Biomedical subjects
C Bailly
Publications and source records attributed to C Bailly.
Diethylpyrocarbonate and osmium tetroxide as probes for drug-induced changes in DNA conformation in vitro.
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Copper-dependent oxidative and topoisomerase II-mediated DNA cleavage by a netropsin/4'-(9-acridinylamino)methanesulfon-m-anisidide combilexin.
A conjugate molecule was synthesized by linking the DNA-intercalative antitumor drug 4'-(9-acridinylamino)methanesulfon-manisidide (mAMSA) via a 4-carboxamide side chain to a dipyrrolecarboxamide moiety structurally related to the minor groove-binding antibiotic netropsin. The molecule (netropsin/ mAMSA) behaves as a threading intercalator. Its netropsin-like tail becomes located in the minor groove of the double helix and serves to drive the hybrid molecule preferentially to AT-rich sites on various DNA fragments as revealed by DNase I footprinting. The hybrid retains the susceptibility to copper-dependent oxidation characteristic of the parent mAMSA moiety as well as its ability to generate oxygen radicals, which can mediate DNA damage, mainly at cytidine and guanosine nucleotides. It also retains the property of stimulating the formation of cleavable complexes with DNA in the presence of topoisomerase II, but its netropsin-like moiety confers little or no influence on the reaction with topoisomerase I. Although netropsin/mAMSA is less potent than mAMSA at producing cleavable complexes with topoisomerase II, it promotes the appearance of cleavage sites at much the same nucleotide sequences as does the parent compound. The dipyrrolecarboxamide tail is not silent, however, since it modifies the concentration-dependence of cleavable complex formation.
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.
[Cytotoxicity and interaction of amsacrine derivatives with topoisomerase II: role of the 1' substitute on the aniline nucleus].
Amsacrine is an intercalating planar polycyclic aromatic molecule that displays antitumor activity. The cytotoxicity of this compound is related to its interaction with topoisomerase II. The substituent at position 1' on the aniline is thought to be essential to the formation of the topoisomerase II-DNA cleavable complex and hence the cytotoxicity of the drug. The influence of three substituents at position 1' on the modulation of the activity of topoisomerase II was investigated. The following observations emerge from our structure-activity relationship study: i) the effects of the drugs on topoisomerase II-mediated DNA cleavage in vitro are correlated with the results of the cytotoxicity assays performed with cells sensitive (DC-3F) and resistant to topoisomerase II inhibitors (DC-3F/9-OH-E); ii) depending on the nature of the 1' substituent of the drugs, the restoration of a normal topoisomerase II alpha catalytic activity in resistant DC-3F/9-OH-E cells transfected with a plasmid carrying a wild type topoisomerase II alpha cDNA (hTOP2) either does not modify the susceptibility of the cells to the drug or partially reverse the resistance phenotype. The molecular and cellular studies reveal that topoisomerase II alpha is implicated in the cytotoxicity of amsacrine and confirm that the substituent at position 1' on the anilino ring of amsacrine governs the interaction with topoisomerase II.
[Metastatic epiduritis with invasiveness of the left renal vein].
We report a case of epidural metastasis with left renal vein invasion. This nonsymptomatic venous involvement was detected by ultrasonography and confirmed at CT and MR imaging. Such an unusual cause of renal vein tumor thrombus occurred through a lumbar vein which represents one of its usual collateral branches.
PCR-based development of DNA substrates containing modified bases: an efficient system for investigating the role of the exocyclic groups in chemical and structural recognition by minor groove binding drugs and proteins.
DNA molecules containing inosine in place of guanosine and/or 2,6-diaminopurine in place of adenine have been synthesized and tested as substrates for binding of sequence-selective ligands, both small and large. Footprinting patterns reveal that the binding sites for AT- or GC-specific antibiotics (distamycin or mithramycin, respectively) are completely changed in the modified DNAs, as expected for direct sequence readout involving contact with the purine 2-amino group. However, we also find large changes in the binding of HMG-D, a member of the HMG-1 family of chromosomal proteins, pointing to an indirect influence of the exocyclic amino group on ligand binding via an effect on the deformability of the double helix. This interpretation is confirmed by the finding that deoxyuridine-containing poly- and oligonucleotides, which lack the exocyclic methyl group of thymidine in the major groove, interact 5-10 times more strongly with HMG-D than do their counterparts containing natural nucleotides.
Melanoma in childhood: an EORTC-MCG multicenter study on the clinico-pathological aspects.
