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Biomedical subjects

M Huchet

Publications and source records attributed to M Huchet.

At least 19 recordsLinked to original sources
PubMed2001

The homocamptothecin BN 80915 is a highly potent orally active topoisomerase I poison.

BN 80915, a lead compound of the homocamptothecin (hCPT) family, has entered clinical trials. BN 80915 is a difluoro-hCPT where the six-membered alpha-hydroxylactone ring of camptothecin (CPT) is replaced by a seven-membered beta-hydroxylactone ring. Preclinical data reported here show that in spite of the modification to the crucial E-ring of CPTs, BN 80915 retains topoisomerase I poisoning activity as shown in living HT29 cells as well as in cell-free assays, where BN 80915 always performs better than SN-38 or TPT. In antiproliferative assays BN 80915 is also very potent as evidenced by IC50s values consistently lower than those of SN38 in sensitive cell lines as well as in their related multidrug-resistant lines overexpressing P-glycoprotein or multidrug resistance-associated protein. Furthermore, in human plasma, in contrast to CPT analogs, the hydrolysis of BN 80915 is slow, leading to improved plasma stability, and irreversible, thus avoiding toxicity related to the accumulation of active principle during excretion in the urinary tract. These findings may account for the good in vivo efficacy observed in PC3 xenograft experiments where BN 80915 administered orally at very low doses doubled the tumor growth delay in comparison to CPT-11 administered i.p. Altogether, these results strongly support further development of BN 80915.

Adenocarcinoma↗
PubMed2001

A novel B-ring modified homocamptothecin, 12-Cl-hCPT, showing antiproliferative and topoisomerase I inhibitory activities superior to SN-38.

We report the synthesis and pharmacological evaluation of a novel homocamptothecin (hCPT) derivative, 12-Cl-hCPT, which contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone found in camptothecin (CPT) and bears a chloro substituent at position 12. The capacity of 12-Cl-hCPT to inhibit DNA topoisomerase I was compared with that of SN-38, the active metabolite of the clinically used antitumour prodrug CPT-11. In the DNA relaxation assay, 12-Cl-hCPT proved to be slightly more potent than SN-38 at stimulating the formation of nicked plasmid DNA molecules. A series of radiolabelled DNA restriction fragments were employed to identify and compare the position of the DNA cleavage sites induced by topoisomerase I in the presence of 12-Cl-hCPT and SN-38. These sequencing studies confirm that both 12-Cl-hCPT and SN-38 strongly promote DNA cleavage by topoisomerase I and reveal that the majority of the cleavage sites are located at the same nucleotide positions for the two drugs. However, a certain number of DNA cleavage sites were found to be specific to 12-Cl-hCPT. These sites, previously characterized with unsubstituted hCPT, generally correspond to 5'-CG sites whereas the sites common to the 12-Cl-hCPT and SN-38 essentially correspond to 5'-TG sites. We also quantified the formation of drug-induced protein-DNA complexes formed in HT29 human colon carcinoma cells. Trapping of endogenous proteins onto DNA was found to be much more efficient with 12-Cl-hCPT than with SN-38. These data provide a molecular basis to account for the enhanced antiproliferative activity of 12-Cl-hCPT compared with that of SN-38. Biological evaluation on a panel of sensitive and drug-resistant cell lines revealed 12-Cl-hCPT to be more cytotoxic to tumour cells than SN-38. 12-Cl-hCPT proved 14- and 23-fold more active than SN-38 toward the K562adr and T24anp multidrug-resistant cell lines, respectively. The marked topoisomerase I inhibitory properties of 12-Cl-hCPT coupled with its interesting antiproliferative activity, in particular against cancer cells presenting multidrug resistance phenotype with overexpression of P-glycoprotein, makes 12-Cl-hCPT a valid candidate for subsequent preclinical evaluation. Collectively, the data strengthen homocamptothecin as an extremely promising template to generate novel and potent antitumour agents.

Antibiotics, Antineoplastic↗
PubMed2000

Topoisomerase I-mediated antiproliferative activity of enantiomerically pure fluorinated homocamptothecins.

Homocamptothecin (hCPT) is an E-ring modified camptothecin (CPT) analogue bearing a methylene spacer between the alcohol and carboxyl functions of the CPT lactone. Combining pronounced inhibitory activity of topoisomerase I (Topo I) with enhanced plasma stability, hCPT constitutes an attractive template for the elaboration of new anticancer agents. Fluorinated hCPT analogues, prepared in enantiomerically pure form, were assayed by their stimulation of Topo I-mediated DNA cleavage. Translation into cytotoxicity against tumor cells was evaluated on HT29 human colon adenocarcinoma and on the multidrug resistant lung and bladder tumor cell lines, A549 and T24r. Good correlation is observed between the ability of the drugs to stimulate Topo I-mediated DNA cleavage and the respective 50% inhibitory concentrations (IC(50) values) of the HT29, A549, and T24r cell growth. Fluorine substitution in the A-ring of hCPT was found to have a pronounced influence on biological activity, providing several compounds which are up to 100-fold more potent than CPT in terms of IC(50). Among these, 10,11-difluoro-hCPT has been selected for further development.

