Estimation of the Barrel TOF Response A.Galoyan, J. Ritman, V. Uzhinsky
RETINOL AND RETINOIC ACID VECTORIZATION WITH A SYNTHETIC PEPTIDE FORMING β-BARREL STRUCTURE
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80 Biology of the Cell RETINOL AND RETINOIC ACID VECTOR.IZATlON WITH A SYNTHETIC PEFTIDE FORMING P-3ARREL STRUCTURE Pierre PELLEGRIN ; Frederic HEITZ ; Jean MERY and RenC BENNES C.R.B.M. (C.N.R.S. I.N.S.E.R.M.) B.P. 5051 34033 Montpellier CCdex FRANCE We built a peptide which autoassociates as beta-barrels (under defined conditions), The secondary structure is elucidated by CD, PTIR and fluorescence spe@oscopy measurements. When phosphate ions are present (> for&f, pH 7.2) and for peptide concentrations below 5mM, the peptide is autoassociated as antiparallel g-sheets. The fluorescence studies concerning all tram retinol and all tram retinoi’c acid with this peptidic structure show that~ten antiparailel strands beta barrels bind retinolds with a dissociation constant (Ko = 3.10” mo1.l’) very close to natural ~Retino’ids Binding Proteins (Cogan LJ., Kopelman M., Mokady S. and Shi&zky M.(1976) Eur.J. &&em. ,65,?1-78 ; LaLonde J. M. , Bemlohr D. A. and Banaszak L. J.(I994) FASEB J. 8, 1240-1247). Thus, we mimic perfectly endogenous proteins which bind retino’ids. We have vectorized the retinol (Vit. A) helping us with these fi- barrels into human fibroblasts ; the retinal stays in a perirmclear situation, The association between the retinoyc acid and the peptide- has been used to vector&e this retindid toward murin leukemic. cells (Ll2IO). The results, ahhough preliminary, show that this dierenciation inducer (Comic M. ,Agadir A., Degos Land Chomienne C.(l994) Anticancer Res. 14, 2339- 2346 ; Moqattash S. Nader GA. Lutton JD. (1994) Leukemia Res.18 : 53 1- 39) have deeply’affectedthe cell multiplication. It remains to show that the observed diminution is due to an enhanced cellular differentiation and not to a toxicity of the used complex. MODULATIONS OF THE HI9 GENE EXPRE#ION IN NORMAL OR MALIGNANT MBMMARY EPITHELlAL CELLS ADRIAENSSENS Eric*, DUMONT Lionel *! FAUQUETIE William * LE BOURHIS X&en *, DU$lIMONT Thmry *‘, COLL Jean **, CURG Jean-Jacques *Unite “Dynamique des cellules embryonnaires et cancereuses”, DRED 1033, Centre de Biobgie Cellulaire, Univemitt des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq cedex, France.**“Ut& d’Oncologie moltculaire”, CNRS URA 1160, Institut Pasteur, I Rue Calmette, 59019 Lille cedex, Frartce.0 Facult6 des Sciences Jean Pen-h-t, Universid d’Artois, 62307 Lens cedex, France. We studied conditions of the expression of this gene during the ontogenesis of the mammary gland in mouse and in normal or tumoral mammary epithelirit cells. HI 9 expression was detected after in situ molecular hybridization (ISH) at 4 stages of the mouse mammary gland development. Ina 14.5 days foetus, H19 signal was scattered over mammary gland outlines and adjacent tissues. At birth and before puberty (25 days), HI 9 transcripts were far less abundant.. After puberty and during tubule organisation, the HI9 signal was important and preferentially localized over epithelial celfs. These~results prompted us to establish the role of the extmeellular niatrix and mesenchymal cells on the HI9 gene expression in epithelial tiells. Thus, @Is were cultured within a collagene matrix (“3-D”cuIture). By HIS, we observed a H19 overexpression for.both normal and tumoral cellsgrown in “3-D” culture when compared with monolayer culture conditions. Adding a fibroblast conditioned medium had an enhancing effect on Hi9 expression only in monolayer conditions. Furthermore, the HI9 gene expression was studied in different proliferating conditions (serum, differentiating or anti-prolifemting factors) and in presence. of inhibitors or activators of transduction pathways including protein l&nases (PKA or PKC). We conclude from these experiments that the transcription level depends on the cell type, but is reduced in cell Iignes treated with TcFpl and constantly raised when normal or pathological ceils were cultured into a collagene matrix. CHARAC’I’ERIZATION OF CENTRIOLAK i’l<O’l’hI$~ DURING CILIATED DfFFERENTIAT16N IN MAMMALS -1, LAOUKILI Jamilal, GENDRON MarieClaudc2, MARANO Francelyne 1. i. Laboratoire de cjrophysiologre et toxicoiogle celirtluwe, 2. Ldoratoire de cytodtrie, lnstitut Jacques Monad. Universitt! Paris 7,2-Place .lussieu, 75005 Paris, France In mosf arurnal cells, centrioles are integral cornuonents of the centrosouic‘ They also form the elementary struct& which bears flagella and cilia Ciliated epithelial cells in mammals possess 200 functional cilia anchored at the apical membrane. During ciliated epithelial differentiation, cilia elongation is preceded by the formation of centrioles which seems to be independent of preexisting centriolar structures. Cenrrioles are made in~e;lc!? cell by waves of successive assembly, developing from procerm-ioles which are anchored on fibrogranular material whose origin and molecular uaturc are unknown (Dirksen, E.R. (1991) Biol. Cell. 72, 31-38). Many anticentrosome antibodies tested in ii?lluunofluoresc~nce on rabbi tracheal epithelial cells and human nasal epithelia recognized both centrosomes from non-ciliated cells and centriolcs/basal bodies of ciliateti cehs. Polyglutamyiation of a- and P-tubulins from cilia and centrioles u’;r\ demonstrated usin$ a monocloual antibody against potyglutamylared tubuiins (Wollf, A: et al. (1992) Eur J. Cell Viol. 59, 425-432) Moreover, this specific antibody allowed a mcasurc of the percentage o: ciliated cells from a given epithelium. hi primary cultures of respirator: epithelial ceils (Baeza-Squiban et ai. (1994), In I%ro Celt. Dev Bir,i.. 30A, 56-67), cell migration and proliferation was correlated with a rapid decrease of the percentage of ciliated cells. Culture conditions werr~ modified to obtain a ciliated differentiation in virro. Ciliogenesis w:i:, quantified by flow cytometry, and centriolar and pericentriolar components analyzed during the kinetic of differentiation. These results open the way of a detaiIed analysis of molecular mechanisms linked to cenuiolar assembl;i during ciliated epithelial differentiation. REPRESSION OF THE HUMAN H19 PROMOTER BY TUMOR SUPPRESSOR WILD TYPE GENE PRODUCT ~53 DUGIMGNTThierrv ‘**, MONTPELLIERClaire”*” ADRIAENSSENS Eric *,*iGTSOVA Violetta**, LAGROU Christian STEHELIN Dominique , COLL Jean **, CURGY Jean-Jacques * ‘The first two authors made an equal contribution to this work *Centre de Biologie Cellulaire, Unit6Dynamique des CelluIes Embryonnarres et Can&reuses (EA 1033), SN3, Universin? des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq cedex France. +Facultt des Sciences Jean Penin, Universite d’Artois, 62307 Lens cedex France. **Unite d’oncologie Moleculaire, CNRS URA 1160, Institut Pasteur, rue Calmette, 59019 Liile cedex France. The developmentally regulated H19.gene displays several remarkable properties: i.e. expression of an apparently non-translated mRNA, genomic imprinting (maternal allele only expressed), relaxation of .this imprinting and/or epigenetic lesions in some tumors. Despite numerous observations on the imprinting status of the gene and its relaxation. data on trans and cis- acting factors required for the H19 gene expression are still missing.~As -a fist approach to address identification of factors involved in the regdation of the gene, we found that cells from a ~53 andsense-aansfected HeLa clone displayed increased amounts of HI9 transcripts, when compared to the non- transfected cells. This ~preliminary indication of the repressing effect ofthe ~53 protein on HZ9 expression was confirmed by transient co:transfection experiments in HeLa cells, using CAT and LUC surrogate constructs under the control of 823 bp of the HI 9 promoter, and different ~53 derived constructs containing sense or antisense wild type p.53 gene, or one mutated (143 VaI>>Ala) sequence conferring- a temperature sensitivity phenotype (ts.). Indeed, we observed a down regufation of the reporter gone after transient co-transfeetions using~a sense wild type ~53 sequence~or the-us. mutant at a permissive temperature (32°C) and an increase of Hj9 prornoter- driven activity with both antisense ps3 sequence and the.pf3 mutated sequence at a non-permissive temperature (37°C). Furthermore, same co- nansfectioriexperiments were performed in a cell type lacking endogenous ~53 (Calu 6 cells); results broqght additional evidences that the. wild type a51 protein exertes a down regulation of the HI9 gene expression.-
