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Effect of sinefungin on leishmania donovani cell cycle progression
Biology
106 lq;l’l’fic’,‘
OF
SINIII~LINGIN
DONOVAN1 CISLI, CYCIS
ON
I,I~;ISIIMANIA
I'ROGiWSS-ION
MOULAY lnmyn I, GENDRON Marie-Claude*, ROBERT-GERO Malkal, TOURNIER F&d&$ 1. 1.C.S.N - CNRS 91198 G(flYvorc. ZI.J.M.CNRS - tctboruroirc & cymn&rie, Paris 7, 75005 Paris. 3. Luborufoire de cyrophysiologie et de mxicologie celluluirc, UniversirP Paris 7, 7SoO5 Paris. Leishmania donovani,a protozoan parasite belonging to
the Ttypatutsotna/i~fac family, is the etiologic agent of the killing disease Kala azar or visceral leishmaniasis. Sinefungin is an antibiotic whose antileishmanial activity was demonstrated in virro and in viva. This molecule is structurallv related to S-adenosvlmethionine. Amount the more important effectsof sinefungin on L Bmavani promastigotesuis a very rapid and significative DNA synthesis inhibition. These cells contain a single and DNA rich mitochondrion whose division cycle interferes with the nuclear division cycle. We have looked for the best orocedure which allowed the most*discriminatine anal&s in flow cytometry of the nuclear cycle. For exponentiallyvgrow&g cells, the duration of each nuclear cvcle Dhase was established (G1:4hr. S:2.5hr. G2/M: 1.5hr). We demonskated that sinefungin blocks‘the eelis in early S phase and in a non reversible manner. According to this result, the release of these cells, after a double thymidine block showed a different relative evolution of the cell cycle progression depending on the presence or absence of the drug. On the other hand, sinefungin did not react with the stationnary growing cells which are arrested in GO/Gl. When these cells were transfered into a fresh medium, without the drug they started a new division cycle. In contrast, when the drug was present, the cells remained blocked in GO/G I, At the used concentration (O,lpg/ml), sinefungin provoked the roundine of the cells and the loss of the external Dart of the flagellum (PHl%&JZA’r, M.A. et al., (1992) Exp parasi&ogy 74, 175187). Surm%in&. taxol induced the same morphological changes but it interfered with the G2/M phase. In si[u*. imm&ofluores&nce and biochemical analysis showed that the microttibule network was very stable for coatrol and treated cells. We have not detected a significant differencein the quantity or the distribution of tubulin in treated cells.
CDKZ AND NEW&Y SYNTKIESIZED CDC2 PROMOTE INlT?ATlON OF SPAASE IN XEN0PVS %:cc EXTPUCTS. TASSAN
jean-pierreI,
COX
rick2,
PHILIPPE
opher.c.2. I D&w~emenr de Biologic el GtWIique du L&eloppemer~t. URA CNRS 256, lhiwmiile de Rennes I. 35042 Retmes ceerlex. 2Depwmettf of Getteti~.andDtw&qmtenopnrenr, school of BiolOgqI Sciences, University of Sussex, Faher,Br&irton 3iW 9QC LI.K. Cydin dependant protein kinases (cdk‘s) are key regulators of the eukaryotic cell cycle Whereas p34cdc2 is active and esse&al at the G2/M tmmitian, a closely related kinese p33cdk2 has been implicated in the initiation of DNA replication. In low-speed supematant extracts made from crushed Xenopus eggs, cxogenously added demembrsnated sperm nuclei decondense, acquire a nuclear membrane and lamina, and subsequently replicate their DNA only once beFore mitosis. In Xenopus egg extracts, antibodies end Sucl-beads have been used to remove cdk2 and cdc2. Depletion of both proteins, or just cdk2, inhibits replication while readdition of the proteins reeluted from Suclbeads restimulates it. Using both the above procedupes to inhibit DNA repficetion we foumi that addition of single mRNA species (either cdkt or cdc2) restimulate DNA synthesis. An ATP-b&ding-site mutant cdk2 mRNA (cdk2R33) ftiled to stimulate replication and inhibited S phase initiation in mock-depieted extracts. Both human and xenopus edc2 mRNAs rescued replication in this system. Human mutant mRNAs have been used to show that the kinase activity of cdc2 can substantially stimulate DNA repli&on in Sucl deplered extracts. This activity is distinct fi-om’the cdiZ/cyclin B kinase active in mitosis. Rescue of replication by p34cdc2 is also observed in extracts depl%tcd with a cdk2 antibody which still retain much of their endogenous cdc2 protein. The fbnctional a&say we have used demonstrates that newly synthesised p34cdc2, but not the inherit& ‘old’ form, can induce S phase and in this c&e mlry overtap in Kmction with p33cdk2.