Antineoplastic Agents↗
PubMed1999

Homocamptothecin, an E-ring-modified camptothecin analogue, generates new topoisomerase I-mediated DNA breaks.

Homocamptothecin (hCPT) contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone ring found in camptothecin and its tumor active analogues, including topotecan and irinotecan. The homologation of the lactone E-ring reinforces the stability of the lactone, thus reducing considerably its conversion into a carboxylate form which is inactive. We have recently shown that hCPT is much more active than the parent compound against a variety of tumor cells in vitro and in xenograft models, suggesting that a highly reactive lactone is not essential for topoisomerase I-mediated anticancer activity [Lesueur-Ginot et al. (1999) Cancer Res. 59, 2939-2943]. In the present study, we provide further evidence that hCPT has superior topoisomerase I inhibition capacities to CPT. In particular, we show that replacement of the camptothecin lactone E-ring with a homologous seven-membered lactone ring changes the sequence-specificity of the drug-induced DNA cleavage by topoisomerase I. Both CPT and hCPT stimulate the cleavage by topoisomerase I at T( downward arrow)G sites, but in addition, hCPT stabilizes cleavage at specific sites containing the sequence AAC( downward arrow)G. At low drug concentrations, the cleavage at the T( downward arrow)G sites and at the hCPT-specific C( downward arrow)G sites is more pronounced and more stable with hCPT than with CPT. The in vitro data were confirmed in cells. Higher levels of protein-DNA complexes were detected in P388 leukemia cells treated with hCPT than those treated with CPT. Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped onto DNA by hCPT in cells. Finally, the use of a leukemia cell line resistant to CPT provided evidence that topoisomerase I is involved in the cytotoxicity of hCPT. Altogether, the results show that the beta-hydroxylactone ring of hCPT plays an important and positive role in the poisoning of topoisomerase I. An explanation is proposed to account for such remarkable changes in the sequence specificity of topoisomerase I cleavage consequent to the modification of the lactone. The study sheds new light on the importance of the lactone ring of camptothecins for the stabilization of topoisomerase I-DNA complexes.

Animals↗
PubMed1999

Reduced antinociception in mice lacking neuronal nicotinic receptor subunits.

Nicotine exerts antinociceptive effects by interacting with one or more of the subtypes of nicotinic acetylcholine receptors (nAChRs) that are present throughout the neuronal pathways that respond to pain. To identify the particular subunits involved in this process, we generated mice lacking the alpha4 subunit of the neuronal nAChR by homologous recombination techniques and studied these together with previously generated mutant mice lacking the beta2 nAChR subunit. Here we show that the homozygous alpha4-/- mice no longer express high-affinity [3H]nicotine and [3H]epibatidine binding sites throughout the brain. In addition, both types of mutant mice display a reduced antinociceptive effect of nicotine on the hot-plate test and diminished sensitivity to nicotine in the tail-flick test. Patch-clamp recordings further reveal that raphe magnus and thalamic neurons no longer respond to nicotine. The alpha4 nAChR subunit, possibly associated with the beta2 nAChR subunit, is therefore crucial for nicotine-elicited antinociception.

Analgesia↗
PubMed1998

The early expression of Rev-erbbeta occurs in the developing nervous system of mouse embryo.

The orphan ligand nuclear receptor Rev-erbbeta acts in vitro as a negative regulator of transcription. However, its precise physiological role is still unknown. As a first attempt to better understand its biological function, we have studied the distribution and the localization of the Rev-erbbeta mRNA transcripts in different mouse embryonal carcinoma cell lines, in mouse embryos and adult tissues. Our results indicated that Rev-erbbeta transcripts are present in both the non-differentiated and differentiated F9 cells into either parietal or visceral endoderm. At 12.5 days of gestation (E12.5) of mouse embryos, Rev-erbbeta transcripts were localized only in the developing nervous system. In contrast, at later stages of development as well as in the adult, its messengers were widely distributed. These results suggest that Rev-erbbeta may have different roles at the different stages of mouse development, with a more specific role in the nervous system at earlier stages.

Animals↗
PubMed1995

Developmental regulation of acetylcholinesterase transcripts in the mouse diaphragm: alternative splicing and focalization.