Melanoma in children is rare. Nevertheless, it is imperative that clinicians be aware that melanoma does occur in childhood. Yet there is very little information available on the clinico-pathologic variations, and the prognostic parameters of melanoma in children. This report presents the results of a multicenter study of 102 lesions originally diagnosed as cutaneous melanoma, conducted among 5 Western European countries and collected during the period 1961-1994. Criteria for inclusion in the study included: (1) diagnosis of cutaneous melanoma; (2) age up to 16 years at diagnosis; and (3) availability of representative microscopic slides. On the basis of the histologic review only, 60 lesions were confirmed as melanoma, and 42 lesions initially diagnosed as melanoma were reclassified as nevi; 31 of the latter contained a predominance of spindle cells. The only significant parameter associated with the development of metatases or fatal outcome was thickness of more than 2.00 mm. The 5-year survival rate observed in this study was 84%. Based on these findings we conclude that considerable over-diagnosis of melanomas in children occurs. In order, therefore, to give consistent epidemiological data on melanomas in children and to improve proper recognition of their diagnostic features, both by clinicians and by pathologists, we propose to set up a central registry of melanomas in children in Europe, under the auspices of the European Organization for Research and Treatment of Cancer.
A simple ligation assay to detect effects of drugs on the curvature/flexibility of DNA.
Circular DNA molecules can readily be formed from the 169 bp tyrT fragment in the presence of T4 DNA ligase. We have analyzed the formation of DNA circles in the presence of the clinically important antitumour drugs amsacrine, mitoxantrone and daunomycin. All three are intercalating agents but they affect the closure reaction differently: daunomycin and mitoxantrone progressively inhibit the formation of circles whereas at low concentrations amsacrine strongly enhances the yield of circles suggesting that this drug can increase the flexibility and/or curvature of DNA. The ligation assay described here may prove useful and widely applicable for investigating the effects of small molecules on the secondary structure of DNA.
DNA sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata.
Ecteinascidin 743 is one of several related marine alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata. It is remarkably active and potent in a variety of in vitro and in vivo systems and has been selected for development as an anticancer agent. The present study investigates the interactions of ecteinascidin 743 with DNA. Ecteinascidin 743 retarded the electrophoretic migration of both supercoiled and relaxed simian virus 40 DNA even in the presence of sodium dodecyl sulfate and after ethanol precipitation, consistent with covalent DNA modifications. Similar results were obtained in a DNA oligonucleotide derived from ribosomal DNA. However, DNA denaturation reversed the DNA modifications. The homopolymeric oligonucleotide dG/dC was modified while neither the dI/dC nor the dA/dT oligonucleotides were, consistent with covalent attachment of ecteinascidin 743 to the exocyclic amino group at position 2 of guanine. Ecteinascidin 743 was then compared to another known DNA minor groove alkylating agent, anthramycin, which has also been shown to alkylate guanine N2. Footprinting analyses with DNase I and 1,10-phenanthroline-copper and exonuclease III digestions showed that ecteinascidin 743 covers three to five bases of DNA and exhibits a different sequence specificity than anthramycin in the Escherichia coli tyrosine tRNA promoter (tyrT DNA). The binding of ecteinascidin to DNA was abolished when guanines were substituted with inosines in this promoter. A band shift assay was designed to evaluate the influence of the bases flanking a centrally located guanine in an oligonucleotide containing inosines in place of guanines elsewhere. Ecteinascidin 743 and anthramycin showed similarities as well as differences in sequence selectivity. Ecteinascidin 743-guanine adducts appeared to require at least one flanking guanine and were strongest when the flanking guanine was 3' to the targeted guanine. These data indicate that ecteinascidin 743 is a novel DNA minor groove, guanine-specific alkylating agent.
Sequence-selective intercalation of antitumour bis-naphthalimides into DNA. Evidence for an approach via the major groove.
LU 79553, a bis-naphthalimide drug highly active against human solid tumour xenografts, has been shown to bis-intercalate into DNA with a helix-unwinding angle of 37 degrees. Footprinting experiments with DNase I reveal that the drug is selective for mixed nucleotide sequences characterised by an alternating purine-pyrimidine motif, particularly those containing GpT (ApC) and TpG (CpA) steps. Derivatives bearing nitro or amino substituents on the naphthalimide chromophores bind at essentially identical sites. The footprinting profiles on tyrT DNA and on two fragments from pBS bear a remarkable resemblance to those determined for nogalamycin, an antibiotic which binds intercalatively leaving bulky carbohydrate substituents blocking both the major and minor grooves of the helix. Several lines of evidence indicate that the bis-naphthalimides recognise their preferred binding sites via the unusual expedient of intercalating from the major groove. Footprints on the complementary DNA strands sometimes appear staggered in the 5'direction. Repositioning the 2-amino group of G.C base pairs, which serves as a critical minor-groove marker, by substitution with inosine and/or 2,6-diaminopurine has little effect on the distribution of binding sites for LU 79553. The bis-naphthalimides affect the guanine-specific reaction with dimethyl sulfate (which reacts with the N7 position of the base located in the major groove) but not reactions with tetrachloropalladinate or methylene blue. Photoactivation of LU 79553-DNA complexes leads to a small amount of strand scission mainly at guanine residues. These observations make a strong case for binding via the major groove of the double helix, in contrast to nearly all common intercalating drugs, which could be important in explaining the unique biological selectivity of bis-naphthalimides.
