12th EUROPEAN CELL CYCLE CONFERENCE: EuroConference on Cell Cycle Control in Normal and Malignant Cells

Cell Biology International 2001, Vol. 25, No. 2, A1–A34 doi:10.1006/cbir.2000.0713, available online at http://www.idealibrary.com on

12th EUROPEAN CELL CYCLE CONFERENCE EuroConference on Cell Cycle Control in Normal and Malignant Cells* 10–14 February 2001. Mayrhofen/Zillertal, Tyrol, Austria

The traditional series of European Conferences on the Cell Cycle was initiated with a ‘Micro-Symposium’ in 1970 by Eric Zeuthen (Copenhagen). Further meetings were held at various places throughout Europe every two to three years. Our last meeting, the 11th ECCC, was organized by Lilia Alberghina at Gardone Riviera, Italy, in 1997 followed now by the 12th European Cell Cycle Conference in the Alpine ski resort of Mayrhofen/Zillertal. Research on the molecular mechanisms governing cell proliferation and division is one of the most dynamic fields in biological science today. Besides revealing the intricate molecular mechanism of a fundamental phenomenon of life, it strongly influences related areas of biomedical research, e.g. molecular aspects of aging and human cancer. The main goal of this Conference is to provide a platform, particularly for young scientists, to present and discuss most recent advances in this fast-moving field of cell cycle research. The scientific programme is designed to convey a comprehensive view of current lines in cell cycle research, including potentially important aspects which are less well recognized, such as ‘Cell Cycle Regulation in Plants’. A special lecture sponsored by the International Federation of Cell Biology is devoted to this topic by Gerd Ju¨rgens (Tu¨bingen), and will be published in this journal (Cell Biology International). Sixteen invited lectures and about 90 free communications cover a wide range of topics related to cell cycle research, from DNA-Replication, Molecular Mechanisms of Checkpoints, Proteolysis in Cell Cycle Control, Molecular Mechanisms of Mitosis, Apoptosis, through to the Cell Cycle and Cancer. All invited speakers are leading experts—among them recipients of prestigious scientific awards—who have largely influenced the continuing acceleration throughout the field of cell cycle research. The close contact between young scientists and leading experts is particularly facilitated by the intimate atmosphere of the conference site and the combination of scientific sessions with an informal social programme including outdoor activities (skiing, etc.), similar to the well-known ‘Keystone Symposia’ in Colorado, U.S.A. The publication of all the abstracts before the meeting would not have been possible without the hard work of the Editor of Cell Biology International, Denys Wheatley, who also created the new logo of the European Cell Cycle Conferences. On behalf of the organizers of the 12th ECCC, I want to thank him for his generous support and keen advice. Wilhelm Sachsenmaier Innsbruck, Austria February 2001

*Supported by the European Commission, DGXII, Human Potential Programme, High-Level Scientific Conferences 1065–6995/01/0100A1+34 $35.00/0

 2001 Academic Press

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Cell Biology International, Vol. 25, No. 2, 2001

SECTION 1 (INVITED SPEAKERS) S-1. G1/S MACHINERY IN CELL CYCLE COORDINATION AND RESPONSE TO DNA DAMAGE J. Bartek, N. Mailand, J. Falck, C. S. So¨ rensen, E. Santoni-Rugiu, R. Syljua˚ sen, J. Bartkova, C. Lukas and J. Lukas Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark (E-mail: [email protected])