of the
Cell
MOLECULAKCLONINGANIICELL CYCLEREGULATORY PROPERTIESOF THECHlCKENE2F-1 TRkNsCRWT~ON FACTtiW , LOiSUU Laurent,ARNAUD Lionel, TREMBLE Al ! Afain et BRUN Gilbert. ChtRS-UMR 49 LA INCA, Ecole Not-male Suptfrieure de Lyon, 46 AlIke d’ltalie, 69364 Lyon cedex 07
In human ceils the transcription of numerous genes, expressed 3: rbz Gl/S transition of the cell cycle, is mostly controled by the reversible association of the transcription factor E2F-1 with the product of the Rb antioncagene (plOSRbj. This suggests that the dereg&tion of E2F-1 activity cafe participate in the genesis of pathological disorders,.especidly in neural cells where the lossof function of plOSRb, resulting in constitutive activation of E2F-I, induces growth alterations and cancers. In view of such a possibility, we have investigate& thecell cycle regulatory properties ofE2F-1 in rhe~avian neuroretina (NR) which enables both, in viva studies during embryonic development, and in vivo studies in NR cell cultures induced to divide by rh’z Rous Sarcoma Virus (RSV). We have isolated a molecular clone containing the complete z:DNA sequence of the E2F-1 chicken gene (chE2F-I). Sequence data shtiw ih;:c, although slirrhtv shorter (403/437aal. the chEZF-1 factor is highly. homol&ous,-in.rhe. basic fiNA binding, helix-loop-helix, leucin zippa dimerisation motifs and in the Rb binding carboxy terminal domain, to the human protein and also to the mouse and drosophila E2F-1 factors cloned recently. Cotransfection of a c%2F-1 expression vector with the adenovirus E2A promoter CAT gene -fusion and eleccrophorede mobility shift assays carried out with an in vitro translated protein indicate that chEZF-I. is a potent transactivator at the 5ZTiTCGCGC site. In addition, it was found thar the chicken protein forms stable hererodimers with the human DPI prote~l, thereby increasing its affinity for the promoter response elements. Northern blot analyses of chE2F-1 in synchronized chicken fibroblasts or d&ng embryonic development indicate that expression of this function is tightly coupled to the proliferative properties of ceti.or tissues, with a peak & activity at the GI/S transition. Using in situ hybridization, we have observed that chEZF-I expression is very high at early stages of NR development wi:E! a subsequent decrease at El0 when most of the neuroblasts enter terminal differentiation. Comparison of Nonhern and Western biot data indicates that il.Zi: I E2F-I availability is mainly determined by transcriptional mechanist% tVr were thus led us to test the effects of the constitutive deregulation of E2F I gene expression, obtained from retroviral vectors, on cell proliferatioil~rrr,d cell transformation. ResuIts on the overexpression of different versions of the chEZF-1 gene on cell growth propenies will be presented and discussed.
