Alkylation of prohibitin by cyclohexylphenyl-chloroethyl urea on an aspartyl residue is associated with cell cycle G(1) arrest in B16 cells.

BACKGROUND AND PURPOSE: Phenyl-chloroethyl ureas (CEUs) are a class of anticancer drugs that mainly react with proteins. Two molecules of this family, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) induced G(1)/S and G(2)/M cell cycle blocks, respectively. We hypothesised that these observations were linked to a differential protein alkylation pattern. EXPERIMENTAL APPROACH: Proteins from B16 cells incubated with [(14)C-urea]-CCEU and [(125)I]-ICEU were compared by 2D-analyses followed by MALDI-TOF identification of modified proteins and characterisation of the CCEU binding. Protein expression was investigated by Western blot analyses and cell cycle data were obtained by flow cytometry. KEY RESULTS: Several proteins (PDIA1, PDIA3, PDIA6, TRX, VDAC2) were alkylated by both ICEU and CCEU but beta-tubulin and prohibitin (PHB) were specifically alkylated by either ICEU or CCEU respectively. Specific alkylation of these two proteins might explain the observed difference in B16 cell cycle arrest in G(2) and G(1) phases respectively. Mass spectrometry studies on the alkylated prohibitin localised the modified peptide and identified Asp-40 as the target for CCEU. This alkylation induced an increased cellular content of PHB that should contribute to the accumulation of cells in G(1) phase. CONCLUSIONS AND IMPLICATIONS: This study reinforces our findings that CEUs alkylate proteins through an ester linkage with an acidic amino acid and shows that PHB alkylation contributes to G(1)/S arrest in CCEU treated B16 cells. Modification of PHB status and/or activity is an open route for new cancer therapeutics.

Molecular cloning of fused, a gene required for normal segmentation in the Drosophila melanogaster embryo.

Using the chromosomal walk technique, we isolated recombinant lambda bacteriophage and cosmid clones spanning 250 kilobases (kb) in the 17C-D region of the X chromosome of Drosophila melanogaster. This region was known to contain the segment polarity gene fused. Several lethal fused mutations were used to define more precisely the localization of this locus. Southern analysis of genomic DNA revealed that all of them were relatively large deficiencies, the smallest one being 40 kb long. None of the 12 viable fused mutations examined possessed detectable alterations. We isolated a cosmid containing an insertion covering the entire smallest fused deletion (40 kb). We injected this DNA into fused mutant embryos and obtained a partial phenotypic rescue of the embryonic pattern, indicating that this region contained all the sequences necessary for the embryonic expression of the fu+ gene. Within this DNA, a subclone of 14 kb codes for poly(A)+ RNAs of 3.5, 2.5, 1.6, and 1.3 kb detected in embryos from various developmental stages as well as in adults. All these transcripts showed the same developmental expression. This transcribed region was injected into fused mutant embryos, and once again we obtained a partial rescue of the embryonic phenotype, confirming that this region contained at least the fused gene.

Sex-specific difference of the galabiosylceramide level in the glycosphingolipids of human thyroid.

The glycosphingolipids of human thyroid were isolated and characterized by gas-liquid chromatography and sequential enzymic hydrolysis. The major purified components were identified as glucosyl- and galactosyl-ceramides, lactosyl- and galabiosylceramides, globotriaosyl- and globotetraosylceramides. The long-chain base analyses showed a high proportion of phytosphingosine in glycosylceramide and galabiosylceramide. Fatty acids in 22:0, 24:0, 24:1 prevailed, especially in the cerebroside fraction, with a significant content of alpha-hydroxylated species in galactosylceramide. Female thyroid had a very low content of galabiosylceramide and a higher content of glucosylceramide, as compared to male. No significant difference was found in the other neutral glycosphingolipids and gangliosides.

Evidence for several cell populations in human thyroid with distinct glycosphingolipid patterns.

Thyrocytes, which are functional cells of human thyroid, have been isolated, and their glycosphingolipid content has been analyzed in the various fractions obtained from the digested gland as well as in the tissue remaining after enzymatic treatment. The ganglioside content was not significantly different in the different fractions, with GM3 and Gd3 as major components. Analysis of neutral glycolipids revealed striking differences between isolated thyrocytes and whole thyroid. The membraneous material released from the proteinase-treated thyroid presented a pattern of monohexosylceramides clearly distinct from that of thyrocytes. The present data suggest the presence of at least two cellular populations with distinct glycolipid patterns in thyroid tissue: accessory cells containing most of the glycolipids, and thyrocytes in which the major neutral glycosphingolipid is phytosphingosine-containing glucosylceramide.

