Serologic responses to peptides of Anaplasma phagocytophilum and Borrelia burgdorferi in dogs infested with wild-caught Ixodes scapularis.

Anaplasma phagocytophilum and Borrelia burgdorferi are both transmitted by Ixodes spp. and are associated with clinical illness in some infected dogs. This study evaluated canine antibody responses to the A. phagocytophilum p44 peptides APH-1 and APH-4 as well as the B. burgdorferi C6 peptide before and after doxycycline treatment. A total of eight dogs were infested with wild-caught I. scapularis for 1 week. Blood was collected prior to tick attachment and from Days 3-77 to 218-302 with doxycycline treatment beginning on Day 218. Blood was assayed for A. phagocytophilum DNA by PCR assay. Sera was assessed for antibodies by immunofluorescent antibody (IFA) test and ELISA. Anaplasma phagocytophilum DNA was amplified from blood of all dogs by Day 7. Antibodies to APH-4 were detected in serum as early as 14days after tick exposure and six dogs had APH-4 antibodies detected 3-7 days before antibodies against APH-1. All dogs were seropositive for A. phagocytophilum from Days 218 to 302. Antibodies to B. burgdorferi were detected in 6/8 dogs beginning 21days after I. scapularis infestation. Among the five dogs that remained seropositive at Day 218, C6 antibody levels declined on average 81% within 84days of initiating treatment. The results suggest that the APH-4 peptide may be more useful than APH-1 for detecting antibodies earlier in the course of an A. phagocytophilum infection. After doxycycline administration, C6 antibody levels but not APH-1 or APH-4 antibody levels decreased, suggesting a treatment effect on C6 antibody production.

In vitro and in vivo pharmacological characterizations of the antitumor properties of two new olivacine derivatives, S16020-2 and S30972-1.

S16020-2, a new olivacine derivative and a topoisomerase II inhibitor, has recently entered clinical trials. New analogues and derivatives have been synthesized from the S16020-2 compound. Preliminary data indicate that S30972-1, one of these S16020-2 derivatives, may exhibit a comparatively higher level of antitumor potency associated with an improved therapeutic index than does S16020-2. The antitumor activities of S16020-2 and S30972-1 were therefore characterized both in vitro and in vivo, with Adriamycin and etoposide chosen as reference compounds. The in vitro data show that S30972-1 is a topoisomerase II inhibitor, mediating its activity through an ATP-dependent mechanism such as S16020-2. The two olivacine derivatives exhibited similar activities in vitro at the levels of the global growth of six human cancer cell lines, of the induction of apoptosis, and of the G2 cell cycle phase arrest. The in vivo antitumor activity characterization included the use of two murine leukemia types (P388-LEU and L1210-LEU), two murine lymphoma-like models (P388-LYM and L1210-LYM), two mammary adenocarcinomas (MXT-HI and MXT-HS), and one melanoma (B16). The data show that S30972-1 is actually more efficient in vivo than S16020-2, a feature that may relate to the fact that S30972-1 is less toxic than S16020-2. The S30972-1 compound exhibited in vivo a level of antitumor activity that was also actually higher than that exhibited by Adriamycin and similar to that exhibited by etoposide.

Cellular resistance to the antitumor DNA topoisomerase II inhibitor S16020-2: importance of the N-[2(Dimethylamino)ethyl]carbamoyl side chain.

The new olivacine derivative S16020-2 (NSC-659687) is a DNA topoisomerase II inhibitor endowed with a remarkable antitumor activity against various experimental tumors. In vitro physicochemical properties of this compound, in particular its interaction with DNA and DNA topoisomerase II, were very similar to those of ellipticine derivatives, except for a strictly ATP-dependent mechanism of cleavable complex induction. From the Chinese hamster lung fibroblast cell line DC-3F, a subline resistant to S16020-2, named DC-3F/S16, was selected by adding stepwise increasing concentrations of the drug to the cell growth medium. Whereas DC-3F/9-OH-E cells, a DC-3F subline resistant to 9-hydroxy-ellipticine, are cross-resistant to S16020-2, DC-3F/S16 cells are only very weakly cross-resistant to ellipticine derivatives, indicating that, despite their structural similarity, these compounds may differ in their mechanisms of action. Uptake and efflux rates of S16020-2 were identical in the resistant and the sensitive cells. Topoisomerase IIalpha was expressed at the same level in both sensitive and resistant cells, whereas expression of the beta-enzyme was approximately 50% lower in the resistant cells. Sequencing of both alpha- and beta-isoform cDNAs revealed a point mutation that converts Arg(486) to a Gly in the alpha cDNA, whereas the beta cDNA was not modified. This amino acid substitution in a highly conserved sequence of the enzyme appears to be responsible for the resistance to S16020-2. Comparative analysis of the properties of the ellipticine and S16020-2-resistant cells suggests that S16020-2, which is a DNA intercalator, might also interact with this enzyme amino acid sequence through its side chain.