The lecture will summarize our most recent data on the molecular pathways governing G1 progression and G1/S transition of the mammalian somatic cell cycle, the role(s) the G1/S machinery plays in coordinating DNA synthesis with G2/M phases, the mechanistic nature of the cell cycle checkpoint responses of G1- and S-phase cells to DNA damage, as well as the ways these pathways become deregulated in human tumors. Key decisions about cellular fate are taken in G1, and our data indicate that G1/S progression is governed jointly by the so-called RB pathway (the p16-cyclin D/CDK-pRB-E2F axis) and a myc-dependent pathway, two cooperating parallel mechanisms which converge on the control of S-phase initiators such as cyclin E and Cdc25A. Components of these two pathways qualify as tumor suppressors or oncogenes, and their aberrations via diverse molecular mechanisms can contribute to oncogenesis. Based on our recent results, we also propose a concept of S/G2/M coordination by the RB and myc pathways, including regulation of DNA replication, centrosome duplication, cell growth, and cell-cycle regulated proteolysis dependent on the anaphase-promoting complex/cyclosome (APC/C). Apart from controlling accumulation of key mitotic regulators, our results indicate that APC/C activity helps to establish proper duration of G1, and to restrict DNA replication to one round per every cell cycle. Whereas defects in the cell cycle machinery provide a direct proliferative advantage to tumor cells, aberrations in the so-called cell cycle checkpoints, signaling pathways which monitor and ensure genome integrity, act indirectly yet affect both tumor progression and response to radiation and chemotherapy of cancer. In terms of molecular pathways activated by DNA damage, our recent data suggest a two-wave response of cell cycle checkpoints at G1/S. The initial, acute response to DNA damage reflects rapid, p53-independent pathways operating through phosphorylation and degradation (inactivation) of the Cdc25A phosphatase, while the maintenance of the G1 checkpoint is controlled by the p53 pathway. In addition to its rapid execution, the p53-independent Cdc25A pathway blocks cell cycle progression not only at G1/S, but also during S phase. Mechanistic details of the rapid G1/S checkpoint pathways including kinase cascades upstream of Cdc25A differ depending on the nature of DNA damaging agents, and specific features of responses to UV light, ionizing radiation and anti-cancer drugs will be presented. Examples of cancer-associated aberrations of both the cell cycle machinery and the DNA damage checkpoint pathways will be briefly summarized, along with the ways this emerging knowledge provides novel tools for differential diagnosis, indicators of prognosis, and particularly leads for design of new strategies to treat cancer.

S-2. ORC-DEPENDENT NUCLEOSOME POSITIONING STIMULATES THE INITIATION OF DNA REPLICATION James R. Lipford and Stephen P. Bell Massachusetts Institute of Technology, Howard Hughes Medical Institute, Department of Biology, Cambridge, MA, U.S.A. (E-mail: [email protected])

The packaging of eukaryotic DNA into nucleosomes is a critical regulator of many nuclear events, including DNA replication. Studies in numerous organisms suggest a connection between the initiation of DNA replication and global changes in chromatin structure. We have begun to address the molecular basis of the interplay between chromatin and replication by studying the determinants and functions of the specialized nucleosomal configuration of S. cerevisiae origins of DNA replication. We have found that the yeast initiator, ORC, positions nucleosomes adjacent to origins in vivo and in vitro. This function appears to be facilitated by a close interaction between ORC

and the nucleosome(s) immediately adjacent to the origin. By shifting the adjacent nucleosomes away from the origin, we have addressed the role of ORC-directed nucleosomal positioning at the ARS1 origin. This change in nucleosome location disrupts initiation from ARS1, indicating that the specialized nucleosomal configuration at yeast origins stimulates origin function. Interestingly, disruption of originproximal nucleosomes does not alter the association of ORC with the origin but instead inhibits assembly of the pre-RC. The mechanism by which correct nucleosome positioning stimulates the initiation of DNA replication and the potential role for nucleosomes in regulating origin function will be discussed.

S-3. REGULATING THE INITIATION OF DNA REPLICATION IN BUDDING YEAST John F. X. Diffley ICRF Clare Hall Laboratories, South Mimms, Herts EN6 3LD, U.K. (E-mail: J.Diffl[email protected])

