Biomedical subjects

C Houssier

Publications and source records attributed to C Houssier.

At least 37 records · Page 2Linked to original sources

PubMed1997

DNA cleavage by topoisomerase I in the presence of indolocarbazole derivatives of rebeccamycin.

DNA topoisomerase I has been shown to be an important therapeutic target in cancer chemotherapy for the camptothecins as well as for indolocarbazole antibiotics such as BE-13793C and its synthetic derivatives NB-506 and ED-110 [Yoshinari et al. (1993) Cancer Res. 53, 490-494]. To investigate the mechanism of topoisomerase I inhibition by indolocarbazoles, we have studied the induction of DNA cleavage by purified mammalian topoisomerase I mediated by the antitumor antibiotic rebeccamycin and a series of 20 indolocarbazole derivatives. The compounds tested bear (i) various functional groups on the non-indolic moiety (X = CO, CH2, CHOH), (ii) a hydrogen or a chlorine atom at positions 1 and 11 (R2), and (iii) different substituents on the maleimido function (R1 = H, OH, NH2, NHCHO). Half of the ligands have the same carbohydrate moiety as rebeccamycin whereas the other ligands have no sugar residue. The inhibitory potency of the test compounds was assessed in vitro by comparing the cleavage of [32P]-labeled restriction fragments by the enzyme in the absence and presence of the drug. In addition, the DNA-binding properties of these compounds were investigated by means of complementary spectroscopic techniques including electric linear dichroism, and the DNA sequence selectivity was probed by DNase I footprinting. The study shows that the sugar residue attached to the indolocarbazole chromophore is critical for the drug ability to interfere with topoisomerase I as well as for the formation of intercalation complexes. Structure-activity relationships indicate that the presence of chlorine atoms significantly reduces the effects on topoisomerase I whereas the substituents on the maleimido function and the functional group on the non-indolic moiety can be varied without reduction of activity. The results suggest that the inhibition of topoisomerase I by indolocarbazoles arises in part from their ability to interact with DNA. Analysis of the base preferences around topoisomerase I cleavage sites in various restriction fragments indicated that, in a manner similar to camptothecin, the rebeccamycin analogue R-3 stabilized topoisomerase I preferentially at sites having a T and a G on the 5' and 3' sides of the cleaved bond, respectively. By analogy with models previously proposed for camptothecin and numerous topoisomerase II inhibitors which intercalate into DNA, a stacking model for the interaction between DNA, topoisomerase I and indolocarbazoles is proposed. These findings provide guidance for the development of new topoisomerase I-targeted antitumor indolocarbazole derivatives.

Aminoglycosides↗

PubMed1997

Effects of compensatory solutes on DNA and chromatin structural organization in solution.

We investigated the effect of glycine and other osmotic effectors on DNA and chromatin precipitation by mono-, di- and multivalent cations and histone H1. The addition of these compounds drastically reduces the precipitation effects with an efficiency in the order taurine > glycine > proline and sorbitol > inositol > betaine. Aminocarboxylic acids with increasing distance between the charged C- and N-terminal groups displayed enhanced efficiency in the protection effect against DNA precipitation. We interpreted these observations on the basis of Manning's counterion condensation theory, taking into account the increase in dielectric constant upon osmotic effector addition. 23Na-NMR was used to evidence sodium counterions release as a result of this increase in dielectric constant.

Animals↗

PubMed1997

Effect of glycine on DNA structural transitions induced by multivalent cationic compounds.

We have investigated the effect of glycine (an organic osmolyte) on several DNA transitions induced by Tb3+, spermidine3+ and spermine4+ addition, using light scattering, circular dichroism, UV spectroscopy and electric linear dichroism techniques. DNA condensation and B-Z transition by the three compounds is perturbed by glycine: more Tb3+, spermidine3+ and spermine4+ must be added to obtain the same extent of condensation or Z-form as compared to the behaviour in the absence of this organic osmolyte. However, according to the light scattering experiments, glycine has also a structural effect on the DNA condensation that could be explained by an influence of the medium dielectric constant on the morphology of particles formed or on the rate of the condensation process. Contrary to these transitions, the particular B-B'-psi transition resulting from the addition of Tb3+ to a DNA solution is not observed in the presence of glycine. Since the chelation of Tb3+ by the phosphate group and the N-7 of guanine is presumably responsible for this transition, the glycine effect could probably be explained by a perturbation of this chelation by the change in solvent polarity and the chelating ability of the organic osmolyte.