Transfection of 9-hydroxyellipticine-resistant Chinese hamster fibroblasts with human topoisomerase IIalpha cDNA: selective restoration of the sensitivity to DNA religation inhibitors.

In the Chinese hamster lung cell line DC-3F/9-OH-E, selected for resistance to 9-OH-ellipticine and cross-resistant to other topoisomerase II inhibitors, the amount of topoisomerase IIalpha is 4-5-fold lower than in the parental DC-3F cells, whereas topoisomerase IIbeta is undetectable. Cloning and sequencing of topoisomerase IIalpha cDNAs from DC-3F and DC-3F/9-OH-E cells revealed an allele polymorphism, one allele differing from the other by the presence of seven silent mutations and three mutations in the noncoding region. In addition, the mutated allele contains three missense mutations located close to the ATP binding site (Thr371Ser) or to the catalytic site (Ala751Gly; Ile863Thr). To analyze the contribution of these topoisomerase IIalpha alterations to their resistance phenotype, DC-3F/9-OH-E cells were transfected with an eukaryotic expression vector containing the human topoisomerase IIalpha cDNA. In one transfected clone, the amount of topoisomerase IIalpha isoform and the catalytic activity were similar to that in the parental DC-3F cells. These cells, which contain only topoisomerase IIalpha, are then a unique mammalian cell line to analyze the physiological and pharmacological properties of this enzyme. However, the restoration of a nearly normal topoisomerase IIalpha activity in the DC-3F/9-OH-E cells did not have the same effect on their sensitivity to different enzyme inhibitors; a 75% reversion of the resistance, associated with a 2-3-fold increased stabilization of the cleavable complex, was observed with both etoposide and m-AMSA, two drugs that inhibit the DNA religation step in the enzyme catalytic cycle; in contrast, the transfected cells remained fully resistant to ellipticine derivatives that did not induce the stabilization of the cleavable complex. We hypothesized that a trans-acting factor, inhibiting the induction of cleavable complex formation by drugs that are not religation inhibitors, might be present in the resistant cells. However, such a factor was not detected in in vitro experiments, and other hypotheses are discussed.

S16020-2, a new highly cytotoxic antitumor olivacine derivative: DNA interaction and DNA topoisomerase II inhibition.

S16020-2 (NSC-659687) is a new olivacine derivative that is highly cytotoxic in vitro and displays remarkable antitumor activity against various experimental tumors, especially some solid tumor models. Its antitumor activity is notably higher than that of 2-methyl-9-hydroxy-ellipticinium (NMHE) and comparable to that of doxorubicin HCl, although with a different tumor specificity. S16020-2 is being tested in phase I clinical trials. A study of the interaction of S16020-2 with DNA showed that it binds through intercalation between adjacent DNA base pairs, inducing an unwinding of 10 degrees of the double helix. Its DNA affinity is approximately equal to that of NMHE and decreases as a function of the salt concentration, indicating a significant electrostatic contribution to the overall binding free energy. S16020-2 did not interfere with the catalytic cycle of DNA topoisomerase I but stimulated DNA topoisomerase II-mediated DNA cleavage via a strictly ATP-dependent mechanism. The interactions of S16020-2 and NMHE with DNA topoisomerase II in vitro are very similar. Both drugs have the same DNA sequence specificity of cleavage and the same biphasic dose-effect response, and neither drug inhibited the rate of DNA religation. In contrast with these observations, in in vivo experiments, S16020-2 was able to induce topoisomerase II-mediated DNA strand breaks at concentrations 500-fold lower than NMHE. We conclude that DNA topoisomerase II most likely is the cellular target involved in the mechanism of cytotoxicity of S16020-2. Its higher biological activity and potency to induce cellular DNA cleavage suggest the involvement of as-yet-unidentified cellular factors.