The Suppressor of fused gene encodes a novel PEST protein involved in Drosophila segment polarity establishment.

Suppressor of fused, Su(fu), was identified as a semi-dominant suppressor of the putative serine/threonine kinase encoded by the segment polarity gene fused in Drosophila melanogaster. The amorphic Su(fu) mutation is viable, shows a maternal effect and displays no phenotype by itself. Su(fu) mutations are often found associated to karmoisin (kar) mutations but two complementation groups can be clearly identified. By using a differential hybridization screening method, we have cloned the Su(fu) region and identified chromosomal rearrangements associated with Su(fu) mutations. Two classes of cDNAs with similar developmental patterns, including a maternal contribution, are detectable in the region. Transformation experiments clearly assigned the Su(fu)+ function to one of these transcription units while the other one can be most likely assigned to the kar+ function. Surprisingly the 5' end of the kar RNA mapped within the 3' untranslated region of the Su(fu) transcribed sequence. The Su(fu) gene encodes a 53-kD protein, which contains a PEST sequence and shows no significant homologies with known proteins. Genetic analysis shows that proper development requires a fine tuning of the genetic doses of fu and Su(fu) both maternally and zygotically. These results, together with previous genetic and molecular data, suggest that fused and Suppressor of fused could act through a competitive posttraductionnal modification of a common target in the hedgehog signaling pathway.

Segmental polarity in Drosophila melanogaster: genetic dissection of fused in a Suppressor of fused background reveals interaction with costal-2.

fused (fu) is a segment polarity gene that encodes a putative serine/threonine kinase. A complete suppressor of the embryonic and adult phenotypes of fu mutants, Suppressor of fused (Su(fu)), was previously described. The amorphic Su(fu) mutation is viable and displays no phenotype by itself. We have used this suppressor as a tool to perform a genetic dissection of the fu gene. Analysis of the interaction between Su(fu) and 33 fu alleles shows that they belong to three different classes. Defects due to class I fu alleles are fully suppressed by Su(fu). Class II fu alleles lead to a new segment polarity phenotype in interaction with Su(fu). This phenotype corresponds to embryonic and adult anomalies similar to those displayed by the segment polarity mutant costal-2 (cos-2). Class II alleles are recessive to class I alleles in a fu[I]/fu[II];Su(fu)/Su(fu) combination. Class 0 alleles, like class I alleles, confer a normal segmentation phenotype in interaction with Su(fu). However class II alleles are dominant over class 0 alleles in a fu[0]/fu[II];Su(fu)/Su(fu) combination. Alleles of class I and II correspond to small molecular events, which may leave part of the Fu protein intact. On the contrary, class 0 alleles correspond to large deletions. Several class I and class II fu mutations have been mapped, and three mutant alleles were sequenced. These data suggest that class I mutations affect the catalytic domain of the putative Fu kinase and leave the carboxy terminal domain intact, whereas predicted class II proteins have an abnormal carboxy terminal domain. Su(fu) enhances the cos-2 phenotype and cos-2 mutations interact with fu in a way similar to Su(fu). All together these results suggest that a close relationship might exist between fu, Su(fu) and cos-2 throughout development. We thus propose a model where the Fu+ kinase is a posterior inhibitor of Costal-2+ while Su(fu)+ is an activator of Costal-2+. The expression pattern of wingless and engrailed in fu and fu;Su(fu) embryos is in accordance with this interpretation.

Functional domains of fused, a serine-threonine kinase required for signaling in Drosophila.

fused (fu) is a segment-polarity gene encoding a putative serine-threonine kinase. In a wild-type context, all fu mutations display the same set of phenotypes. Nevertheless, mutations of the Suppressor of fused [Su(fu)] gene define three classes of alleles (fuO, fuI, fuII). Here, we report the molecular analysis of known fu mutations and the generation of new alleles by in vitro mutagenesis. We show that the Fused (Fu) protein functions in vivo as a kinase. The N-terminal kinase and the extreme C-terminal domains are necessary for Fu+ activity while a central region appears to be dispensable. We observe a striking correlation between the molecular lesions of fu mutations and phenotype displayed in their interaction with Su(fu). Indeed, fuI alleles which are suppressed by Su(fu) mutations are defined by inframe alterations of the N-terminal catalytic domain whereas the C-terminal domain is missing or altered in all fuII alleles. An unregulated FuII protein, which can be limited to the 80 N-terminal amino acids of the kinase domain, would be responsible for the neomorphic costal-2 phenotype displayed by the fuII-Su(fu) interaction. We propose that the Fu C-terminal domain can differentially regulate the Fu catalytic domain according to cell position in the parasegment.