Chemical Precipitation↗

PubMed1997

Synthesis, DNA-binding and cytotoxic properties of a bis(netropsin)-anthracenedione conjugate.

A combilexin molecule containing two netropsin moieties attached to the aminoalkyl side chains of mitoxantrone has been synthesized and evaluated for cytotoxic activity towards murine L1210 leukaemia and human MCF7 carcinoma cells in vitro. It is marginally less cytotoxic than mitoxantrone but much more growth-inhibitory than netropsin. Various spectroscopic and biochemical techniques have been employed to characterize the interaction of the drug, NetMitox, with DNA. Circular dichroism (CD) and electric linear dichroism (ELD) data indicate that binding of the netropsin moiety or moieties within the minor groove of the double helix impedes the intercalation of the adjacent anthracenedione ring. ELD and footprinting experiments reveal a certain amount of mutual interference between the two functionalities of the conjugate molecule but the selective recognition of AT-rich sequences by netropsin largely dominates the recognition pattern. The lack of interaction with GC-rich sequences is attributable to steric hindrance occasioned by the 2-amino group of guanine which impedes access of the netropsin moiety into the minor groove, as is evident by the good binding of the hybrid to poly(dI-dC) x poly(dI-dC) as well as by the redistribution of its binding sites on DNA molecules substituted with inosine and/or 2,6-diaminopurine. The difficulty of the anthracenedione system in intercalating correlates with the lack of effect of the drug on cleavable complex formation with topoisomerase II as well as its diminished cytotoxicity compared to mitoxantrone. However, the finding that the drug retains significant toxicity towards leukaemia cells may suggest that DNA is perhaps not the unique molecular target.

Animals↗

PubMed1996

Structural and functional aspects of chloride binding to Alteromonas haloplanctis alpha-amylase.

Chloride is the allosteric effector of vertebrate pancreatic and salivary alpha-amylases and of the bacterial alpha-amylase from Alteromonas haloplanctis. Activation experiments of A. haloplanctis alpha-amylase by several monovalent anions show that a negative charge, not restricted to that of Cl-, is essential for the amylolytic reaction. Engineering of the chloride binding site reveals that a basic residue is an essential component of the site. The mutation K337R alters the Cl--binding properties, whereas the mutation K337Q produces an active, chloride-independent enzyme. Comparison of the Kd values for Cl- in three homologous alpha-amylases also indicates that the binding affinity is dependent on the chloride coordination mode by this basic residue. Analysis of substrate and chloride binding according to the allosteric kinetic model shows that the chloride effector is not involved in substrate binding. By contrast, the pH dependence of activity and experiments of chemical modifications and Ca2+ inhibition show that the chloride ion is responsible for the pKa shift of catalytic groups and interacts with active site carboxyl groups.

Amino Acid Sequence↗

PubMed1996

The binding mode of drugs to the TAR RNA of HIV-1 studied by electric linear dichroism.

For the first time, the interaction between a series of small molecules and the TAR RNA of HIV-1 has been investigated by electric linear dichroism (ELD). The compounds tested include the DNA intercalating drugs proflavine and ethidium bromide and an amsacrine-4-carboxamide DNA-threading intercalator as well as the AT-specific DNA minor groove binders netropsin, Hoechst 33258, berenil and DAPI. In all cases except for netropsin, negative reduced dichroism signals were measured in the drug absorption band. In agreement with previous studies, the results indicate that both classical and threading intercalation can occur with the TAR RNA. The ELD data show that the mode of binding of the drugs Hoechst 33258, berenil and DAPI to the TAR RNA is similar to their binding mode in GC-rich regions of DNA and likely involves intercalation into the A-form TAR RNA helix. The wide and shallow minor groove of the TAR RNA is apparently not accessible to DNA minor groove binding drugs such as netropsin. The ELD technique appears uniquely valuable as a means of investigating the interaction of drugs with the TAR RNA.

Antiviral Agents↗

PubMed1996

Interaction of a DNA-threading netropsin-amsacrine combilexin with DNA and chromatin.