X CHROMOSOME INACTIVATlON AND DNA METHYLATION,
AN
IN
SITU
STUDY
BERNARDINO Jacqueline), MALFOY Bernad’, DUTRILLAUX Bemardl, NIVELEAU Alain et BOURGEOIS Claire A.1 ‘URA 620 CNRS, Institur Curie, Secrion de Biologic, 75231 Paris Cedex OS. Fax : 40.51.66.74 2URA 1459 CNRS, I&itut Pasteur de Lyon, Avenue 69635 Lyon, Fax : 7X72.39.89
26 we d’l,h, Tony tiarmer,
Inactivation is the means of regulating gene dosage for compensating for the difference in the number of X chromosomes between both sexes in mammals. Inactive X chromosomes remain highly condensed throughout interphase, show minimal transcriptional activity and are late replicating (MIGEON B.R.-(1994). Trends Goner.. 10, 230-235). DNA methyiation has been shown to participate to X chromosome inactivation (SINGER-SAM I. andRIGGS A.D. (1993). In “DNA methylation : Molecular Biology and Biological Significance”. JOST J.P. and SALUZ H.P., eds.. Birktx&ser Veriag, Basel). However, data~on X chromosome methvlation/inactivatiQn are limited to a few loci, the actual methyiation s&us of the whale chromosome being rarely investigated. The results obtained on mctaphase chromosom& using either methyl sensitive restiiction enzymes or antibodies raised against 5-methylcytosine (anti-5mC) gave conflicting results. To further investigate the relationship between X inactivation and DNA methylatioo in human primary cultures of fibroblastes and lymphocyt&s, interacticins of anti;fimCZ with metaphase chromosomes were evidenced by a sensitive immunofhmrescent method (BARBIN A., MONTPELLIER C..KOKALJVOKAC N., GIBAUD A., NIVELEAU A., MALFOY B.. DUTRLLLALJX 8.. BOURGEOIS CA. (1994). Hum. &net., 94, 684-692). The fliiorescent signals were quantified after digitalisation using a cooled CCD camera. The analysis of the relative methylation of X chromosomes shows that most often, if not in all cases, the X inactivated or late r~plicatingchrotiosome is hypemethylated in comparison to the active one. Thus. Xmchromosome inactivation does not seems to be associated with a global. hypermethylation, as observed in constitutive heterochromatin.
106 lq;l’l’fic’,‘
OF
SINIII~LINGIN
DONOVAN1 CISLI, CYCIS
ON
I,I~;ISIIMANIA
I'ROGiWSS-ION
MOULAY lnmyn I, GENDRON Marie-Claude*, ROBERT-GERO Malkal, TOURNIER F&d&$ 1. 1.C.S.N - CNRS 91198 G(flYvorc. ZI.J.M.CNRS - tctboruroirc & cymn&rie, Paris 7, 75005 Paris. 3. Luborufoire de cyrophysiologie et de mxicologie celluluirc, UniversirP Paris 7, 7SoO5 Paris. Leishmania donovani,a protozoan parasite belonging to
the Ttypatutsotna/i~fac family, is the etiologic agent of the killing disease Kala azar or visceral leishmaniasis. Sinefungin is an antibiotic whose antileishmanial activity was demonstrated in virro and in viva. This molecule is structurallv related to S-adenosvlmethionine. Amount the more important effectsof sinefungin on L Bmavani promastigotesuis a very rapid and significative DNA synthesis inhibition. These cells contain a single and DNA rich mitochondrion whose division cycle interferes with the nuclear division cycle. We have looked for the best orocedure which allowed the most*discriminatine anal&s in flow cytometry of the nuclear cycle. For exponentiallyvgrow&g cells, the duration of each nuclear cvcle Dhase was established (G1:4hr. S:2.5hr. G2/M: 1.5hr). We demonskated that sinefungin blocks‘the eelis in early S phase and in a non reversible manner. According to this result, the release of these cells, after a double thymidine block showed a different relative evolution of the cell cycle progression depending on the presence or absence of the drug. On the other hand, sinefungin did not react with the stationnary growing cells which are arrested in GO/Gl. When these cells were transfered into a fresh medium, without the drug they started a new division cycle. In contrast, when the drug was present, the cells remained blocked in GO/G I, At the used concentration (O,lpg/ml), sinefungin provoked the roundine of the cells and the loss of the external Dart of the flagellum (PHl%&JZA’r, M.A. et al., (1992) Exp parasi&ogy 74, 175187). Surm%in&. taxol induced the same morphological changes but it interfered with the G2/M phase. In si[u*. imm&ofluores&nce and biochemical analysis showed that the microttibule network was very stable for coatrol and treated cells. We have not detected a significant differencein the quantity or the distribution of tubulin in treated cells.
