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Biomedical subjects

M Leng

Publications and source records attributed to M Leng.

At least 37 records · Page 2Linked to original sources
PubMed1997

Chemical versatility of transplatin monofunctional adducts within multiple site-specifically platinated DNA.

The first step of the reaction between DNA and the antitumor drug cisplatin or its clinically inactive isomer transplatin yields monofunctional adducts. Most of the cisplatin monofunctional adducts further react and rather rapidly (t(1/2) smaller than a few hours) to form intrastrand and interstrand cross-links. It is generally accepted that the clinical activity of cisplatin is related to the formation of bifunctional lesions. As concerns transplatin, several studies disagree on the rate of closure of the monofunctional adducts and the nature of the bifunctional lesions. In order to explain these discrepancies, we have prepared several duplexes containing a single monofunctional trans-[Pt(NH3)2(dG)Cl]+ adduct and zero to two monofunctional [Pt(dien)(dG)]2+ adducts at defined positions. In these duplexes, the inert [Pt(dien)(dG)]2+ adducts mimic the presence of transplatin monofunctional adducts. We show that the closure of the transplatin monofunctional adducts is strongly affected by the presence of other adducts and by the length of the duplexes. These findings suggest that the discrepancies in the literature originate from the nature of the platinated samples (molar ratio of bound platinum per nucleotide, length of the DNA fragments). Our general conclusion is that within transplatin-modified DNA, at a low level of platination, the monofunctional adducts evolve slowly (t(1/2) > 24 h) into bifunctional lesions and that these bifunctional lesions are mainly interstrand cross-links. This could explain, at least in part, the clinical inefficiency of transplatin.

Base Sequence↗
PubMed1997

Transplatin-modified oligo(2'-O-methyl ribonucleotide)s: a new tool for selective modulation of gene expression.

In the reaction between trans-diamminedichloroplatinum(II) and single-stranded oligo(2'-O-methyl ribonucleotide)s containing the sequence GNG (N being a nucleotide residue), the 1,3-trans-{Pt-(NH3)2[GNG]} cross-links are formed. The 1,3-intrastrand cross-links are inert within the single-stranded oligonucleotides. By contrast, they rearrange into interstrand cross-links when the platinated oligonucleotides are paired with their complementary RNA strands. The rate of the interstrand cross-linking reaction depends upon the sequence facing the intrastrand cross-links. When the complementary sequences are 5'-CN'C (N' being a nucleotide), the rates are rather slow (T1/2 >/= 3 h at 37 degrees C). The rearrangement of the intrastrand cross-links into interstrand cross-links can be achieved in a few minutes when the triplets facing the intrastrand cross-links are replaced by doublet 5'-UA or 5'-CA. In vitro, the specificity of the cross-linking reaction between a platinated oligo(2'-O-methyl ribonucleotide) and its target sequence (containing the 5'-CA doublet) located within the coding region of Ha-ras mRNA is demonstrated by steric blocking of reverse transcriptase and translation machinery. Within the HBL100ras1 cells, this platinated oligonucleotide binds specifically and irreversibly to the cognate Ha-ras mRNA. It also inhibits the proliferation of the HBL100ras1 cells in a dose-dependent manner. The fast and specific interstrand cross-linking reaction triggered by the formation of a double helix between platinated oligo(2'-O-methyl ribonucleotide)s and RNA enhances the potential of the oligonucleotides which do not induce mRNA cleavage by RNase H, to modulate gene expression by steric blocking of the translation machinery.

Cell Division↗
PubMed1997

Rearrangement of interstrand cross-links into intrastrand cross-links in cis-diamminedichloroplatinum(II)-modified DNA.