Stabilization of triple helical DNA by a benzopyridoquinoxaline intercalator.
Biophysical, footprinting, and chemical probing experiments are described which characterize the triple helix-stabilizing effects of a benzo[f]pyridoquinoxaline derivative BfPQ-4,3 structurally related to the previously reported benzo[f]pyridoindole compound BePI [Mergny et al. (1992) Science 256, 1681-1684]. Two parallel triple helix model systems have been investigated; one in which the third strand matched perfectly a 27 base pair purine-pyrimidine motif in target DNA and another in which the third strand was one nucleotide longer, i.e., a 28-mer. In the latter system, the pairing of the (Y)28 third strand to the (Y.R)27 target induces the formation of a bulge containing at least one unpaired base, which can be evidenced by chemical probing experiments with osmium tetroxide. BPQ, which uinwinds a duplex DNA by 17 degrees as judged by viscometric experiments and otherwise behaves as a typical nonspecific intercalculating drug, promotes the formation of Y.R.Y parallel triple helix containing both T.A.T and C.G.C+ triplets. Both DNase I and MPE.FeII footprinting experiments concur that triplex formation with the target (Y.R)27 sequence can be detected in the presence of BPQ at about 10-fold lower oligonucleotide concentrations than are required to produce an equivalent footprint in the absence of the drug. In addition, BPQ will promote binding to the polypurine-polypyrimidine target sequence by the longer mismatched oligonucleotide, providing significant stabilization of the parallel bulge-containing(Y.R)27,(Y)28 triplex with nearly the same efficiency as the bulge-free (Y.R)27.(Y)28 triplex. Thus in vivo BPQ might enhance the formation of both undesired and desired DNA triplexes. By performing an MPE*FeII probing reaction with a 5'-32 P-labeled oligonucleotide third strand, we have obtained evidence that BPQ is actually bound to the triplex region and may distort in a sequence-specific fashion.
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.
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.
DNA recognition by two mitoxantrone analogues: influence of the hydroxyl groups.
The clinically useful anticancer drug mitoxantrone intercalates preferentially into 5'-(A/T)CG and 5'-(A/T)CA sites on DNA. The 5,8 hydroxyl substituents on its anthracenedione chromophore are available to interact with the double helix. Footprinting experiments with two anthraquinone derivatives structurally related to mitoxantrone and ametantrone have been undertaken to assess the influence of the hydroxyl groups on the DNA recognition process. The results confirm that they do play a role in the recognition of preferred nucleotide sequences and suggest that the binding of anthraquinones to a 5'-(A/T)CG site is dependent on the presence of the 5,8 hydroxyl substitutes whereas binding to 5'-(A/T)CA sites appears to proceed just as well without them.
Germ line mutation at BRCA1 affects the histoprognostic grade in hereditary breast cancer.
Histoprognostic grade is a determinant parameter to select the initial therapeutic strategy in breast cancer. Our aim was to analyze the grade repartition in BRCA1-associated breast cancer (BC) and to explore the possible connections between grade and the BRCA1 gene function. We first compared 27 BRCA1-associated BCs from 14 families with 4,461 cases from an administrative district registry and 242 cases from a hospital-based registry, matching for grade and constitutive elements, and then considered their repartition in families. We observed a prevalence of grade 3 (P < 0.0001) in BRCA1-associated BC. This was attributed mainly to nuclear polymorphism (P < 0.0001), mitotic activity (P < 0.0001), and tubular differentiation (P = 0.0004), implying that BRCA1-associated BCs are highly proliferating tumors. Moreover, it is suggested that grade segregates as a genetic trait within families (P = 0.0015), and this was attributed to the mitotic index segregation only (P = 0.0005). Therefore grade, through its components, could be interpreted as the morphological translation of the BRCA1 germ line mutation. Thus, a genotype-phenotype correlation may exist between the type of mutation and the aggressiveness of the disease. These findings are bound to have an important impact in the care management of hereditary breast cancer at the individual and at the familial level and in the comprehensive approach of breast cancer development.
Cooperativity in the binding of echinomycin to DNA fragments containing closely spaced CpG sites.
Quantitative footprinting has been used to investigate cooperative binding of the antitumor antibiotic echinomycin to DNA fragments containing closely spaced CpG steps. The sequences of the designed DNA fragments contained two pairs of strong echinomycin binding sites: a pair of ACGT sites together with an ACGT site and a TCGA site, either directly adjacent or separated by two or four A.T base pairs. The results demonstrate that the binding of echinomycin to the sequences ACGTACGT and TCGAACGT is highly cooperative. The extent of cooperativity depends on the nature of the sequences clamped by the antibiotic and diminishes as the distance between the binding sites is increased. Various methods of extracting the information necessary to establish cooperativity have been compared. Beyond the specific interest in echinomycin-DNA interaction, the present quantitative footprinting study provides a model that may be generally applicable for designing investigations into cooperativity in drug-DNA recognition.
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.