CDKZ AND NEW&Y SYNTKIESIZED CDC2 PROMOTE INlT?ATlON OF SPAASE IN XEN0PVS %:cc EXTPUCTS. TASSAN
jean-pierreI,
COX
rick2,
PHILIPPE
opher.c.2. I D&w~emenr de Biologic el GtWIique du L&eloppemer~t. URA CNRS 256, lhiwmiile de Rennes I. 35042 Retmes ceerlex. 2Depwmettf of Getteti~.andDtw&qmtenopnrenr, school of BiolOgqI Sciences, University of Sussex, Faher,Br&irton 3iW 9QC LI.K. Cydin dependant protein kinases (cdk‘s) are key regulators of the eukaryotic cell cycle Whereas p34cdc2 is active and esse&al at the G2/M tmmitian, a closely related kinese p33cdk2 has been implicated in the initiation of DNA replication. In low-speed supematant extracts made from crushed Xenopus eggs, cxogenously added demembrsnated sperm nuclei decondense, acquire a nuclear membrane and lamina, and subsequently replicate their DNA only once beFore mitosis. In Xenopus egg extracts, antibodies end Sucl-beads have been used to remove cdk2 and cdc2. Depletion of both proteins, or just cdk2, inhibits replication while readdition of the proteins reeluted from Suclbeads restimulates it. Using both the above procedupes to inhibit DNA repficetion we foumi that addition of single mRNA species (either cdkt or cdc2) restimulate DNA synthesis. An ATP-b&ding-site mutant cdk2 mRNA (cdk2R33) ftiled to stimulate replication and inhibited S phase initiation in mock-depieted extracts. Both human and xenopus edc2 mRNAs rescued replication in this system. Human mutant mRNAs have been used to show that the kinase activity of cdc2 can substantially stimulate DNA repli&on in Sucl deplered extracts. This activity is distinct fi-om’the cdiZ/cyclin B kinase active in mitosis. Rescue of replication by p34cdc2 is also observed in extracts depl%tcd with a cdk2 antibody which still retain much of their endogenous cdc2 protein. The fbnctional a&say we have used demonstrates that newly synthesised p34cdc2, but not the inherit& ‘old’ form, can induce S phase and in this c&e mlry overtap in Kmction with p33cdk2.
of the
Cell
MOLECULAKCLONINGANIICELL CYCLEREGULATORY PROPERTIESOF THECHlCKENE2F-1 TRkNsCRWT~ON FACTtiW , LOiSUU Laurent,ARNAUD Lionel, TREMBLE Al ! Afain et BRUN Gilbert. ChtRS-UMR 49 LA INCA, Ecole Not-male Suptfrieure de Lyon, 46 AlIke d’ltalie, 69364 Lyon cedex 07
In human ceils the transcription of numerous genes, expressed 3: rbz Gl/S transition of the cell cycle, is mostly controled by the reversible association of the transcription factor E2F-1 with the product of the Rb antioncagene (plOSRbj. This suggests that the dereg&tion of E2F-1 activity cafe participate in the genesis of pathological disorders,.especidly in neural cells where the lossof function of plOSRb, resulting in constitutive activation of E2F-I, induces growth alterations and cancers. In view of such a possibility, we have investigate& thecell cycle regulatory properties ofE2F-1 in rhe~avian neuroretina (NR) which enables both, in viva studies during embryonic development, and in vivo studies in NR cell cultures induced to divide by rh’z Rous Sarcoma Virus (RSV). We have isolated a molecular clone containing the complete z:DNA sequence of the E2F-1 chicken gene (chE2F-I). Sequence data shtiw ih;:c, although slirrhtv shorter (403/437aal. the chEZF-1 factor is highly. homol&ous,-in.rhe. basic fiNA binding, helix-loop-helix, leucin zippa dimerisation motifs and in the Rb binding carboxy terminal domain, to the human protein and also to the mouse and drosophila E2F-1 factors cloned