In the reaction of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, bifunctional intrastrand and interstrand cross-links are formed. In this work, we show that at 37 degrees C interstrand cross-links (ICL) are labile and rearrange into intrastrand cross-links. The ICL instability was first studied with a 10 base pairs (bp) double-stranded oligonucleotide containing a unique site-specific ICL resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(GC/GC) site by a cis-diammineplatinum(II) residue. The bonds between the platinum and the N7 of guanine residues within the interstrand adduct are cleaved. In 50 mM NaCl or NaClO4, this cleavage results in the formation of monofunctional adducts which subsequently form intrastrand cross-links. One cleavage reaction takes place per cross-linked duplex in either of both DNA strands. Whereas the starting cross-linked 10 bp duplex is hydrogen bonded, the two complementary DNA strands separate after the cleavage of the ICL. Under these conditions, the cleavage reaction is irreversible allowing its rate measurement (t1/2= 29+/-2 h) and closure of monofunctional adducts to intrastrand cross-links occurs within single-stranded DNA. Within a longer cross-linked oligonucleotide (20 bp), ICL are apparently more stable (t1/2= 120+/-12 h) as a consequense of monofunctional adducts closure back to ICL. We propose that the ICL cleavage is reversible in DNA and that these adducts rearrange finally into intrastrand cross-links. Our results could explain an 'ICL unhooking' in previously reported in vivo repair studies [Zhenet al. (1993)Carcinogenesis14, 919-924].

Antineoplastic Agents↗
PubMed1997

Structural transitions of a GG-platinated DNA duplex induced by pH, temperature and box A of high-mobility-group protein 1.

[1H, 15N] and 1H NMR, and CD spectroscopy are used to show that the duplex d(A-T-A-C-A-T-Pt 7G-Pt7G-T-A-C-A-T-A).d(T-A-T-G-T-A-C-C-A-T-G-T-A-T), where Pt7G is platinated guanine, containing the cis-[Pt(NH3)2]2+ adduct, undergoes reversible temperature-induced (T0.5 310 K) and pH-induced (pKa approximately 4.8) transitions between kinked-duplex and distorted forms, with the latter forms predominating at high temperature and low pH. A related pH-induced structural change was observed for the unplatinated duplex (pKa 4.69, Hill coefficient n = 1.4) but was less cooperative than for the platinated duplex (n = 2). The pH-induced transition is attributed to protonation of cytosine residues and has wider implications, since many reported NMR studies of DNA are carried out near pH 5 to minimize NH-exchange rates. The [Pt(en)]2+ (where en is 1,2-ethanediamine) GG chelate of the same duplex is shown to exist in kinked and distorted forms, and the [1H,15N]-NMR shifts for the kinked form are indicative of the presence of highly stereospecific interactions with the Pt-NH protons. On binding of the duplex platinated with [Pt(NH3)2]2+ to high-mobility-group protein 1 (HMG1) box A, similar changes in shifts of the Pt-NH3 resonances to those induced by raising the temperature or lowering the pH were observed. The specific changes in 1H-NMR chemical shifts of HMG1 box A are consistent with binding of the platinated duplex (intermediate exchange rate on the 1H-NMR time-scale) to the concave face of the protein via helices I and II and the intervening loop.

Base Composition↗
PubMed1997

The Drosophila DSP1 gene encoding an HMG 1-like protein: genomic organization, evolutionary conservation and expression.

The gene that encodes the dorsal switch protein (DSP1) has been isolated from a Drosophila melanogaster cosmid library. It is organized into seven exons and six introns. The relative position of the introns within the region coding for the high mobility group (HMG) domains are identical to those of vertebrate HMG 1/2 genes. The close similarity between DSP1 and HMG 1/2 genes strongly suggests that these genes derived from a common ancestral gene. DSP1 encodes, at least, two distinct mRNAs that differ in the length of their 5'-untranslated region and coding sequence. Detailed sequence analysis shows that alternative splicing of precursor mRNA gives rise to the two isoform mRNAs found in Drosophila cells.

Alternative Splicing↗
PubMed1997

Distortions of the DNA double helix induced by 1,3-trans-diamminedichloroplatinum(II)-intrastrand cross-link: an internal coordinate molecular modeling study.

A trans-diamminedichloroplatinum(II) (trans-DDP) intrastrand adduct within the sequence d(TCTG*TG*TC).d(GACACAGA) (where G* represents a platinated guanine) is modeled on the basis of qualitative experimental data concerning global unwinding and curvature as well as information on base pairing. Modeling is performed using the internal coordinate JUMNA program, specific to nucleic acids, and modified to include the possibility of covalently bound ligands. Calibration of the energy functions representing the Pt-N7 bond with guanine is described. The platinum atom and the platinum-nitrogen bonds are parameterized for use in the Hückel Del Re method to calculate monopoles at each atom. These monopoles are consistent with the Flex force field included in Jumna. By developing an appropriate minimization protocol we are able to generate stable, distorted three-dimensional structures compatible with the experimental data and including an unusually high global unwinding. No a priori geometric assumptions are made in generating these structures.