Enhanced topoisomerase II-induced DNA breaks and free radical production by a new anthracycline with potent antileukemic activity.

In a previous study we reported that a new anthracycline derivative (moflomycin) exhibited a higher antileukemic activity compared to other anthracyclines, such as daunorubicin and doxorubicin. To explain the superior antileukemic effect of moflomycin and to disclose a possible structure-activity relationship, we investigated the three main mechanisms by which anthracyclines are though to exert their antitumor effect: DNA binding, free radical production and topoisomerase II inhibition. The DNA interaction was assessed both by DNA binding and DNA unwinding assays, free radical generation was studied by electron spin resonance, and topoisomerase II interaction by analysis of the stimulation of enzyme-induced DNA breaks. The results showed a higher free radical production and a greater stimulation of topoisomerase II-mediated DNA cleavage by moflomycin than doxorubicin, associated with a lower DNA affinity. The different biochemical characteristics of moflomycin, particularly its interaction with topoisomerase II, are related to the structural modifications performed on the chromophore. These properties, associated with a higher stability of the molecule induced by the presence of an iodine atom on the sugar moiety, are probably responsible for the higher antileukemic activity of this compound.

Genistein resistance in human leukaemic CCRF-CEM cells: selection of a diploid cell line with reduced DNA topoisomerase II beta isoform.

Genistein, an isoflavonoid derivative initially described as an in vitro protein tyrosine kinase inhibitor, also inhibits mammalian DNA topoisomerase II both in vitro and in vivo. From a human leukaemic T cell line (CCRF-CEM), two genistein-resistant cell lines, which grow in the presence of 50 and 150 microM genistein, respectively, were selected and designated CEM/GN50 and CEM/GN150. Flow cytometry and karyotype analyses revealed that more than 95% of the parental cells were tetraploid whereas both resistant sublines were essentially diploid and were likely derived from the diploid fraction in the initial population. The CEM/GN cells were 3- to 4-fold resistant to genistein, and highly cross-resistant to certain metabolic inhibitors such as cytosine-arabinoside (50-fold) and 5-fluoro-2'-deoxyuridine (5000-fold). This resistance was associated with a markedly decreased uptake of thymidine and a 10-fold reduction in thymidine kinase activity. The CEM/GM cells were also 15- to 30-fold cross-resistant to topoisomerase inhibitors (etoposide, m-AMSA, 2-Me-9-OH-ellipticinium). Comparison of topoisomerase II activities in the sensitive and resistant cells showed: (i) an approximately 2-fold reduced decatenation activity in nuclear extracts from the resistant cells; (ii) an approximate 30% reduction in DNA-protein cross-links in etoposide-treated resistant cells; and (iii) a markedly reduced expression of the topoisomerase II beta isoform. These data, consistent with our previous results, indicate that the cytotoxicity of genistein is at least in part related to its capacity to inhibit DNA topoisomerase II.

Stimulation of site-specific topoisomerase II-mediated DNA cleavage by an N-methylpyrrolecarboxamide-anilinoacridine conjugate: relation to DNA binding.