Combilexins are a group of DNA ligands having a sequence-specific minor groove binding element combined with an intercalating chromophore which stabilizes the DNA complex and can interfere with topoisomerases. In this study, complementary methods of spectroscopy (absorption, circular dichroism, electric linear dichroism) and biochemistry (viscometry, footprinting) have been applied to explore the nature of the complex formed between a new amsacrine-4-carboxamide-netropsin combilexin and DNA or chromatin. Collectively, the structural and kinetic data concur that the conjugate threads through the DNA double helix so as to intercalate its acridine chromophore, leaving the netropsin moiety and the methanesulfonanilino group positioned within the minor and major grooves of the double helix, respectively. The hybrid retains the AT selectivity conferred by the netropsin moiety. The threading-type intercalation process, evidenced by stopped-flow measurements, is affected when the DNA is wrapped around histones. The composite drug can bind to both the DNA linker segments and the nucleosomal cores in chromatin though, unlike its constituents, it antagonizes the salt-induced condensation of chromatin. As far as its mode of binding to DNA is concerned, the netropsin-amsacrine hybrid molecule exhibits structural features reminiscent of the antitumor antibiotics nogalamycin and pluramycin. The design of DNA-threading combilexins provides an original route for the development of sequence-specific ligands capable of forming stable complexes with DNA.

Amsacrine↗

PubMed1996

Polyelectrolyte counterion condensation theory explains differential scanning calorimetry studies of salt-induced condensation of chicken erythrocyte chromatin.

The salt-induced chromatin condensation in chicken erythrocyte nuclei is studied by differential scanning calorimetry (DSC). The degree of chromatin condensation is measured for condensation induced by monovalent, divalent, trivalent, or tetravalent cations and by a mixture of sodium and magnesium. These last two cations show an evident competition effect. Salt-induced chromatin condensation is shown to be an entropy-driven process. A simple model of chromatin based on the polyelectrolyte counterion condensation theory is used in order to compute the charge neutralized by the cations in each chromatin domain. The degree of chromatin condensation is shown to be related to the weighed sum of the square of the phosphate charge of each domain. The model predicts the salt and the chromatin concentration dependence of the condensation and the effect of H1 removal.

Animals↗

PubMed1996

Electric linear dichroism as a new tool to study sequence preference in drug binding to DNA.

An original approach using electric linear dichroism (ELD) and natural DNAs and polynucleotides of differing base composition has been developed with the aim to investigate the sequence-dependent recognition of DNA by drugs. Both intercalators and minor groove binders have been studied as well as certain hybrid molecules. The results indicate that the orientation of drugs upon binding to nucleic acids can change markedly according to the target sequence. Among the intercalators tested, only actinomycin D and hycanthone show a clear preference for GC- and AT-rich sequences, respectively. For minor groove binders, the linear dichroism showing a strong dependence on base composition of the DNA and polynucleotides is most pronounced. Netropsin and distamycin bind to DNA with a marked AT specificity. Hoechst 33258, berenil and DAPI exhibit positive and negative dichroism signals at AT and GC sites respectively, suggesting that at least two types of drug-DNA interaction are involved depending on the AT/GC content of the DNA. Further investigations using polynucleotides with inosine substituted for guanosine, and competition experiments with intercalative drugs suggest that Hoechst 33258, berenil and DAPI interact with GC sequences via a non-classical intercalation process.

Amino Acid Sequence↗

PubMed1996

23Na NMR study of the effect of organic osmolytes on DNA counterion atmosphere.

The effect of different organic osmolytes on the DNA counterion condensation layer has been investigated by 23Na NMR relaxation measurements. The zwitterionic compounds glycine, beta-alanine, 4-aminobutyric acid, and 6-aminocaproic acid have shown an increasing capacity to decrease the amount of sodium ions in the vicinity of the macromolecule. The experimental data have been correlated with the dielectric constant increase in their corresponding solutions and have been compared with the prediction of counterion condensation theory. Polyols (sorbitol and mannitol) did not display the same effect. These compounds largely increase the relaxation rate of sodium ions in the proximity of DNA, unlike the zwitterionic compounds. This probably results from a perturbation of the water dynamic around the macromolecule, of the primary or secondary hydration shell of the sodium nuclei involved, or both.