Algorithms↗
PubMed1997

Interstrand crosslinking reaction in transplatin-modified oligo-2'-O-methyl ribonucleotide-RNA hybrids.

In the context of developing an approach to irreversibly and specifically link oligonucleotides to RNA, the purpose of this work was to determine the factors interfering with the rate of the rearrangement of the transplatin 1,3-intrastrand crosslinks into interstrand crosslinks, rearrangement triggered by the formation of a double helix between platinated oligo-2'-O-methyl-ribonucleotides and their complementary strands. The rate of the rearrangement has been studied as a function of the length of the hybrids, the location of the intrastrand crosslinks, the nature of the oligonucleotide backbone, and the nature of the doublet replacing the triplet complementary to the intrastrand crosslinks. The thermal stability of the platinated hybrids has been determined in various salt conditions. The results are discussed in relation to the mechanism of the rearrangement. It is shown that the cellular proteins present weaker nonspecific interactions with single-stranded platinated oligo-2'-O-methyl-nucleotides than with the isosequential oligodeoxyribonucleotides.

Base Sequence↗
PubMed1996

Interstrand cross-linking reaction in triplexes containing a monofunctional transplatin-adduct.

Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.

Alkylating Agents↗
PubMed1996

High resolution mapping of E.coli transcription elongation complex in situ reveals protein interactions with the non-transcribed strand.

We have used chemical probes and UV light to perform a high resolution mapping of an Escherichia coli transcription elongation complex that was arrested in vivo by a protein readblock at a position distal to the promoter. The in situ probing data provide a precise picture of a constrained ternary complex in which the front edge of the polymerase is located at <6 bp from the catalytic center. Furthermore, our analyses reveal protein contacts with the non-transcribed strand within the arrested ternary complex. Thus, these results contribute substantially to the emerging view of a flexible transcription elongation complex in which the non-transcribed strand is an important regulatory element.

Acetaldehyde↗
PubMed1996

Large deletions induced in the white gene of Drosophila melanogaster by the antitumoral drug cis-dichlorodiammineplatinum(II): influence of non-homologous recombination.

We have studied two mutants carrying large deletions induced in the white gene of Drosophila by the antitumoral drug cisplatin. The breakpoints of the deletions were located by southern analysis and the sequences of the deletion junctions were determined. Two base-pair repeats are associated with the ends of these deletions; one of the repeats is preserved in the new junction after the deletion. DNA sequences such as A-T rich, alternating purine/pyrimidine tracts, polypurine-polypyrimidine tracts and topoisomerase I and II cleavage sites are found near the junctions. These results suggest that illegitimate recombinational processes are involved in the generation of cisplatin-induced large deletions.

ATP-Binding Cassette Transporters↗
PubMed1996

NMR solution structure of a DNA decamer containing an interstrand cross-link of the antitumor drug cis-diamminedichloroplatinum (II).

A 10 base pairs double-stranded oligonucleotide with the sequence d(CCTCG.CTCTC). d(GAGAG.CGAGG) containing a single interstrand cross-link resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(G.C/G.C) site by a cis-diammineplatinum(II) residue was analyzed by 1H NMR spectroscopy. All the exchangeable and nonexchangeable protons resonance lines (except some H5'-H5") were assigned. NOESY spectra and chemical shifts indicated that the cross-linkage of the guanines of G.5 and G.6 induced extrahelicity of C5 and C6. Moreover, several unusual proximities were observed such as: (i) NOE cross-peaks between the H2'-H2" of G.5 or G.6 and the aromatic proton of their 5' neighbor C4 or A7 (ii) the absence of cross-peak for the steps G.5-C6, C6-T7 and C5-G4 (iii) a strong NOESY connectivity between H8(G.5) and H2(A7). All these data allowed us to describe the head to tail arrangement of the two cross-linked guanines as well as their stacking with flanking neighbor nucleotides (G.5 with T7.A7 base pair and G.6 with C4.G4 base pair). Using all the NOESY and TOCSY data (208 constraints), we have obtained a solution structure of the cross-linked duplex by using the NMR-constrained molecular mechanics program JUMNA. The reversal position of the two cross-linked guanines placed the cis-diammineplatinum(II) residue in the minor groove. The stacking of the two cross-linked guanines with the surrounding bases induced a bend of 40 degrees toward the minor groove. The locally left-helix formation, the extrusion of the cytosines and the stacking of the platinated guanines led to an unwinding of 76 degrees. This value is in good agreement with the values deduced from gel electrophoresis experiments.