Drosophila null slimb clones transiently deregulate Hedgehog-independent transcription of wingless in all limb discs, and induce decapentaplegic transcription linked to imaginal disc regeneration.

Drosophila Slimb (Slmb) is a F-box/WD40 protein which potentially participates in the ubiquitin proteolysis machinery. During development, Slmb is required in limb discs to repress Hedgehog (Hh) target genes, i.e. wingless (wg) and decapentaplegic (dpp), as well as the Wg signal transduction pathway. These repression functions have been proposed from studies using weak slmb alleles. Interestingly, experiments with strong slmb alleles have revealed additional mechanisms in which slmb is required, such as leg dorsal-ventral restriction. We have isolated new alleles of the slmb gene in a screen for new negative regulators of dpp: several amorphs (characterized by genetic and molecular criteria) and a cold-sensitive hypomorph. By performing somatic clone experiments with these new amorphic slmb alleles, we have determined that regulation of Dpp and Wg morphogens by Slmb could be different from what has already been published. We show here that in leg discs, lack of slmb function derepresses the transcription of wg independently of Hh signaling. We present evidence that ectopic legs resulting from slmb(-) clone induction only come from wg misexpression in the normal dpp domain, as ectopic proximo-distal axis are induced dorsally, and adult ectopic legs are often perfect with respect to antero-posterior polarity. In wing discs, transcription of wg, which is normally independent of Hh signaling, is also derepressed in the absence of slmb function. We also describe, in discs bearing amorphic slmb clones and in discs of two other slmb(-) contexts, a novel pattern of dpp expression consisting of an enlargement of the normal dpp domain. Strong evidence indicates that this dpp modification can be linked to imaginal disc regeneration following slmb(-) cell elimination. We have investigated the fate of slmb(-) clones, which disappear before adulthood, and found that two mechanisms of cell elimination can account for imaginal cell regeneration: an early apoptosis and a mechanism of sorting-out which excludes all slmb(-) clones from all imaginal discs. This result suggests that Slmb is likely to be involved, in addition to its repression role on Dpp and Wg, in some other essential cellular mechanism, as in the absence of Slmb, cell affinities are dramatically modified regardless of the deregulated morphogen and of the type of imaginal disc.

Production of dominant female sterility in Drosophila melanogaster by insertion of the ovoD1 allele on autosomes: use of transformed strains to generate germline mosaics.

We have cloned a 7 kb genomic fragment containing the dominant female-sterile mutation ovoD1. This fragment confers to transgenic females a sterility phenotype, the severity of which depends both on the genetic background and the ratio of ovoD1 product to ovo+ product. Females containing two copies of the ovoD1 transgene, or those containing one recessive null allele at the ovo locus, are about as sterile as ovoD1 females. Twenty transformed strains were obtained and five of them were tested and shown to be excellent tools for identifying a germline clone of cells sustaining mitotic recombination on the autosomes. One of the tested strains carries an insert on chromosome 4, which enabled us to show that mitotic recombination on that chromosome is not a rare event: it is in fact frequent enough for the maternal effects of the zygotic lethal mutations cubitus interruptus Dominant (ciD) and l(4)29 to be studied.

Modulation of Hedgehog target gene expression by the Fused serine-threonine kinase in wing imaginal discs.

The Fused (Fu) serine-threonine kinase and the Suppressor of fused (Su(fu)) product are part of the Hedgehog (Hh) signaling pathway both in embryos and in imaginal discs. In wing imaginal discs, the Hh signal induces Cubitus interruptus (Ci) accumulation and activates patched (ptc) and decapentaplegic (dpp) expression along the anterior/posterior (A/P) boundary. In this paper, we have examined the role of the Fu and Su(fu) proteins in the regulation of Hh target gene expression in wing imaginal discs, by using different classes of fu alleles and an amorphic Su(fu) mutation. We show that, at the A/P boundary, Fu kinase activity is involved in the maintenance of high ptc expression and in the induction of late anterior engrailed (en) expression. These combined effects can account for the modulation of Ci accumulation and for the precise localization of the Dpp morphogen stripe. In contrast, in more anterior cells which do not receive Hh signal, we show that Fu plays a role independent of its kinase function in the regulation of Ci accumulation. In these cells, Fu may be involved in the stabilization of a large protein complex which is probably responsible for the regulation of Ci cleavage and/or targeting to nucleus. We propose that the Fused function is necessary for the activation of full-length Ci and counteracts the negative Su(fu) effect on the pathway, leading to en, ptc and dpp expression.

Molecular organisation and expression pattern of the segment polarity gene fused of Drosophila melanogaster.