Alcohols↗

PubMed1996

Dielectric constant and ionic strength effects on DNA precipitation.

We have investigated the effect of different zwitterionic compounds on DNA precipitation induced by spermine4+. Glycine, beta-alanine, 4-aminobutyric acid, and 6-aminocaproic acid have shown an increasing capacity to attenuate DNA precipitation. This protection effect has been correlated with the dielectric constant increase of their corresponding solutions. Calculations based on these experimental data and counter-ion condensation theory have confirmed the importance of this parameter for DNA-ion interactions and precipitation mechanisms. We have also observed a resolubilization of DNA in the presence of 6-aminocaproic acid at high spermine4+ concentration and in the presence of glycine at high spermidine3+ concentration. This could be explained by an increase of screening effect with polyamine concentration.

Aminocaproic Acid↗

PubMed1996

Interaction of DNA-threading peptide-amsacrine conjugates with DNA and chromatin.

The SPKK peptide motif which functions as a DNA minor groove binding unit in various chromosomal and gene regulatory proteins has been attached to the antitumour drug amsacrine in order to reinforce interaction with DNA. Two bifunctional molecules in which an amsacrine-4-carboxamide derivative is linked to one or two SPKK motifs via a diaminopropyl tether have been designed and synthesized. We have applied various spectroscopic methods (absorption, circular and linear dichroism) to delineate the role of the peptide moiety as opposed to the chromophoric acridine moiety in the interaction of the conjugates with both DNA and chromatin. The structural and kinetic data concur that the two peptide conjugates thread through the DNA double helix so as to intercalate their acridine chromophore, leaving the SPKK motif and the methanesulphonanilino group positioned within the minor and major grooves of the double helix respectively. The threading-type intercalation process, evidenced by stopped-flow measurements, is not affected when the DNA is wrapped around histones. Linkage of the SPKK peptide motif to intercalating drugs represents an efficient system to stabilize drug-DNA complexes.

Amsacrine↗

PubMed1995

Tolerance to osmotic shocks in rats kidney cortex and medulla.

Kidney medulla cells of mammals have to cope with large changes in environmental osmolarity, a challenge most other mammalian cells never have to experience. In these last cells, application of osmotic shocks induces dramatic modifications in chromatin organization. The present paper reports on the changes of medulla cell chromatin in situ, in rat kidney slices submitted to osmotic challenges and in vitro, on preparations of extracted chromatin submitted to changes in environmental ion concentrations. Our results show that the chromatin of kidney medulla cells: (1) does not behave differently from the other mammalian chromatins when submitted in situ or in vitro to osmotic challenges; (2) presents in vitro physico-chemical characteristics similar to those of the other mammalian chromatins; and (3) is protected in vitro, as the other mammalian chromatins, from the disrupting effects of increases in inorganic ion concentrations by different compensatory organic solutes. The ability of kidney medulla cells to adapt to large increases in osmolarity could thus be related to a rapid control of the level of such compounds rather than to some rather specific, intrinsic molecular adaptations of macromolecules.

Animals↗

PubMed1995

Osmotic effectors and DNA structure: effect of glycine on precipitation of DNA by multivalent cations.

We have investigated the effect of glycine (an organic osmolyte) on DNA precipitation induced by spermine4+, spermidine3+ and Tb3+ addition, using circular dichroism (CD), UV spectroscopy (UV), and electric linear dichroism (ELD) techniques. DNA precipitation by the three compounds is perturbed by glycine: more spermine4+, spermidine3+ and Tb3+ must be added to obtain the same extent of precipitation as compared to the behaviour in absence of this organic osmolyte. It seems that glycine has a general effect on the DNA environment. Calculations based on experimental results and Manning's counterion condensation theory show that glycine could modify the electrostatic environment of DNA as a consequence of a change in dielectric constant.

Animals↗

PubMed1995

Use of electric linear dichroism and competition experiments with intercalating drugs to investigate the mode of binding of Hoechst 33258, berenil and DAPI to GC sequences.