Cyclin dependent kinases (CDK’s) play an essential role in triggering the initiation of DNA replication during S phase. In addition, CDK’s also prevent re-replication, in part by preventing the assembly of prereplicative complexes (pre-RCs) at origins after origin firing. Two pre-RC components, Cdc6p and the MCM complex are negatively regulated by CDK activity. The Cdc6 protein is targeted for SCFCDC4mediated rapid degradation at the end of G1 by CDKs and the export of the MCM proteins from the nucleus is activated by CDKs. Surprisingly, in both cases, the S phase promoting cyclins, Clb5 and Clb6 are not required. In Clb5,6
strains, the rapid degradation of Cdc6 and the export of MCM proteins which are not bound to chromatin occurs at the same time as it does in wild type cells. This has led us to propose that the G1 cyclins (CLNs) play a role in preventing re-replication. We have begun to address this question more directly by examining the effects of CLN overproduction on pre-RC assembly. Our results indicate that CLNs are sufficient to prevent pre-RC assembly and DNA replication, at least when expressed to high levels. These results will be discussed with respect to the mechanisms which ensure once per cell cycle replication. To gain a deeper understanding of the mechanism and regulation of initiation, we have recently established a cell free system for the assembly of pre-RCs. In this system, origin-containing DNA coupled to paramagnetic beads is incubated in extracts prepared from G1arrested cells. After retrieval of the beads, proteins bound to DNA are examined by immunoblotting. ORC, Cdc6 and the MCM complex are loaded a sequentially in a reaction that requires ATP for several distinct steps. Interestingly, extracts prepared from nocodazolearrested cells are competent to load ORC but not Cdc6 or the MCM complex. Possible reasons for this will be discussed.

S.4. GROWTH-LINKED AND PATTERN-LINKED CELL CYCLES IN DROSOPHILA B. A. Edgar, J. Britton, L. A. Johnston1, T. Reis, D. Prober and A. de la Cruz Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle WA 98109, U.S.A. (E-mail: [email protected]); 1Dept. Genetics, College of Physicians and Surgeons, Columbia University, New York, NY, U.S.A.

Cell cycles in Drosophila are regulated in distinct ways at different developmental stages, and in different tissues. Developmentally programmed transcriptional control of the CDK regulators string (Cdc25), cyclin E, and dacapo (a CDI) is the primary regulatory mode used in embryogenesis. In the larva, terminally differentiated tissues that undergo extensive growth utilize a DNA endoreplication cycle that is nutritionally regulated. These endocycles employ conserved G1/S machinery including Cyclin E/Cdk2, E2F, and RBF. Their progression normally requires external nutrition (feeding), but this requirement can be bypassed by forced expression of G1/S regulators such as Cyclin E or E2F, or by factors that promote cell growth such as the insulin receptor (Inr), phosphatidyl-inositide 3-kinase (Pi3K), or the dMyc transcription factor. Inr/Pi3K signaling is essential for cell growth and cell cycle progression in endocycling cells, and suppression of this activity phenocopies starvation. Studies in the imaginal discs,

Cell Biology International, Vol. 25, No. 2, 2001 which generate the adult structures, reveal a more complex cell cycle that is regulated at both G1/S and G2/M transitions, and which integrates both growth and developmental signals. Cell growth appears to affect this cycle primarily at G1/S via Cyclin E, which is upregulated at the protein level in response to increased growth. In contrast, developmental signals related to pattern formation affect the imaginal disc cycle at least in part at G2/M transitions via String/ Cdc25. In this case the regulatory mechanism is transcriptional. Data concerning cross-regulation between G1/S and G2/M controls in the discs will also be presented.

S-5. COMPARING THE SUBSTRATE SPECIFICITY AND DESTRUCTION OF CYCLINS A AND B Stephan Geley1, Tod Duncan2, Chizuko Tsurumi2, Yoshimi Tanaka2, Edgar Kramer3, Julian Gannon2 and Tim Hunt2 1 Institute for General and Experimental Pathology, University of Innsbruck Medical School, Fritz PreglStrasse 3, A-6020 Innsbruck, Austria (E-mail: [email protected]); 2ICRF Clare Hall Laboratories, South Mimms, Herts EN6 3LD, U.K. (E-mail: [email protected]); 3IMP, Dr Bohr-Gasse 7, A1030, Vienna, Austria

