affinity in vitro and induction of DNA breaks in cells, although a relatively high affinity seemed to be a necessary condition, since the most active compounds have the highest affinities and compounds having a very low affinity are totally inactive. The close correlation between cytotoxicity and extent of induction of DNA breaks suggests that these breaks may be in fact the lethal lesions responsible for cell death and thereby for the antitumor properties of these tricyclic intercalators.

In this work we study the effects of an intercalating antitumoral agent: 9-hydroxy-2-methyl ellipticinium (NMHE) on the structure of chromatin, using micrococcal nuclease and DNase 1 as structural probes. The binding of the drug to chromatin, either in vitro or in the nuclei, induces two structural changes of chromatin: (a) an unfolding of the overall structure which results in an activation of the rate of degradation of chromatin by micrococcal nuclease and (b) a disorganisation of the core particle structure leading to the unwrapping of the DNA from the histone core. Moreover, by studying the interaction of MMHE with nuclei labeled in the active regions of the genome through a nick-translation reaction, it appears that the drug is overconcentrated in these regions and does not induce any new structural changes. The interaction of NMHE with DNase 1-sensitive regions of chromatin indicates that these regions are already "open" or relaxed and represent a preferential target for the drug.

The interaction of 3-nitrobenzothiazolo (3,2-a) quinolinium (NBQ) perchlorate with DNA was studied by u.v.-visible and fluorescence spectrophotometry as well as by hydrodynamic methods. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts, and the fluorescence was quenched. Binding parameters, determined from spectrophotometric measurements by Scatchard analysis according to an excluded-site model, indicated a binding constant of 2.4 × 105 M−1 for calf thymus DNA at ionic strength 0.01. The interaction was markedly suppressed by increasing the salt concentration. Binding to the GC-rich DNA of Micrococcus lysodeikticus was weaker than the binding to calf thymus DNA at ionic strength 0.01. NBQ increased the viscosity of sonicated rod-like DNA fragments, producing a calculated increment in length of 2.4 A/bound drug molecule. It removed and reversed the supercoiling of closed circular duplex plasmid pBR322 DNA by virtue of a helix-unwinding angle estimated as approximately 13°/bound ligand molecule. We conclude that the binding of NBQ to DNA occurs by a mechanism of intercalation, which probably accounts for its reported antitumor activity.

Interactions of Mitoxantrone and Ametantrone with natural and synthetic nucleic acids in aqueous medium [0.15 NaCl, 5 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes), pH 7.0, 25 degrees] have been studied using computer-aided spectrophotometric techniques. Absorption spectra of the drugs in monomeric and dimeric form and their complexes with DNAs at low drug/phosphate ratios (D/P) have been established. The latter were red-shifted and had lower amplitude as compared with the spectra of the free ligand's monomer; the change is consistent with the already well-established intercalative mode of drug-nucleic acid interaction. Drug-DNA equilibria have been studied using the McGhee-von Hippel model of noncooperative ligand-polymer interaction, with the correction for dimerization of drugs. Although Mitoxantrone is two orders of magnitude more potent an antitumor drug than Ametantrone, the intrinsic association constants (Ki) of both drugs were of similar magnitude. Also, no significant DNA-base specificity for either of the drugs (measured as Ki value for various homopolymers) was observed. Therefore, no correlation was apparent between the intercalative mode of binding to DNA, regardless of base composition, and the pharmacological activity of these drugs. At higher D/P ratios, a secondary mode of binding was detected by both spectroscopy and light-scattering measurement. Homopolymer-pairs and polymers containing only dI and dC were especially susceptible to this secondary type of binding. The possibility that this secondary type of binding may be responsible for the antitumor properties of the drugs is considered.