The DNA binding properties and effects on topoisomerase II of MePyGA, an anilinoacridine derivative bearing an N-methylpyrrolecarboxamide unit at position 1', have been compared with those of its precursor glycylanilinoacridine and the structurally related antileukaemic drug amsacrine. Electric linear dichroism spectroscopy reveals that MePyGA intercalates its acridine chromophore between DNA base pairs with a preference for GC-rich sequences, whereas both its structural analogue lacking the N-methylpyrrole unit and amsacrine intercalate into DNA without any strong sequence preference. The effects of the test drug on the catalytic activities of topoisomerase II were studied in vitro using purified calf thymus enzyme and 32P-labeled DNA. MePyGA stabilizes the topoisomerase II-DNA covalent complex and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. The removal of the N-methylpyrrole unit abolishes both the GC-preferential binding to DNA and the topoisomerase II-mediated DNA cleavage. MePyGA and amsacrine stimulate the cleavage of DNA by topoisomerase II at different places: cleavage stimulated by amsacrine is consistent with the expected adenine requirement at position +1 whereas the predominant sites of DNA cleavage stimulated by MePyGA contain a cytosine at position +/- 1. This is the first instance where an anilinoacridine derivative differing only by the nature of the substituent at position 1' has been found to affect the catalytic activity of topoisomerase II differently. The spectroscopic and biochemical data lead to the conclusion that two functional domains can be identified in MePyGA: its anilino group can be regarded as a skeletal core to which are connected (i) the tricyclic acridine moiety which represents the DNA-binding domain and (ii) the N-methylpyrrole moiety which constitutes the topoisomerase II-targeted domain. The structure of the substituent at position 1' of the anilinoacridine chromophore evidently determines the location of the sites of DNA cleavage by topoisomerase II. These findings provide guidance for the synthesis and development of new topoisomerase II-targeted antitumor anilinoacridine derivatives.

Inhibition of DNA topoisomerases I and II and induction of apoptosis by erbstatin and tyrphostin derivatives.

Inhibitors of protein tyrosine kinases (PTK) and DNA topoisomerases are potential antitumour agents. Drugs which bind to the ATP site of PTK, such as genistein, are common inhibitors to both types of enzymes. Eleven erbstatin and tyrphostin derivatives, which inhibit epidermal growth factor receptor PTK activity by competing with both the peptide substrate and ATP were tested for their capacity to inhibit DNA topoisomerases I and II. Erbstatin, two synthetic derivatives with a modified side chain and the tyrphostin AG 786 inhibited both topoisomerases in the same range of concentrations (20-50 microM). The tyrphostin AG 213 inhibited only topoisomerase II. In this series, absence of PTK inhibitory effect was correlated with the absence of DNA topoisomerase inhibition, while the detection of PTK inhibition may or may not be associated with DNA topoisomerase inhibition. In contrast to genistein, none of these molecules induced the stabilization of the topoisomerase-DNA cleavable complex, either in vitro or in vivo. Alcaline elution analysis revealed that erbstatin did not induce the formation of protein associated DNA strand breaks. However, an extensive degradation of the cellular DNA was observed which was shown to result from an internucleosomal fragmentation. Furthermore, typical morphological modifications associated with apoptosis were observed in the erbstatin treated cells by electron microscopy. These data indicate that erbstatin induces an apoptotic cell death.

[Cellular resistance to DNA-topoisomerase II inhibitors]. / Résistance cellulaire aux inhibiteurs de l'ADN topo-isomérase II.

Chinese hamster lung cells resistant to 9-OH-ellipticine (DC-3F/9-OH-E) present a complex phenotype. These cells, which are about 150-fold resistant to 9-OH-E, display a cross-resistance to other topo-II inhibitors, such as m-AMSA or VP-16, which stabilize the cleavable complex. In addition, these cells display also a cross-resistance to suramin, which is also a topo-II inhibitor, but does not stabilize the cleavable complex. Finally, DC-3F/9-OH-E present a multidrug-resistant phenotype (MDR) which confers a cross-resistance to natural products such as actinomycin D, taxol or vincristine, due to a decrease of cellular accumulation of these drugs. Analysis of expression of the genes encoding topo-II alpha and beta, and the evaluation of both enzyme forms by immunoblotting, revealed that DC-3F cells contained about 20-fold less of the beta form than of the alpha form. The alpha form was decreased by about 4-5-fold in DC-3F/9-OH-E, whereas the beta form became undetectable. Purification and characterization of topo-II activities in sensitive and resistant cells is presently in progress. Analysis of the expression of pgp1, 2, 3 genes, involved in the MDR phenotype in hamster, by Northern blotting or by immunoblotting, has shown that the MDR phenotype in DC-3F/9-OH-E cells is due to the overexpression of pgp1 gene. In these cells, pgp3 expression is positively regulated by myc oncogene expression. Overexpression of the myc gene is followed by an overexpression of the pgp3 gene and is associated to a reversal of the MDR phenotype.