While it is well-established that cyclin B is essential for cells to enter mitosis, the role of cyclin A in mitosis is not so clear, and many investigators have favoured a primary role in S-phase, or at least a dual role in both S-phase and mitosis. We have started to compare the substrate specificities of cyclin A/CDK2 with cyclin B1/CDK1 by screening expression libraries. We are also testing the specificity of the RxL-binding ‘hydrophobic patch’ on the surface of cyclin A by phage display, and plan to do the same for cyclin B. It is intriguing that Wee1 appears to be a good substrate for both forms of the kinase, although most of the targets of cyclin A are nuclear proteins. We have been trying to find out how the cyclin ‘destruction boxes’ (D-boxes) are recognised for a long time, and recently made some progress by comparing the destruction of cyclin A with that of cyclin B. It turns out that cyclin A is a much preferred substrate for the CDC20-dependent form of the APC/C, and can be polyubiquitylated and degraded even when the spindle checkpoint is active, and cyclin B is stable. Overexpression of cyclin A delays the metaphase to anaphase transition, but only when it possesses an intact destruction box (D-box). Indestructible forms of cyclin A lacking a D-box cause almost no arrest at metaphase, but rather an anaphase arrest. Cyclin A can be degraded when cyclin B is stable under conditions of ubiquitin limitation imposed by addition of inappropriate ubiquitin E2 ligases. We don’t think that cyclin A has a special dedicated form of CDC20 or APC/C, but rather, that the combination of a D-box which is much larger than that of cyclin B, together with additional recognition of the main body of the cyclin (or possibly of the CDK) means that cyclin A binds much more tightly to the APC/C, allowing it to be modified by polyubiquityl chains that are long enough for efficient delivery to the proteasome even when ubiquitylation is significantly impaired, whether by the Mad2-dependent mechanism or by low ubiquitin levels.

S-6. GENETIC ANALYSIS OF CYTOKINESIS IN ARABIDOPSIS G. Ju¨ rgens, U. Mayer, M. Heese, A. Vo¨ lker and G. Strompen ZMBP, Entwicklungsgenetik, Universita¨ t Tu¨ bingen, Auf der Morgenstelle 1, D-72076 Tu¨ bingen, Germany (E-mail: [email protected])

Cytokinesis partitions the cytoplasm of a dividing cell, enabling the daughter cells to initiate their distinct cell cycles. Yeast and animal cells display common features of cytokinesis, such as the formation and contraction of an actomyosin ring that guides the expansion of the plasma membrane from the periphery to the centre of the cell division plane. This process also involves addition of membrane material by vesicle delivery to the base of the ingrowing furrow. By contrast, plant cells have evolved a different strategy of cell division, with targeted vesicle fusion generating the partitioning plasma membrane de novo. A dynamic cytoskeletal array, the phragmoplast, initially targets

A3 membrane vesicles to the centre of the division plane where they fuse with one another to form a transient membrane compartment, the cell plate. Lateral expansion of the cell plate is accompanied by a reorganisation of the phragmoplast from a compact structure to an expanding hollow cylinder that delivers vesicles to the growing margin of the cell plate until it reaches the parental plasma membrane. The expanding cell plate also undergoes a complex process of maturation, including the secretion of cell wall material into its lumen. Cytokinesis has been studied genetically in the model plant Arabidopsis whose genome has been sequenced. Consistent with the unique mode of plant cytokinesis, Arabidopsis lacks a gene for a type-II myosin that would participate in the formation of a contractile actomyosin ring. Conversely, Arabidopsis encodes a cytokinesis-specific syntaxin that has no close homologue in yeast, Drosophila or C. elegans. To analyse the mechanism of plant cytokinesis, we have isolated Arabidopsis mutants that display celldivision defects in embryogenesis. These mutants fall into two broad categories. One class of mutants is defective in the microtubule cytoskeleton that is a prerequisite for proper delivery of cytokinetic vesicles. Mutants of the other class affect the formation of the cell plate by vesicle fusion. Molecular analysis of the genes affected is beginning to give insight into the mechanism of plant cytokinesis.

