SBCF/Euroconference Meeting on ‘Cell Proliferation and Tumour Suppressors’, November 1996 71

MPF AND PROCAINE-INDUCED MATURATION OF XENOPUS OOCYTE

BODART Jean-Francois’, FLAMENT..&?phanc’. BROWAEYS Edith’. BERTOUT ~arcl, ROUSSEAU Arlettel, GANNON Julian2 et VILAIN Jean-Pierrel. 1 - Centre de Biologie Cellulaire, Unit6 de Dynamique des cellules embryonnaires et cancereuses, Laboratoire de Biologie du Developpcment. EA DRED 1033, UniversitC de Lille 1, SN3, F-59655 Villeneuve d’Ascq cedex, France. 2 - Imperial Cancer Research Fund Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD. U.K.

The incubation of Xenopus luevis oocytes in procaine containing solutions induces germinal vesicle breakdown without white spot formation and, in some cases, with appearance of spindle and chromosomes in the cytoplasm. The present study was performed to determine if the M-phase promoting factor was involved in this unusual maturation. Procaine failed to induce maturation in the presence of 6-dimethylamino purine or roscovitine which are both known to inhibit p34cdc2 kinase. Histone Hl kinase activity was detected in procaine-treated oocytes but it was always lower than in progesterone-treated controls (three times). A shift in p34cdc2 was observed in oocytes that had been exposed to procaine for 16 hours but it was not detected in those exposed for 24 hours. Finally, cytoplasm transfer experiments demonstrated that the maturation promoting activity that occured in oocytes incubated in procaine for 16 hours could induce maturation of the recipients stage VI oocytes. This transferable activity was weaker than those from progesterone controls since only 30% of the recipients underwent germinal vesicle breakdown and only a few spindles were observed which were not always correctly located. Taken together these results demonstrate that a transient activation of the M- phase promoting factor is involved in procaine maturing effect despite some differences with progesterone-treated oocytes which might explain the particular type of maturation induced by this substance. The discovery of the mechanisms by which procaine is able to activate the M-phase promoting factor might now help to understand some steps of progesterone-induced maturation that have still to bc elucidated.

REGULATION OF p21wAFr’C”‘r EXPRESSION IN HUMAN OVARIAN CARCINOMA

-1, MAZARS Philippel, BALDIN Vtroniquel, VIDAL Sirnonel, JOZAN Suzanne~ et VALE’ITE Anniel.

‘Instinct de Phartnacologie et de Biologic Structurale, CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex et 2Centre Claudius Rgaud, 20-24 rue

du pont Saint-Pierre 31052 Toulouse Cedes FRANCE.

The growth-inhibitory protein p21 wA~1’C1P1 is a potent inhibitor of various cyclin-dependent kinases the expression of which is regulated at the transcriptional level by p53-dependent and -independent mechanism. A direct effect of p21 as an inhibitor of cell proliferation has been demonstrated by transient expression in tumor cells. Experiments on primary specimen from different types of human malignancies revealed p21 gene mutations to be rare events. Although genetic changes associated with the development of human ovarian carcinoma are poorly understood, p53 gene alterations have been detected in 79% of ovarian cancers. Consequently, dysregulation of p21 gene expression providing growth advantage to tumor cells could exist in human ovarian cancers.

Our results show in ovarian cancer cell lines as well as in samples from ovarian tumors that the heterogeneous expression of ~21 could not be correlated to the proliferation rate of the cells. Moreover, they suggest that, in vivo, p21 expression could not be strictly correlated with the ~53 phenotype. Then we compared p21 expression in tumor and normal cells and observed the hiehest level of ~21 mRNA in normal cells. On the other hand. in some cell li;es. the p21 p&in was expressed at a very high level. The different 021 characteristics have been studied: half life. subcellular localization and &rctionality (in vivo ability to bind to cdk2 and to inhibit cdk2 activity in vitro). In consequence of this study we haven’t been able to distinguish p21 in normal cells from p21 in tumor cells. However, our results indicated that not only p21 but also proteins involved in cell cycle progression (cyclins. cdks and PCNA) were highly expressed which could explain why a tumor cell can escape from the negative control of ~21. We hypothesized that ~21 overexpression in tumor cells could allow a normal response to inhibition of proliferation to be restored.