Chemoresistance to doxorubicin and cisplatin in a murine cell line. Analysis of P-glycoprotein, topoisomerase II activity, glutathione and related enzymes.

Resistance to antineoplastic drugs has often been associated with P-glycoprotein overexpression, this certainly being not the sole mechanism. In order to characterize resistance to doxorubicin and cisplatin, we have analysed P-glycoprotein expression, topoisomerase II activity, glutathione and related enzymes in murine leukemic cells (doxorubicin or cisplatin-resistant). The doxorubicin-resistant cells contained P-glycoprotein, showed lower activities of glutathione S-transferase well as of glutathione reductase and topoisomerase II. The modifications observed in the most cisplatin-resistant cell line were a higher activity of glutathione S-transferase isoenzyme pi and topoisomerase II. These results suggest that drug uptake, glutathione metabolism as well as topoisomerase II activity are all characteristic of multidrug resistance.

[In vitro stimulation by ellipticine derivatives of DNA cleavage induced by DNA topoisomerase II: a structure-activity relationship]. / Stimulation in vitro par des dérivés de l'ellipticine des coupures de l'ADN induites par l'ADN topo-isomérase II: relation structure-activité.

Ellipticines are intercalating planar polycyclic aromatic molecules that display antitumor activity. The cytotoxicity of these compounds is related to the presence of an hydroxy group at position 9 of the pyridocarbazole ring system and to their interaction with DNA topoisomerase II. The ability of 13 ellipticine derivatives to stabilize the topoisomerase II-DNA covalent complex in vitro is reported. The following observations emerge from our structure-activity relationship study: i) the hydroxy group at position 9 is essential for stabilizing the covalent complex, ii) the replacement of the methyl group at position 5 by an ethyl group (EPC) enhances the complex stabilization. The interaction of EPC and three other ellipticine analogues with DNA shows that the covalent complexes which are most stable have the lowest drug-DNA binding constants. In addition our study suggests that ellipticines induce covalent complex stabilization by a cooperative mechanism. A model is proposed to explain this stabilization by ellipticines. This study supports the idea that topoisomerase II is the primary target involved in the mechanisms of action of ellipticines.

Intoplicine (RP 60475) and its derivatives, a new class of antitumor agents inhibiting both topoisomerase I and II activities.

Intoplicine (RP 60475, NSC 645008) is an antitumor derivative in the 7H-benzo[e]pyrido[4,3-b]indole series which is now being tested in clinical trials. Intoplicine strongly binds DNA (KA = 2 x 10(5) M-1) and thereby increases the length of linear DNA. These properties are consistent with DNA unwinding by intoplicine. Intoplicine was found to be a dual topoisomerase I and II inhibitor, with DNA sites of enzyme inhibition being different for these two enzymes. In this study, 22 analogues of intoplicine were evaluated for their effects on topoisomerase I- and II-mediated DNA cleavage reactions by using enzymes purified from calf thymus. Site-specific DNA cleavage mediated by topoisomerase I was observed with 7H-benzo[e]pyrido[4,3-b]indole derivatives but not with 11H-benzo[g]-pyrido[4,3-b]indole derivatives. Site-specific DNA cleavage mediated by topoisomerase II occurred with derivatives having hydroxyl groups at the 3-position on the 7H-benzo[e]pyrido[4,3-b]indole ring or at the 4-position on the 11H-benzo[g]pyrido[4,3-b]indole ring. Study of the relationships between the in vivo antitumor activity on P388 leukemia and the topoisomerase I- and/or II-mediated DNA cleavage activity revealed that the most highly active antitumor compounds possessed both topoisomerase I-and II-inhibitory properties. Compounds selectively inhibiting either topoisomerase I or II were less active. These results suggest that dual topoisomerase I and II inhibition is critical for the antitumor activity of this new series of antitumor compounds.

Analysis of the DNA topoisomerase-II-mediated cleavage of the long terminal repeat of Drosophila 1731 retrotransposon.