S-7. REGULATION AND FUNCTION OF SCFSKP2 UBIQUITIN PROTEIN LIGASE C. Wirbelauer, M. Gstaiger, H. Sutterlu¨ ty and W. Krek Friedrich Miescher Institut, Maulbeerstrasse 66, 4058 Basel, Switzerland (E-mail: [email protected])

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin protein ligase complexes that has emerged as a central regulator of the G1 to S phase transition in mammalian cells. An increase in Skp2 protein expression allows cells to enter S phase and a decrease is necessary for proliferating cells to exit the cell cycle. Furthermore, it has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G1 phase progression including cyclin E, the transcription factor E2F1 and the cyclin-dependent kinase (Cdk) inhibitor p27, a dosage-dependent tumor suppressor protein. Abnormally low amounts of the latter occurs frequently in many human cancers and is associated with the aggressive tumor phenotype and a poor prognosis in cancer patients. We found that Skp2 is overexpressed in a variety of primary human cancers and that in these cancers, increased Skp2 protein expression correlates well with reduced p27 protein levels, raising the possibility that differences in p27 proteolysis activity among human tumors may be achieved, at least in part, through upregulation of Skp2 expression. In this regard, Skp2 cooperates with activated Ras to cause malignant transformation of primary rodent fibroblasts. Taken together, these results qualify Skp2 as an oncoprotein and focus attention on the importance of increased Skp2 protein expression in human tumors. Currently, we are focusing our efforts on understanding the mechanisms of Skp2 regulation. New results suggest that Skp2 is degraded by the proteasome in Go/G1 and is stabilized when cells re-enter the cell cycle. Interestingly, rapid degradation of Skp2 in quiescent cells involves Skp2-bound Cul1. Consistent with a role for Cu1l in Skp2 degradation, recombinant Cul1-Roc1/Rbx1-Skp1 complexes can catalyze Skp2 ubiquitination in vitro. These results suggest a model in which degradation of Skp2 in Go/G1 is mediated, at least in part, by an autocatalytic mechanism involving a Skp2-bound Cul1-based core ligase and imply a role for this mechanism in the suppression of SCFSkp2 ubiquitin protein ligase function during the Go/G1-phases of the cell cycle.

S-8. TRANSCRIPTIONAL CONTROL DURING THE p53 RESPONSE Constantinos Demonacos, Marija Krstic-Demonacos and Nicholas B. La Thangue Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ. U.K. (E-mail: [email protected])

The p53 protein is a stress-responsive transcription factor the activation of which causes cell cycle arrest or apoptosis. The transcription of p53 target genes involves p300/CBP co-activators, which are

A4 multiprotein complexes that interact with the p53 activation domain. Whilst phosphorylation is believed to alter the stability of p53 and influence the interaction of p53 with p300, little is known about the role and regulation of other components in the p300 co-activator complex during a stress response. Here we report a new component of the p300 co-activator complex, Strap, which plays an important role in regulating p300. Strap has an unusual structure, being composed almost entirely of a tandem series of six tetratricopeptide repeat (TPR) motifs. The TPR motif functions as a protein interaction domain, and it is consistent with this property that we find that Strap harbours distinct and dedicated domains that allow it to interact with different components of the p300 complex. Thus, Strap can augment the interaction between p300 and the p300 co-factor JMY, thereby favouring the p53 response. Most importantly, we find that Strap facilitates p300 activity in response to stress treatment, which is caused in part by the stress-responsive accumulation of Strap protein. These results define a novel level of control in regulating the activity of the p300 co-activator complex, and suggest that Strap may function in maintaining p300 co-activator activity and therefore the p53 response in conditions of cellular stress.