Transcript of RETINOL AND RETINOIC ACID VECTORIZATION WITH A SYNTHETIC PEPTIDE FORMING β-BARREL STRUCTURE
80 Biology of the Cell
RETINOL AND RETINOIC ACID VECTOR.IZATlON WITH A SYNTHETIC PEFTIDE FORMING P-3ARREL STRUCTURE
Pierre PELLEGRIN ; Frederic HEITZ ; Jean MERY and RenC BENNES
C.R.B.M. (C.N.R.S. I.N.S.E.R.M.) B.P. 5051 34033 Montpellier CCdex FRANCE
We built a peptide which autoassociates as beta-barrels (under defined conditions), The secondary structure is elucidated by CD, PTIR and fluorescence spe@oscopy measurements. When phosphate ions are present (> for&f, pH 7.2) and for peptide concentrations below 5mM, the peptide is autoassociated as antiparallel g-sheets. The fluorescence studies concerning all tram retinol and all tram retinoi’c acid with this peptidic structure show that~ ten antiparailel strands beta barrels bind retinolds with a dissociation constant (Ko = 3.10” mo1.l’) very close to natural ~Retino’ids Binding Proteins (Cogan LJ., Kopelman M., Mokady S. and Shi&zky M.(1976) Eur.J. &&em. ,65,?1-78 ; LaLonde J. M. , Bemlohr D. A. and Banaszak L. J.(I994) FASEB J. 8, 1240-1247). Thus, we mimic perfectly endogenous proteins which bind retino’ids.
We have vectorized the retinol (Vit. A) helping us with these fi- barrels into human fibroblasts ; the retinal stays in a perirmclear situation, The association between the retinoyc acid and the peptide- has been used to vector&e this retindid toward murin leukemic. cells (Ll2IO). The results, ahhough preliminary, show that this dierenciation inducer (Comic M. ,Agadir A., Degos Land Chomienne C.(l994) Anticancer Res. 14, 2339- 2346 ; Moqattash S. Nader GA. Lutton JD. (1994) Leukemia Res. 18 : 53 1- 39) have deeply’affectedthe cell multiplication. It remains to show that the observed diminution is due to an enhanced cellular differentiation and not to a toxicity of the used complex.
MODULATIONS OF THE HI9 GENE EXPRE#ION IN NORMAL OR MALIGNANT MBMMARY EPITHELlAL CELLS
ADRIAENSSENS Eric*, DUMONT Lionel *! FAUQUETIE William * LE BOURHIS X&en *, DU$lIMONT Thmry *‘, COLL Jean **,
CURG Jean-Jacques
*Unite “Dynamique des cellules embryonnaires et cancereuses”, DRED 1033, Centre de Biobgie Cellulaire, Univemitt des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq cedex, France.**“Ut& d’Oncologie moltculaire”, CNRS URA 1160, Institut Pasteur, I Rue Calmette, 59019 Lille cedex, Frartce.0 Facult6 des Sciences Jean Pen-h-t, Universid d’Artois, 62307 Lens cedex, France.
We studied conditions of the expression of this gene during the ontogenesis of the mammary gland in mouse and in normal or tumoral mammary epithelirit cells.
HI 9 expression was detected after in situ molecular hybridization (ISH) at 4 stages of the mouse mammary gland development. Ina 14.5 days foetus, H19 signal was scattered over mammary gland outlines and adjacent tissues. At birth and before puberty (25 days), HI 9 transcripts were far less abundant.. After puberty and during tubule organisation, the HI9 signal was important and preferentially localized over epithelial celfs. These~results prompted us to establish the role of the extmeellular niatrix and mesenchymal cells on the HI9 gene expression in epithelial tiells. Thus, @Is were cultured within a collagene matrix (“3-D”cuIture). By HIS, we observed a H19 overexpression for.both normal and tumoral cellsgrown in “3-D” culture when compared with monolayer culture conditions. Adding a fibroblast conditioned medium had an enhancing effect on Hi9 expression only in monolayer conditions. Furthermore, the HI9 gene expression was studied in different proliferating conditions (serum, differentiating or anti-prolifemting factors) and in presence. of inhibitors or activators of transduction pathways including protein l&nases (PKA or PKC).
We conclude from these experiments that the transcription level depends on the cell type, but is reduced in cell Iignes treated with TcFpl and constantly raised when normal or pathological ceils were cultured into a collagene matrix.