The interaction of DNA topoisomerase II with the long terminal repeat (LTR) of the Drosophila melanogaster 1731 retrotransposon was studied. The covalent binding of topoisomerase II to the LTR was strongly stimulated by different inhibitors of the enzyme 4'-demethylepipodophyllotoxin-9-(4,6-O-2-ethylidene-beta-D-glucopy ranoside (VP-16), 4'-(9-acridinylamino)methanesulfon-m-anisidine) (m-AMSA) and an ellipticine derivative. Enzyme-mediated DNA cleavage could be observed in the absence of inhibitors and was stimulated in their presence. Cleavage occurred predominantly at sites located within or at the boundary of alternating purine/pyrimidine tracts in agreement with previous observations [Spitzner, J. R., Chung, I. K. & Muller, M. T. (1990) Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats, Nucleic Acids Res. 18, 1-11]. In addition, all of the cleavage sites observed in the absence of inhibitor were located in the U3 region of the LTR. The site specificity of drug-induced cleavage was studied and the conformity of the cleavage sites with previously established consensus sequences was examined. Our results suggest that DNA topoisomerase II, through its ability to alter the degree of DNA supercoiling, might be involved in the control of different functions of the LTR.

A carboline derivative as a novel mammalian DNA topoisomerase II targeting agent.

The DNA intercalating, ellipticine analog drug, 5,11-dimethyl-5H-indol[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.

Stimulation of topoisomerase II-mediated DNA cleavage by ellipticine derivatives: structure-activity relationship.

Ellipticines are aromatic compounds that intercalate between DNA base pairs and display significant antitumor activity. The cytotoxicity of these compounds is mediated by DNA topoisomerase II, and the presence of a hydroxy group at position 9 of the pyridocarbazole ring system of ellipticines has been found to be essential for high levels of cytotoxicity. The ability of 13 ellipticine derivatives to stabilize the topoisomerase II-DNA covalent complex in vitro was studied, and the data obtained with five pairs of hydroxylated and nonhydroxylated analogues indicate that the hydroxy group at position 9 plays a crucial role in the stabilization of the complex. The influence, upon the complex stabilization, of various substituents at positions 1, 2, 5, and 6 of the pyridocarbazole ring system was investigated. The interaction with DNA of four ellipticine derivatives was studied in the topoisomerase II standard medium. Results suggest that the degree of unwinding might be a determinant of topoisomerase II-DNA-drug complex stability. In addition, the 5-ethyl derivative was observed to induce covalent complex stabilization by a cooperative mechanism.

Biological activity and molecular interaction of a netropsin-acridine hybrid ligand with chromatin and topoisomerase II.

A hybrid molecule, which combines an anilinoacridine chromophore related to the antitumour drug amsacrine (m-AMSA) and a bispyrrole moiety analogous to the antiviral agent netropsin, has been examined for its ability to bind chromatin and to modulate the activity of topoisomerase II. The results show that the presence of histones does not alter the bimodal DNA binding process. Intercalation of the acridine and groove binding of the netropsin part of the drug are both observed with chromatin preparations. Moreover, the hybrid has a clear topoisomerase II-DNA cleavable complex-inducing activity close to that of m-AMSA. The role of the two parts of the hybrid ligand is discussed in relation to ternary complex formation. Two cell lines (L1210 leukemia and MCF7 mammary carcinoma) were compared in their sensitivity to the tested ligand. The drug, which appears to be an efficient growth inhibitor of leukemic cells in vitro, reveals moderate activity against P388 leukemia in vivo. The biological activity of the hybrid may derive from a mechanism that involves DNA binding and topoisomerase II inhibition. This study demonstrates that agents which intercalate and bind to the minor groove of DNA simultaneously represent a new class of drugs interfering with topoisomerase II and provide opportunities for the development of new antitumour agents.

Sequence requirements for mammalian topoisomerase II mediated DNA cleavage stimulated by an ellipticine derivative.

