Daniel Locker

In vitro and in vivo antitumour activity and cellular pharmacological properties of new platinum–iminoether complexes with different configuration at the iminoether ligands

In order to widen our knowledge on antitumour trans-[PtCl2(iminoether)2] complexes, we have synthesised two new derivatives, trans-[PtCl2¿E-HN = C(OEt)Me¿2] (1) and trans-[PtCl2¿Z-HN = C(OEt)Me¿2] (2), which differ in the configuration of the iminoether ligands. Isomer 1 showed an in vitro cytotoxicity similar to that of cisplatin in a panel of human tumour cell lines (mean IC50 = 8 and 7.7 microM, respectively), whereas isomer 2 showed a lower activity (IC50 = 14.3 microM). Both 1 and 2 isomers overcame cisplatin resistance of ovarian cancer cell line A2780/Cp8. In agreement with the n-octanol/saline partition ratios, intracellular platinum content (and DNA platination) after a 2-h exposure to equimolar drug concentrations was in the order 1 > 2 >> cisplatin, thus indicating that substitution of imminoethers for ammines determines a major lipophilicity and cellular uptake of the platinum drug. Both 1 and 2 showed a major toxic effect towards an excision repair-defective Drosophila strain, thus indicating cellular DNA as cytotoxic target. Finally, both 1 and 2 were active in vivo against the murine P388 system, but, contrary to the in vitro activity, isomer 2 was slightly more active than 1. On the whole, the results confirm the antitumour activity of trans-[PtCl2(iminoether)2] complexes, and indicate that the configuration of the iminoether ligands may affect the pharmacological properties of this class of complexes.

Corto and DSP1 interact and bind to a maintenance element of the Scr Hox gene: understanding the role of Enhancers of trithorax and Polycomb

BackgroundPolycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements (ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos. To understand better the role of ETP, we addressed genetic and molecular interactions between corto and dsp1.ResultsWe show that Corto and DSP1 proteins co-localize at 91 sites on polytene chromosomes and co-immunoprecipitate in embryos. They interact directly through the DSP1 HMG-boxes and the amino-part of Corto, which contains a chromodomain. In order to search for a common target, we performed a genetic interaction analysis. We observed that corto mutants suppressed dsp11 sex comb phenotypes and enhanced AntpScx phenotypes, suggesting that corto and dsp1 are simultaneously involved in the regulation of Scr. Using chromatin immunoprecipitation of the Scr ME, we found that Corto was present on this ME both in Drosophila S2 cells and in embryos, whereas DSP1 was present only in S2 cells.ConclusionOur results reveal that the proteins Corto and DSP1 are differently recruited to a Scr ME depending on whether the ME is active, as seen in S2 cells, or inactive, as in most embryonic cells. The presence of a given combination of ETPs on an ME would control the recruitment of either PcG or TrxG complexes, propagating the silenced or active state.

Lethal and sublethal effects of imidacloprid, after chronic exposure, on the insect model Drosophila melanogaster.

Neonicotinoids are subjected to vigilance because of environmental contaminations and deleterious effects on bees. Imidacloprid (IMI) is one of the most representative insecticides of this family. At chronic exposure, concentration-effect relationships are non linear. An insect model should allow a better description of this toxicity. We compared the lethal concentration 50% (LC50) of IMI for a Drosophila-field strain, after acute and chronic exposure. Relative to the acute LC50, the chronic LC50 was lowered by a factor of 29 for males (1.3 mM/45 μM), 52 for larvae (157 μM/3 μM) and more than 172 for females (>3.1 mM/18 μM). Chronic exposure also revealed significant lethal and sublethal effects, at concentrations 3-5 orders of magnitude lower than the chronic LC50. Mean mortalities reached 28% (at 3.91 nM) and 27% (at 39.1 nM) for females and males, respectively. Fecundity decreased of 16% at 1.96 nM. Mating increased of 30% at 0.391 nM. The LOEC (lowest observed effect concentration: 0.391 nM) was 46 000 times lower than the chronic LC50 for males; it was 115 000 times lower than the chronic LC50 for females. This study illuminates effects that neonicotinoids can induce at very low concentrations. This is of particular interest for nontarget insects and for insect dependent species.

DSP1 gene of Drosophila melanogaster encodes an HMG-domain protein that plays multiple roles in development.

DSP1 is an HMG-box containing protein of Drosophila melanogaster which was first identified as a co-repressor of the Dorsal protein. Recently, the analysis of the structure of the gene has led us to propose that DSP1 is the Drosophila equivalent of the ubiquitous vertebrate HMG 1/2 proteins. In the present paper, the patterns of expression of DSP1 protein and RNA in adult flies and during development are reported. In the adults DSP1 protein is located in nurse cells of ovaries and in brain. During eggs development uniform expression of DSP1 protein persists until the end of germband retraction. At later stages, expression is restricted to the ventral nerve chord and brain. Using P-element mutagenesis, we have isolated a mutant deficient in DSP1 functions. Genetic studies of this mutant show that DSP1 protein is essential for the growth and the development of Drosophila. In addition to be a co-repressor of the transcriptional activator Dorsal our results provide compelling evidence that DSP1 is a regulator involved in several pathways necessary for the development of the fly.