Antineoplastic Agents↗
PubMed1995

Intrastrand cross-links are not formed in the reaction between transplatin and native DNA: relation with the clinical inefficiency of transplatin.

The reaction between trans-diamminedichloroplatinum(II) and single-stranded oligonucleotides containing the sequence d(GXG) (X being an adenine, cytosine or thymine residue) yields trans-[Pt(NH3)2[(GXG)-GN7,GN7]] intrastrand cross-links. These cross-links do not prevent the pairing of the platinated oligonucleotides with their complementary strands but they decrease the thermal stability of the duplexes. The thermal stability is not much affected by the chemical nature of the X residue and its complementary base. By gel electrophoresis, it is shown that the trans- [Pt(NH3)2[d(GTG)-GN7,GN7]] cross-link bends the DNA double helix (26 degrees) and unwinds it (45 degrees). The pairing of the platinated oligonucleotides with their complementary strands promotes the rearrangement of the 1,3-intrastrand cross-links into interstrand cross-links. At a given temperature, the nature of the X residue, its complementary base and of the base pairs adjacent to the adducts do not dramatically affect the rate of the reaction. To know whether trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links do not rearrange in some sequences, the location of these adducts was searched in double-stranded DNA after reaction with trans-diamminedichloroplatinum(II) by means of the 3'-5' exonuclease activity of T4 DNA polymerase. At low level of platination, trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links were not detected. Monofunctional adducts and interstrand cross-links were mainly formed. These results are discussed in relation with the clinical inefficiency of trans-diamminedichloroplatinum(II).

Base Sequence↗
PubMed1995

Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides.

The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex.

Base Sequence↗
PubMed1995

Interaction between cisplatin-modified DNA and the HMG boxes of HMG 1: DNase I footprinting and circular dichroism.

The interactions between the two boxes A and B of HMG 1 and cis-diamminedichloroplatinum(II)-modified DNA containing a single intrastrand cross-link at the d(GpG) site were studied by DNase I footprinting and circular dichroism. The DNAase I cleavage patterns of the HMG box-platinated DNA complexes are identical, the two boxes inhibiting the DNase I cutting over at least 15 and 12 nucleotide residues in the platinated strand and the complementary strand, respectively. As judged by circular dichroism, the two boxes have the same alpha-helical content (56%) and they induce the same conformational changes in the platinated DNA.

Amino Acid Sequence↗
PubMed1995

The size of the topological domain modulates the B-Z transition of a (TG)n containing repeat.

Under negative superhelical stress, long (TG)n containing repeats experience a stepwise multiple B-Z transitions. We have investigated the effect of the plasmid size on this transitional behavior. A 66-bp (TG)n containing repeat from the 5'-untranscribed region of mouse ribosomal DNA was inserted in a 3-kb, a 6.5-kb and a 12.5-kb plasmids and its supercoil-driven B-Z transition was followed by OsO4 probing of topoisomer-populations. Our results show a clear correlation between the size of the topological domain and the extent of the region that converts cooperatively into Z-DNA at the initial transition.

Animals↗
PubMed1994

Deletions induced in the white and vermilion genes of Drosophila melanogaster by the antitumor drug cis-dichlorodiammineplatinum(II).

This paper describes the analysis of cisplatin induced mutations at the white (w) and vermilion (v) loci located on the X chromosome of Drosophila melanogaster. Twenty-eight w and eight v mutants have been found in a male genetic context and 42 w mutants in a female genetic context. In these latter experiments, genetic analysis showed the presence of multi-locus deficiencies in 18 out of 42 w mutants. Eighteen w and three v intragenic mutations were analyzed at the molecular level. Seventeen w and three v mutants carry deletions within the gene, ranging in size from 4 to 109 base pairs. Sequence analysis of the mutants indicates that most of them were produced by non-homologous recombinational events occurring between short (2-5 bp) sequence repeats on both sides of the deletion, one repeat being retained at the new junction. These results differ largely from those obtained in prokaryotic and other eukaryotic cells.

ATP-Binding Cassette Transporters↗