Various antitumor drugs stabilize DNA topoisomerase II-DNA transient covalent complexes. The complexes distribution along pBR322 DNA was shown previously to depend upon the nature of the drug (Tewey et al. (1984) Science 226, 466-468). The position in pBR322 of DNA cleavage by calf DNA topoisomerase II for 115 such sites stabilized by an ellipticine derivative and the relative frequency of cleavage at most of these sites were determined. The nucleotide sequence surrounding the 25 strongest sites was analyzed and the following ellipticine specific consensus sequence was deduced: 5'-ANCNT(A/G)T.NN(G/C)N(A/G)-3' where cleavage occurs at the indicated mark. A thymine is always present at the 3' end of at least one strand of the strong cleavage sites, and the dinucleotide AT or GT at the 3' end of the break plays a major role in the complex stabilisation. The predictive value of cleavage of the consensus was tested for two regions of SV40 DNA and cleavage was indeed detected at the majority of the sites matching the consensus. Some complexes stabilized by ellipticine are resistant to salt dissociation and this property seems to be correlated with the presence of symmetrical sequences in the cleavage site with a center of symmetry staggered relatively to the center of symmetry of cleavage.

DNA-drug recognition and effects on topoisomerase II-mediated cytotoxicity. A three-mode binding model for ellipticine derivatives.

Cytotoxic effects and topoisomerase II-mediated DNA breaks induced in vitro by ellipticine derivatives were examined in connection with 1H NMR and circular dichroism (CD) studies on molecular structures and interactions of drugs with DNA. The compounds included four 9-hydroxyellipticine and two 7-hydroxyisoellipticine derivatives. Structure-activity relationships indicated that a change in nitrogen atom position in the pyridinic ring greatly affected drug effects both on topoisomerase II action and cytotoxicity to L1210 cells. The four 9-hydroxyellipticine derivatives yielded bell-shaped curves in in vitro topoisomerase II-mediated DNA break assays, whereas the two 7-hydroxyisoellipticine derivatives demonstrated an almost linear increase at the same concentration (0-10 microM). In both cases, the intensity of cleavage was modulated by the position and the degree of methylation on the pyridinic ring, and results were correlated with cytotoxic activity expressed as the in vitro ID50 values for L1210 leukemia cells. 1H NMR experiments performed on free drug molecules in solution revealed that the two protons (alpha and beta) contiguous to the biologically important hydroxy group were sensitive to changes in electron distribution produced by the distant chemical modifications and methylations of the pyridinic ring. A linear relationship was observed between the differences in chemical shifts of alpha and beta protons (delta delta alpha-beta) versus ID50 values. CD experiments indicated that, at weak ionic strength I = 0.02 and at pH 7, drugs interact with the poly[d(A-T)] duplex according to a "three-mode binding model" which is governed by the drug structure and the drug to DNA ratio. The intercalation mode was related to the induction of topoisomerase II-mediated DNA cleavage, while the external binding mode consecutive to intercalation was related to cleavage suppression. These two modes concerned the good intercalators 9-hydroxyellipticines. The third was found for the weak intercalators 7-hydroxyisoellipticines and was characterized by self-stacked molecules bound "outside" DNA, presumably in the minor groove. Ligands either could be intercalated partially or linked at the edge of bases with a small number of molecules filling intercalation sites, for the second alternative. In addition to having different binding modes, 9-hydroxyellipticines were better inducers of DNA distortions than 7-hydroxyisoellipticines. The incidence of the drug binding modes on DNA-topoisomerase II recognition was discussed in connection with the in vitro cytotoxic activity exhibited by the drugs.

A CD study of interactions of ellipticine derivatives with DNA. Relations with the in vitro cytotoxicity.

UV-absorption and circular dichroism (CD) experiments showed that ellipticine derivatives may interact with DNA according to 3 possible binding modes depending on their structure and concentration. The first mode concerned intercalation of 1-methyl-9-hydroxyellipticine (1-Me-HE) with its long axis perpendicular to the long axis of base pairs. The same drug was able to bind to external sites (second mode) once the intercalation sites were saturated at high concentration. The third mode illustrated by 1,2-dimethyl-9-hydroxyisoellipticinium (1-Me-isoNMHE), concerned self-stacked molecules interacting at the surface of DNA. Biological significance of these different binding modes was then discussed in connection with in vitro cytotoxic activity of compounds.