The Drosophila segment-polarity gene fused (fu) is required for pattern formation within embryonic segments and imaginal discs. We previously reported that the 5' part of the fused gene is homologous to the catalytic domain of serine/threonine kinases. We present here the sequence of the complete transcription unit, which predicts a 805 amino acid long protein. The kinase domain actually corresponds to 268 amino acids in the N-terminal part, and no known function can be attributed to the rest of the putative FUSED protein. Transcripts from the fused gene have been characterized: a unique 3.2 kb fused transcript is produced in nurse cells, in low abundance, from stage 8 of oogenesis, and persistently through the rest of oogenesis. In embryos, this transcript is evenly distributed in all embryonic cells until the extended germ band stage, after which its amount strongly decreases. Ubiquitous expression is detected later in imaginal wing and leg discs. Possible roles of the FUSED protein in signal transduction pathways required for intercellular communication at different stages of development are discussed.

Cloning and characterization of Neisseria meningitidis genes encoding the transferrin-binding proteins Tbp1 and Tbp2.

Genes tbp1 and tbp2, encoding the transferrin-binding proteins Tbp1 and Tbp2, have been isolated from two strains of Neisseria meningitidis. The tbp2 and tbp1 open reading frames are tandemly arranged in the genome with an 87-bp intergenic region, and the DNA region upstream from the tbp2-coding sequence contains domains homologous to Escherichia coli promoter consensus motives. Nucleotide sequence analysis suggests the existence of a Tbp1 precursor carrying an N-terminal signal peptide with a peptidase I cleavage site and of a Tbp2 precursor with N-terminal homology to lipoproteins, including a peptidase II cleavage site. Comparison of the Tbp1 deduced amino acid (aa) sequences from both strains showed about 76% aa homology, while those of Tbp2 revealed only about 47% aa homology. These comparisons should be extended to other Neisseria strains in order to evaluate further this genetic divergence further.

Characterization of a schistosome T cell-stimulating antigen (Sm10) associated with protective immunity in humans.

Parasite antigens that are strong T cell immunogens represent potential candidates for vaccines against pathogens susceptible to T cell-mediated immunity. We have previously shown that chromatographic fractions of schistosomula extracts contain components that are major T cell immunogen(s) in natural schistosome infections in humans and might contribute to the induction of human protective immunity against this parasite. In the present study, we report on the molecular cloning and on the biochemical characterization of the active components of these fractions. The screening of a schistosomula cDNA expression library with antibodies raised against the fractions allowed the cloning of a cDNA that hybridized to a 0.56-kb mRNA of schistosomula and adult worms. This cDNA contains an open reading frame of 267 base pairs (bp) which encodes a 10-kDa polypeptide. The analysis of the cDNA sequence revealed 70% homology with the sequences of previously reported proteins of unknown function. The native molecules in the active fractions were analyzed by mass spectrometry after additional purification by reverse phase high-performance liquid chromatography (HPLC). This procedure revealed two components in the fractions of molecular mass 10383 +/- 2 Da and 10401 +/- 9 Da. Both polypeptides stimulated immune T cells and yielded tryptic peptides whose sequences matched the sequence of the cloned molecule. These two polypeptides probably correspond to different post-translationally modified forms of the polypeptide encoded by the cloned cDNA.

Characterization of pro-invasive mechanisms and N-terminal cleavage of ANXA1 in melanoma.

Annexin A1 deregulation is often associated with cancer. Indeed we have shown that annexin A1 is overexpressed in melanoma and promotes metastases by formyl peptide receptor stimulation and MMP2 expression. Here, we demonstrated in different melanoma cell lines that annexin A1-MMP2 induction is mediated by MAPK and STAT3 pathways. To decipher endogenous annexin A1 action mode, we showed that annexin A1 is externalized in A375 cells and cleaved by a membrane-associated serine protease, allowing the release of a pro-invasive annexin A1 peptide in the extra cellular environment. Finally, a biochemical and proteomic approach allowed to enrich eight out of 12 members of the annexin family and to identify an original annexin A1 cleavage site localized between Ser(28) and Lys(29). Altogether, these data identify signaling pathways involved in annexin A1 pro-invasive role and suggest that externalized full-length annexin A1 interacts with formyl peptide receptors in a juxtacrine manner while ANXA 2-28 release allows autocrine and paracrine interaction.

N-(4-iodophenyl)-N'-(2-chloroethyl)urea as a microtubule disrupter: in vitro and in vivo profiling of antitumoral activity on CT-26 murine colon carcinoma cell line cultured and grafted to mice.