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.

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.

Detection of minor adducts in cisplatin‐modified DNA by transcription footprinting

Two DNA restriction fragments containing either a d(GC)5 or a d(TTGCTTGATTAGTTGTGTT) insert were subjected to reaction with cis‐diamminedichloroplatinum(II) and were then used as templates for RNA synthesis by T7 RNA polymerase. Within the d(GC)5 insert, interstrand cross‐links are preferentially formed. Within the second insert, the reactivity order of the potential binding sites is d(ApG) > d(GpC/ GpC) = d(GpA) > d(GpTpG). In the presence of cyanide ions, the adducts are much less stable at the d(GpA) sites than at the d(GpCpG) sites, in double‐stranded DNA.

In vivo magnetic resonance microscopy of Drosophilae at 9.4 T

In preclinical research, genetic studies have made considerable progress as a result of the development of transgenic animal models of human diseases. Consequently, there is now a need for higher resolution MRI to provide finer details for studies of small animals (rats, mice) or very small animals (insects). One way to address this issue is to work with high-magnetic-field spectrometers (dedicated to small animal imaging) with strong magnetic field gradients. It is also necessary to develop a complete methodology (transmit/receive coil, pulse sequence, fixing system, air supply, anesthesia capabilities, etc.). In this study, we developed noninvasive protocols, both in vitro and in vivo (from coil construction to image generation), for drosophila MRI at 9.4 T. The 10 10 80-μm resolution makes it possible to visualize whole drosophila (head, thorax, abdomen) and internal organs (ovaries, longitudinal and transverse muscles, bowel, proboscis, antennae and optical lobes). We also provide some results obtained with a Drosophila model of muscle degeneration. This opens the way for new applications of structural genetic modification studies using MRI of drosophila.

Genomic organization and nucleotide sequence of a long mosaic repetitive DNA in the mouse genome

A long mosaic repetitive sequence (LMRS) was isolated from a mouse liver genome library using a mouse repetitive DNA as a probe. LMRS exhibits the following features: (1) it is almost 15 kb in length; (2) it is partly organized in tandem array and frequently interrupted by other repeated sequences; and (3) it is located predominantly on the A3 band of the mouse X Chromosome (Chr). One fragment of LMRS (B6) shows restriction fragment length polymorphism (RFLP) between different mouse strains, and is thus potentially useful for mapping studies. The nucleotide sequence confirms a mosaic organization of LMRS which includes three repeats in the 5′ part, showing similarity with the 5′ end of L1Md-A2, and seven long A+T rich segments in the central part of the element. Our findings suggest that this sequence may have arisen from the duplication of an ancestral motif and has expanded by successive waves of amplification and invasion by foreign sequences.

BIOTECHNOCENTRE: an overview of the 16th Annual Meeting

BIOTECHNOCENTRE is a well-established 18year-old organization that has emerged as the necessary hub to facilitate interactions between scientists of academic laboratories with biotechnological and industrial research institutes in France, ‘Region Centre’ (http://www.biotechnocentre.regioncentre.fr). Every year BIOTECHNOCENTRE organizes a 2-day conference with an emphasis on co-operation between these diverse organizations that are active in the areas of biology, biochemistry, and cosmetic and pharmaceutical sciences. In 2003, 150 scientists met in Seillac, France, in the Loire Valley, for the 16th Annual Meeting which was subsidized by the Council of the Region Centre. Prestigious invited speakers presented high-quality seminars in the areas of nanosciences, regulation of cell cycle and cell death, gene therapy and drug metabolism. The presentations of Dr M. Faupel (Novartis, Basel, Switzerland), Dr G. Marguerie (Clinigenetics, Nimes, France) and Dr M. Hoummady (Nanobiogene, Belfort, France) focused on the multiple applications of nanotechnology to the life sciences, while Dr J.-C. Beloeil (CNRS, Orléans, France) discussed imaging technologies. Dr O. Danos (Genethon, Evry, France) reviewed new developments and perspectives in gene therapy, while Dr Y. Bigot (Tours, France) presented work showing that the MLE (mariner-like element) is involved in eukaryotic gene insertion. Dr A. Tedgui (INSERM, Paris, France), Dr P. Auberger (INSERM, Nice, France) and Dr R. Roberts (Aventis, Paris, France) described the role of apoptosis in atherosclerosis, leukaemia and in PPAR (peroxisome proliferator-activated receptor) signalling in the liver. In addition, Dr A. Legrand (CNRS, Orléans,