recently. Cotransfection of a c%2F-1 expression vector with the adenovirus E2A promoter CAT gene -fusion and eleccrophorede mobility shift assays carried out with an in vitro translated protein indicate that chEZF-I. is a potent transactivator at the 5ZTiTCGCGC site. In addition, it was found thar the chicken protein forms stable hererodimers with the human DPI prote~l, thereby increasing its affinity for the promoter response elements. Northern blot analyses of chE2F-1 in synchronized chicken fibroblasts or d&ng embryonic development indicate that expression of this function is tightly coupled to the proliferative properties of ceti.or tissues, with a peak & activity at the GI/S transition. Using in situ hybridization, we have observed that chEZF-I expression is very high at early stages of NR development wi:E! a subsequent decrease at El0 when most of the neuroblasts enter terminal differentiation. Comparison of Nonhern and Western biot data indicates that il.Zi: I E2F-I availability is mainly determined by transcriptional mechanist% tVr were thus led us to test the effects of the constitutive deregulation of E2F I gene expression, obtained from retroviral vectors, on cell proliferatioil~rrr,d cell transformation. ResuIts on the overexpression of different versions of the chEZF-1 gene on cell growth propenies will be presented and discussed.
X CHROMOSOME INACTIVATlON AND DNA METHYLATION,
AN
IN
SITU
STUDY
BERNARDINO Jacqueline), MALFOY Bernad’, DUTRILLAUX Bemardl, NIVELEAU Alain et BOURGEOIS Claire A.1 ‘URA 620 CNRS, Institur Curie, Secrion de Biologic, 75231 Paris Cedex OS. Fax : 40.51.66.74 2URA 1459 CNRS, I&itut Pasteur de Lyon, Avenue 69635 Lyon, Fax : 7X72.39.89
26 we d’l,h, Tony tiarmer,
Inactivation is the means of regulating gene dosage for compensating for the difference in the number of X chromosomes between both sexes in mammals. Inactive X chromosomes remain highly condensed throughout interphase, show minimal transcriptional activity and are late replicating (MIGEON B.R.-(1994). Trends Goner.. 10, 230-235). DNA methyiation has been shown to participate to X chromosome inactivation (SINGER-SAM I. andRIGGS A.D. (1993). In “DNA methylation : Molecular Biology and Biological Significance”. JOST J.P. and SALUZ H.P., eds.. Birktx&ser Veriag, Basel). However, data~on X chromosome methvlation/inactivatiQn are limited to a few loci, the actual methyiation s&us of the whale chromosome being rarely investigated. The results obtained on mctaphase chromosom& using either methyl sensitive restiiction enzymes or antibodies raised against 5-methylcytosine (anti-5mC) gave conflicting results. To further investigate the relationship between X inactivation and DNA methylatioo in human primary cultures of fibroblastes and lymphocyt&s, interacticins of anti;fimCZ with metaphase chromosomes were evidenced by a sensitive immunofhmrescent method (BARBIN A., MONTPELLIER C..KOKALJVOKAC N., GIBAUD A., NIVELEAU A., MALFOY B.. DUTRLLLALJX 8.. BOURGEOIS CA. (1994). Hum. &net., 94, 684-692). The fliiorescent signals were quantified after digitalisation using a cooled CCD camera. The analysis of the relative methylation of X chromosomes shows that most often, if not in all cases, the X inactivated or late r~plicatingchrotiosome is hypemethylated in comparison to the active one. Thus. Xmchromosome inactivation does not seems to be associated with a global. hypermethylation, as observed in constitutive heterochromatin.