The antitumoral profile of the microtubule disrupter N-(4-iodophenyl)-N'-(2-chloroethyl)urea (ICEU) was characterised in vitro and in vivo using the CT-26 colon carcinoma cell line, on the basis of the drug uptake by the cells, the modifications of cell cycle, and beta-tubulin and lipid membrane profiles. N-(4-iodophenyl)-N'-(2-chloroethyl)urea exhibited a rapid and dose-dependent uptake by CT-26 cells suggesting its passive diffusion through the membranes. Intraperitoneally injected ICEU biodistributed into the grafted CT-26 tumour, resulting thus in a significant tumour growth inhibition (TGI). N-(4-iodophenyl)-N'-(2-chloroethyl)urea was also observed to accumulate within colon tissue. Tumour growth inhibition was associated with a slight increase in the number of G2 tetraploid tumour cells in vivo, whereas G2 blockage was more obvious in vitro. The phenotype of beta-tubulin alkylation that was clearly demonstrated in vitro was undetectable in vivo. Nuclear magnetic resonance analysis showed that cells blocked in G2 phase underwent apoptosis, as confirmed by an increase in the methylene group resonance of mobile lipids, parallel to sub-G1 accumulation of the cells. In vivo, a decrease of the signals of both the phospholipid precursors and the products of membrane degradation occurred concomitantly with TGI. This multi-analysis established, at least partly, the ICEU activity profile, in vitro and in vivo, providing additional data in favour of ICEU as a tubulin-interacting drug accumulating within the intestinal tract. This may provide a starting point for researches for future efficacious tubulin-interacting drugs for the treatment of colorectal cancers.

Membrane properties of cultured glial cells obtained mechanically or by enzymatic dissociation: differential alterations by gangliosides.

Rat astroglial cells were obtained either mechanically or by enzymatic dissociation and some properties of the primary and secondary cultures were studied. Differences in the ganglioside amount, taurine uptake, membrane fluorescence anisotropy, or their respective modulation by total brain gangliosides confirmed that although the two types of cultures are morphologically similar, they are biochemically distinct.

Alterations of the cerebroside fraction of human thyroid glycosphingolipids in Graves' disease.

The influence of Graves' disease in human thyroid neutral glycosphingolipids was investigated. The major alteration was in the cerebroside fraction. Although the total amount of cerebroside was not different, the relative proportion of the bands of glucosylceramide separated on borated thin-layer plates was greatly modified in the disease. The band of glucosylceramide containing phytosphingosine and hydroxylated fatty acids decreased strongly, whereas the band with C18 sphingosine and normal fatty acids increased simultaneously. No change was observed in the content of galabiosylceramide. A slight elevation was seen in the amount of globoside at the expense of globotriaosylceramide.

Major gangliosides in normal and pathological human thyroids.

The major gangliosides of human thyroids were extracted, purified and then analyzed by gas-liquid chromatography. In normal thyroid, GM3 and GD3 represented about 80% of lipid-bound sialic acid. GM3 contained more than 50% of long-chain fatty acids, whereas GD3 contained mostly short ones. 4D hydroxy sphinganine represented 20% of long-chain base content in both cases. In pathological thyroids (Graves' disease, cancer, toxic adenoma), GM3 represented about 60% of lipid-bound sialic acid; its fatty acid content was mostly short chain fatty acids, as in normal GD3. 4D hydroxy sphinganine proportion was decreased.

[Injection of DNA from wild-type strains into the eggs of mutant Drosophila melanogaster]. / Injection d'ADN extrait de souche sauvage dans des oeufs mutants de Drosophila melanogaster

DNA was extracted from wild-type Drosophila Melanogaster or from v; bw mutant and injected into the eggs of v; bw strain. Progeny of flies obtained from injected eggs was examined for several generations. Colored eyes appeared occasionally in the progeny of flies obtained after injection of either DNA. A recovery of colored strains was possible in progeny of flies obtained after injection of wild type DNA (4 out of 15), and not in progeny of 34 flies obtained after injection of v; bw DNA. Genetic alteration inducing color was localized in three of the recovered strains at Su (s) site and in one strain at v site.

Selective enrichment of phytosphingosine in glycosphingolipids of isolated human thyrocytes as compared to the whole thyroid.

The glycosphingolipids of isolated human thyrocytes have been analyzed. As compared to the total thyroid gland, the pattern of gangliosides was found to be similar, whereas the neutral glycolipid profile was quite different, with glucosylceramide as the major glycosphingolipid of thyrocytes. Moreover, this glucosylceramide contains almost exclusively phytosphingosine (4-D-hydroxy-sphinganine) which is only a minor component in the long-chain bases of the glycosphingolipids extracted from the whole thyroid gland.