IDENTIFICATION OF A DYNAMIN RELATED PROTEIN IN ROLE OF CYCLIN DEPENDENT KINASE IN Gl-->S THE FISSION YEAST SCHIZOSACCHAROMYCES POMBE. TRANSITION AND IN S PHASE OF THE FIRST SEA

URCHIN EMBRYONIC CYCLE. PELLGGUIN Laetr ‘tia, BELENGUER Pascale, OUSTRIN Marie-Louise et Jean-Luc Moreau, Gerard Peaucellier, Andre Picard. and Anne Marie

DUCOMMUN Bernard. Genevitre. Laboratoire Arago, CNRS-URA 2156, Universitt de Paris VI, Institut de Pharmacologic et de Biologie Structurale, CNRS 6665 1 Banyuls sur mer cedex. France

205. route de Narbonne 31077 TOULOUSE cedex. FRANCE. Cell cycle progression is mainly regulated by the Cyclin Dependent

Kinases (CDKs). In particular, the cdks appear to play a dual role in the control of DNA replication: they are essential for the firing. of origins and they

Close connections seem to exist between extra-cellular signals and the cell cycle machinery. In fission yeast the cdc2 protein kinas< a universal cell cycle regulator, controls both the Gl/S and G2/M transitions. The niml protein kinase is a dose dependent inducer of mitosis that acts upstream of cdc2. It has been shown that although niml is not essential, its function is required for an efficient cellular adaptation to nutritional starvation. We have undertaken a study on the regulation of niml and its role in the transduction of external signals, such as lack of nitrogen, to the cell cycle machinery. One of the approach was to search for partners or regulators using a two-hybrid screen in the budding yeast. Screening of an S. pombe cDNA library led to the identification of a truncated clone that was subsequently fully isolated using an organized genomic library. This gene (2709 bp) named Mspl encodes a 903 amino-acids protein that presents 58 % homology with the S. cerevisae MGMl dynamin. The interaction between niml and mspl was reconstituted in vifro using a GST-msul fusion protein produced in E. co/i. Other experiments are c&ently under way to investigate the physiological significance of this interaction. In parallel we have undertaken the characterisation of the mspl function, the only dynamin identified so far in fission yeast. Strains overexpressing mspl or in which mspl has been deleted are currently being characterised. The identification of the molecular connection between the cdc2 regulation and this new dynamin may provide new insight in the deciphering of the signalling pathways that connect nutritional parameters to the major cellular functions.

act to block the formation of ihe pre-replicative compl&es (p&-R&), thus preventing the re-initiation of DNA replication during G2.

In-complex eucaryotes different cdks seem to ensure these two functions but the precise role of each of them is not clearly understood.

Unfertilized sea urchin eggs are arrested in G I phase of the first mitotic cell cycle. Fertilization resumes the block and induces S phase, making fertilized eggs a good model to study G I-->S transition and early steps of DNA replication.

We previously observed that cycline B/cdQ kinase is activated at the beginning of S phnsc and we showed that cyclin B and cyclirr B-dcpendaut kinase are present in the nucleus during replication (Cenevicre-Garrigues A.M. etal. 1995. J. Cell Sci. 108: 2693-2703). Ada&i and Laemmli (I994 EMBO J. 13: 41534164) demonstrated in xenouus eso extracts that an absence of cvclin B/cdc2 kinase activitv is essential IO allow uost-mitotic assembly of pre-replication centers. Thus we hypothesized that pre-replication centers take place in vivo during the first twenty miuutex after fertilization when cyclin B/cdcZ kinase is tightly inhibited. We report here our first attempt to verify this idea: RPA. a protein associated to the decondensing chromatin was purified. and antibodies were produced against the two largest subunits. These antibodies were used to local& RPA in unfertilized and fertilized eggs and to study the association of RPA with the chromatin in the male and female pronuclei in relation with the cyclin B/cdQ kinase activity.

To further analyse the relationship between cycliulcdk and initiation of DNA replication, we identified the homologue of cdk2 and characterized its activity. We demonstrated that cdk2 is associated with cyclin A and we are studying now the biological role of this complex in S phase.