CHARAC’I’ERIZATION OF CENTRIOLAK i’l<O’l’hI$~ DURING CILIATED DfFFERENTIAT16N IN MAMMALS -1, LAOUKILI Jamilal, GENDRON MarieClaudc2, MARANO Francelyne 1. i. Laboratoire de cjrophysiologre et toxicoiogle celirtluwe, 2. Ldoratoire de cytodtrie, lnstitut Jacques Monad. Universitt! Paris 7,2-Place .lussieu, 75005 Paris, France
In mosf arurnal cells, centrioles are integral cornuonents of the centrosouic‘ They also form the elementary struct& which bears flagella and cilia Ciliated epithelial cells in mammals possess 200 functional cilia anchored at the apical membrane. During ciliated epithelial differentiation, cilia elongation is preceded by the formation of centrioles which seems to be independent of preexisting centriolar structures. Cenrrioles are made in~e;lc!? cell by waves of successive assembly, developing from procerm-ioles which are anchored on fibrogranular material whose origin and molecular uaturc are unknown (Dirksen, E.R. (1991) Biol. Cell. 72, 31-38). Many anticentrosome antibodies tested in ii?lluunofluoresc~nce on rabbi tracheal epithelial cells and human nasal epithelia recognized both centrosomes from non-ciliated cells and centriolcs/basal bodies of ciliateti cehs. Polyglutamyiation of a- and P-tubulins from cilia and centrioles u’;r\ demonstrated usin$ a monocloual antibody against potyglutamylared tubuiins (Wollf, A: et al. (1992) Eur J. Cell Viol. 59, 425-432) Moreover, this specific antibody allowed a mcasurc of the percentage o: ciliated cells from a given epithelium. hi primary cultures of respirator: epithelial ceils (Baeza-Squiban et ai. (1994), In I%ro Celt. Dev Bir,i.. 30A, 56-67), cell migration and proliferation was correlated with a rapid decrease of the percentage of ciliated cells. Culture conditions werr~ modified to obtain a ciliated differentiation in virro. Ciliogenesis w:i:, quantified by flow cytometry, and centriolar and pericentriolar components analyzed during the kinetic of differentiation. These results open the way of a detaiIed analysis of molecular mechanisms linked to cenuiolar assembl;i during ciliated epithelial differentiation.
REPRESSION OF THE HUMAN H19 PROMOTER BY TUMOR SUPPRESSOR WILD TYPE GENE PRODUCT ~53
DUGIMGNTThierrv ‘**, MONTPELLIERClaire”*” ADRIAENSSENS Eric *,*iGTSOVA Violetta**, LAGROU Christian
STEHELIN Dominique , COLL Jean **, CURGY Jean-Jacques *
‘The first two authors made an equal contribution to this work *Centre de Biologie Cellulaire, Unit6Dynamique des CelluIes Embryonnarres et Can&reuses (EA 1033), SN3, Universin? des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq cedex France. +Facultt des Sciences Jean Penin, Universite d’Artois, 62307 Lens cedex France. **Unite d’oncologie Moleculaire, CNRS URA 1160, Institut Pasteur, rue Calmette, 59019 Liile cedex France.
The developmentally regulated H19.gene displays several remarkable properties: i.e. expression of an apparently non-translated mRNA, genomic imprinting (maternal allele only expressed), relaxation of .this imprinting and/or epigenetic lesions in some tumors. Despite numerous observations on the imprinting status of the gene and its relaxation. data on trans and cis- acting factors required for the H19 gene expression are still missing.~As -a fist approach to address identification of factors involved in the regdation of the gene, we found that cells from a ~53 andsense-aansfected HeLa clone displayed increased amounts of HI9 transcripts, when compared to the non- transfected cells. This ~preliminary indication of the repressing effect ofthe ~53 protein on HZ9 expression was confirmed by transient co:transfection experiments in HeLa cells, using CAT and LUC surrogate constructs under the control of 823 bp of the HI 9 promoter, and different ~53 derived constructs containing sense or antisense wild type p.53 gene, or one mutated (143 VaI>>Ala) sequence conferring- a temperature sensitivity phenotype (ts.). Indeed, we observed a down regufation of the reporter gone after transient co-transfeetions using~a sense wild type ~53 sequence~or the-us. mutant at a permissive temperature (32°C) and an increase of Hj9 prornoter- driven activity with both antisense ps3 sequence and the.pf3 mutated sequence at a non-permissive temperature (37°C). Furthermore, same co- nansfectioriexperiments were performed in a cell type lacking endogenous ~53 (Calu 6 cells); results broqght additional evidences that the. wild type a51 protein exertes a down regulation of the HI9 gene expression.-