We studied the splicing and compartmentalization of acetylcholinesterase (AchE) mRNAs during muscle differentiation in the mouse, both in vitro and in vivo. We used the polymerase chain reaction (PCR) to analyse AChE mRNAs in cultures of the myogenic C2 and Sol8 cell lines, and in the developing diaphragm, from embryonic day 14 (E14). We characterized three types of alternatively spliced AChE mRNAs, encoding catalytic subunits that differ by their C-terminal regions (R, H and T). The T transcript is predominant in all cases and represents the only AChE mRNA in the adult muscle. We detected the presence of the minor R and H transcripts in the myogenic cell lines, both as myoblasts and differentiated myotubes, and also in the diaphragm from E14 until birth. At E14 the R transcript represents approximately 1% of AChE mRNA and the level of the H transcript is still lower. By in situ hybridization, we found that the T AChE mRNAs begin to preferentially accumulate at the level of the first neuromuscular contacts in the mouse diaphragm and other muscles as early as E14, e.g. concomitantly with mRNAs encoding the receptor subunits. This suggests that a common control mechanism ensures the synaptic focalization of mRNAs encoding the cholinergic proteins AChE and acetylcholine receptor during muscle development.

Acetylcholinesterase↗
PubMed1995

Developmental regulation of membrane traffic organization during synaptogenesis in mouse diaphragm muscle.

In innervated adult skeletal muscles, the Golgi apparatus (GA) displays a set of remarkable features in comparison with embryonic myotubes. We have previously shown by immunocytochemical techniques, that in adult innervated fibers, the GA is no longer associated with all the nuclei, but appears to be concentrated mostly in the subneural domain under the nerve endings in chick (Jasmin, B. J., J. Cartaud, M. Bornens, and J.-P. Changeux. 1989. Proc. Natl. Acad. Sci. USA. 86:7218-7222) and rat (Jasmin, B. J., C. Antony, J.-P. Changeux, and J. Cartaud. 1995. Eur. J. Neurosci. 7:470-479). In addition to such compartmentalization, biochemical modifications take place that suggest a functional specialization of the subsynaptic GA. Here, we focused on the developmental regulation of the membrane traffic organization during the early steps of synaptogenesis in mouse diaphragm muscle. We investigated by immunofluorescence microscopy on cryosections, the distribution of selected subcompartments of the exocytic pathway, and also of a representative endocytic subcompartment with respect to the junctional or extrajunctional domains of developing myofibers. We show that throughout development the RER, the intermediate compartment, and the prelysosomal compartment (mannose 6-phosphate receptor-rich compartment) are homogeneously distributed along the fibers, irrespective of the subneural or extrajunctional domains. In contrast, at embryonic day E17, thus 2-3 d after the onset of innervation, most GA markers become restricted to the subneural domain. Interestingly, some Golgi markers (e.g., alpha-mannosidase II, TGN 38, present in the embryonic myotubes) are no longer detected in the innervated fiber even in the subsynaptic GA. These data show that in innervated muscle fibers, the distal part of the biosynthetic pathway, i.e., the GA, is remodeled selectively shortly after the onset of innervation. As a consequence, in the innervated fiber, the GA exists both as an evenly distributed organelle with basic functions, and as a highly differentiated subsynaptic organelle ensuring maturation and targeting of synaptic proteins. Finally, in the adult, denervation of a hemidiaphragm causes a burst of reexpression of all Golgi markers in extrasynaptic domains of the fibers, hence showing that the particular organization of the secretory pathway is placed under nerve control.

Animals↗
PubMed1993

Compartmentalized expression of the alpha- and gamma-subunits of the acetylcholine receptor in recently fused myofibers.

The mRNAs encoding the subunits of the acetylcholine receptor are clustered at the level of the neuromuscular junction in adult muscle fibers. We have followed the distribution of the mRNAs encoding the alpha- and gamma-subunits during development of the diaphragm muscle in the mouse by whole-mount in situ hybridization. We show that the mRNAs encoding both subunits display a nonhomogeneous distribution as early as Day 13.5, when the first neuromuscular contacts are formed. Extrajunctional mRNAs disappear during the following days with a concomitant increase in contrast of the synaptic domains. gamma-subunit mRNAs become undetectable at the end of the first postnatal week, together with the appearance of epsilon-subunit mRNAs. Our results imply that the expression of the acetylcholine receptor genes, including the gamma-subunit gene, is compartmentalized soon after neuromuscular contacts have been established. This has important implications for the understanding of the molecular mechanisms of neuromuscular junction formation.

Animals↗
PubMed1992

Localization of mRNAs coding for CMD1, myogenin and the alpha-subunit of the acetylcholine receptor during skeletal muscle development in the chicken.