The drugs Hoechst 33258, berenil and DAPI bind preferentially to the minor groove of AT sequences in DNA. Despite a strong selectivity for AT sites, they can interact with GC sequences by a mechanism which remains so far controversial. The 2-amino group of guanosine represents a steric hindrance to the entry of the drugs in the minor groove of GC sequences. Intercalation and major groove binding to GC sites of GC-rich DNA and polynucleotides have been proposed for these drugs. To investigate further the mode of binding of Hoechst 33258, berenil and DAPI to GC sequences, we studied by electric linear dichroism the mutual interference in the DNA binding reaction between these compounds and a classical intercalator, proflavine, or a DNA-threading intercalating drug, the amsacrine-4-carboxamide derivative SN16713. The results of the competition experiments show that the two acridine intercalators markedly affect the binding of Hoechst 33258, berenil and DAPI to GC polynucleotides but not to DNA containing AT/GC mixed sequences such as calf thymus DNA. Proflavine and SN16713 exert dissimilar effects on the binding of Hoechst 33258, berenil and DAPI to GC sites. The structural changes in DNA induced upon intercalation of the acridine drugs into GC sites are not identically perceived by the test compounds. The electric linear dichroism data support the hypothesis that Hoechst 33258, berenil and DAPI interact with GC sites via a non-classical intercalation process.

Base Sequence↗

PubMed1995

Design of composite drug molecules: mutual effects on binding to DNA of an intercalator, amsacrine, and a minor groove binder, netropsin.

A variety of spectroscopic and biochemical techniques have been employed to investigate the extent to which binding to DNA of an intercalator (amsacrine or its 4-carboxamide derivative SN16713) affects the binding of netropsin, a minor groove-targeted ligand, and vice versa. In general, rather little mutual interference has been found and the binding of one drug is compatible with binding of the other. The anilinoacridines exert little or no effect on the positioning of netropsin in the minor groove, judged by circular dichroism spectroscopy and electric linear dichroism, whereas netropsin has a perceptible effect on the intercalative binding of amsacrine, but not that of SN16713. Neither acridine drug prevents the netropsin-induced Z-->B structure reversion observed with poly(dG-dC).poly(dG-dC) in buffer containing 60% ethanol. The kinetics of dissociation of any one drug from its DNA complex are affected little, if at all, by the simultaneous presence of the other. Footprinting experiments with the several drugs singly or in combination reveal a certain amount of mutual interference, but the selective recognition of AT-rich sequences by netropsin tends to dominate the recognition pattern and is largely maintained in the presence of a considerable excess of amsacrine or its 4-carboxamide derivative.

Amsacrine↗

PubMed1995

Influence of the methyl substituents of a thiazole-containing lexitropsin on the mode of binding to DNA.

We have studied the DNA-binding properties of two thiazole-containing analogues of netropsin which differ by the absence (Thia-Nt) or presence (Methia-Nt) of methyl groups on the thiazole rings. The mode of binding to DNA of the two lexitropsins was investigated by circular dichroism, electric linear dichroism and viscosity measurements. The spectroscopic and hydrodynamic results indicate that the non-methylated lexitropsin binds to the minor groove of DNA, whereas the methylated analogue behaves as an intercalator. Our findings led to the notion that the methyl substituents on the thiazole rings might play a significant part in the intercalation process.

Animals↗

PubMed1995

Binding to DNA and cytotoxic evaluation of ascididemin, the major alkaloid from the Mediterranean ascidian Cystodytes dellechiajei.

The isolation of ascididemin from the Mediterranean ascidian Cystodytes dellechiajei is described. This alkaloid consists of a planar pentacyclic chromophore which was investigated for its DNA-binding and cytotoxic properties. Spectroscopic measurements provided evidence that the drug intercalates into DNA. DNase I footprinting assays indicated that the binding of ascididemin to GC-rich sequences is favoured over binding to AT-rich and mixed sequences. Chemical probes were used to detect ligand-induced structural changes in DNA. The alkaloid induces a hyper-reactivity of the DNA towards potassium permanganate, but not towards diethylpyrocarbonate, just as is the case with ethidium bromide; it has little effect on the catalytic activities of topoisomerases I and II. Ascididemin exhibits marked cytotoxicity towards human leukaemic cells in vitro and appears to be practically equally toxic for drug-sensitive and multidrug-resistant cell lines. The results suggest that DNA, but not topoisomerases, may represent the critical cellular target at which this marine alkaloid exhibits its potent cytotoxic properties in vitro.

Alkaloids↗