Myogenin and CMD1, the chicken homologue of MyoD, transactivate the promoter of the alpha-subunit of the acetylcholine receptor (AChR) in chicken fibroblasts. The expression of these three genes was followed by in situ hybridization. In two-day-old embryos the CMD1 gene is expressed shortly before the AChR alpha-subunit and the myogenin genes. At day 19 extrajunctional AChR mRNA clusters have disappeared and myogenin mRNAs are no longer detected in PLD muscle. Moreover, both myogenin and CMD1 mRNA levels increase after muscle denervation in chicks. These data are compatible with a role for myogenic factors in the induction and maintenance of extra-junctional expression of the AChR genes during early muscle development. Using digoxygenin labelled RNA probes, we also show that the mRNAs for the AChR alpha-subunit display a punctated, probably perinuclear distribution, whereas mRNAs for myogenic genes accumulate in the sarcoplasm around subsets of nuclei in the muscle fiber.

Animals↗
PubMed1991

Occurrence of neuropeptide K-like immunoreactivity in ventral horn cells of the chicken spinal cord during development.

The possible occurrence of NPK-LI in the ventral horns of the embryonic chicken spinal cord was investigated by means of the indirect immunofluorescence method. The results showed a transient appearance of NPK-LI in cells of the lateral motor column between day 5 of incubation and hatching. After this they disappeared and in the ventral horns NPK-LI remained only in fibers. The results are discussed in terms of a possible trophic action of NPK during development.

Animals↗
PubMed1991

Survival in vitro of motoneurons identified or purified by novel antibody-based methods is selectively enhanced by muscle-derived factors.

Motoneurons were identified in vitro by a new method using the SC1 monoclonal antibody. They constituted up to 30% of total neurons in cultures of whole spinal cord from 4.5-day chicken embryos, and survived for at least 5 days in the presence of muscle extract, but not in its absence. By contrast, other neurons and floor-plate cells survived without muscle-derived factors. Motoneurons were purified to homogeneity by 'panning' on dishes coated with SC1 antibody; they developed rapidly even in the absence of other spinal cells. Concentrations of muscle extract required for half-maximal motoneuron survival were indistinguishable in pure and mixed cultures, suggesting that muscle-derived factors act directly on motoneurons. Other purified growth factors tested, including ciliary neurotrophic factor, did not have the survival-promoting activity of muscle.

Animals↗
PubMed1990

Two adjacent MyoD1-binding sites regulate expression of the acetylcholine receptor alpha-subunit gene.

Several genes encoding putative myogenic regulatory factors have been isolated on the basis of their ability to convert nonmuscle cells into myoblasts. Four of these genes code for nuclear proteins that belong to a larger family characterized by a conserved helix-loop-helix motif required for DNA-binding and dimerization. At least one protein, MyoD1, can function as a transcription factor and activate muscle-specific genes during differentiation. But the promoter of the delta-subunit gene of the mouse acetylcholine receptor (AChR) was recently reported to be functional in the absence of MyoD1 binding sites and it has been suggested that the genes coding for the AChR could be regulated independently of MyoD1 protein. Here, we identify two functional MyoD1-binding sites in the muscle-specific enhancer of the chicken AChR alpha-subunit gene that are essential for full activity in transfected myotubes.

Animals↗
PubMed1988

Existence and coexistence of calcitonin gene-related peptide, vasoactive intestinal polypeptide- and somatostatin-like immunoreactivities in spinal cord motoneurons of developing embryos and post-hatch chicks.

By use of immunocytochemical methods, it is shown that immunoreactive calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and somatostatin (SOM) are present in motoneurons in the chicken spinal cord. While CGRP-like immunoreactivity (LI) is present in numerous motoneurons both before and after hatching, SOM- and VIP-LI markedly decline at the end of the embryonic period. Evidence is also provided for coexistence of some of these peptides in certain spinal motoneurons.

Animals↗
PubMed1987

A subpopulation of embryonic telencephalic neurons survive and develop in vitro in response to factors derived from the periphery.

Denervated chick muscle contains factors that enhance neurite outgrowth in cultures of embryonic chicken spinal neurons. Chromatography of muscle extract on a column of DEAE-Sepharose yielded a fraction which retained most of the starting neurite-promoting activity. This DEAE fraction was tested for its activity on neurons from other regions of the central nervous system of 5-day-old chicken embryos. Both neurite outgrowth and survival of telencephalic neurons in vitro were greatly enhanced when the DEAE fraction was added at protein concentrations around 1 microgram/ml. When cultures were prepared from embryos later than 6 days in ovo, the effects of the DEAE fraction progressively diminished with age. Neurons from the embryonic diencephalon, mesencephalon and rhombencephalon were not responsive to the DEAE fraction, although they all developed neurites on a laminin substratum. Similar neurite-promoting activities for telencephalic neurons were found in extracts of neonatal brain, liver and heart, but not lung.

Animals↗