S-9. THE CONTROL OF SISTER CHROMATID SEPARATION IN DROSOPHILA C.F. Lehner, S. Heidmann, A. Herzig, H. Ja¨ ger and O. Leismann Department of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany (E-mail: [email protected])

Sister chromatid separation during mitosis is controlled by a proteolysis pathway which requires the function of the anaphase-promoting complex/cyclosome (APC/C) and Fizzy/Cdc20. This pathway results in the removal of anaphase inhibitors which prevent premature sister chromatid separation. The Drosophila Pimples (PIM) protein functions as an anaphase inhibitor. PIM has a region with similarity to the destruction boxes (d-box) which mediate the APC/C-dependent degradation of mitotic cyclins. In addition, PIM has also a KEN-box, a second motif known to target proteins for APC/C-dependent degradation. Mutations in these regions prevent PIM destruction and sister chromatid separation during mitosis. Moreover, overexpression of wild-type PIM inhibits sister chromatid separation as well. However, PIM acts not only as an anaphase inhibitor. It has also a positive role required for sister separation. PIM binds to Three rows (THR) which is also required for sister chromatid separation. Surprisingly, these proteins have no structural similarity (except for the d- and KENboxes in PIM) to previously characterized proteins and the corresponding genes show little conservation during insect evolution. PIM and THR bind to the Drosophila Separin homolog which is the most divergent member of the separin protein family. Separin proteins have been proposed to act as proteases which cleave the Scc1 subunit of the cohesin complex, thereby dissolving sister chromatid cohesion and allowing anaphase. We will describe the phenotype resulting from mutations in the Drosophila Separin gene, as well as observations suggesting that THR might be a substrate of Separin activity.

S-10. TRANSLATING THE GENOME J. M. Mitchison I.C.A.P.B., University of Edinburgh, West Mains Rd., Edinburgh EH9 3JT, U.K. (E-mail: [email protected])

It is now possible using DNA microarrays to identify the genes which are transcribed periodically during the cell cycle of budding yeast. These are more than 10% of the genome. Other parts of the genome concerned with more specialised activities like meiosis and sporulation will not be transcribed during the normal cell cycle. However, there will be a considerable majority of the genome transcribed continuously during the cycle and responsible for the main components of growth such as many enzymes and structural elements. What is known about the pattern of transcription and translation of these growth genes? The normal assumption is exponential synthesis but most available techniques are not sufficiently sensitive to detect more subtle patterns. One approach is to measure the cell cycle pattern of the rate of total protein synthesis which will detect a divergence

Cell Biology International, Vol. 25, No. 2, 2001 from exponential. If a large majority of the genes are translated exponentially then the overall pattern of the rate will also be exponential or near to it. In fission yeast, however, the pattern is clearly not exponential, showing that the majority of growth genes and of the whole genome is cell cycle regulated. The rate of protein synthesis, as well as other bulk properties, appears to follow patterns of linear (constant rate) increase with a change of rate at the S period.

S-11. THE ROLES OF PROTEOLYSIS IN SEPARATING SISTER CHROMATIDS DURING MITOSIS AND MEIOSIS Kim Nasmyth, Frank Uhlmann, Sarah Buonomo, Attila Toth and Kirsten Rabitsch IMP, Dr. Bohr-gasse 7, A-1030 Vienna, Austria (E-mail: [email protected])

In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase. This ‘sister chromatid cohesion’ is essential for the alignment of chromosomes on the mitotic spindle during metaphase. Cohesion depends on a multisubunit protein complex called cohesin, which possibly forms the physical bridges that connect sisters. Proteolytic cleavage of cohesin’s Scc1 subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin, which is a cysteine protease related to caspases. Cleavage of Scc1 in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles. It has been proposed but never proven that cohesion between sister chromatids distal to chiasmata is responsible for holding homologous chromosomes together while spindles attempt to pull them towards opposite poles during metaphase of meiosis I. Meanwhile, the mechanism by which disjunction of homologues is triggered at the onset of anaphase I has remained a complete mystery. In yeast, cohesion between sister chromatid arms during meiosis depends on a meiosisspecific cohesin subunit called Rec8 which replaces Scc1. Cleavage of Rec8 by separin at one of two different sites is necessary for the resolution of chiasmata and the disjunction of homologous chromosomes during meiosis. The orderly reduction in chromosome number that occurs during meiosis depends on two crucial aspects of chromosome behaviour that are specific to the first meiotic division. These are the retention of cohesion between sister centromeres and their attachment to microtubules that extend to the same pole (monopolar attachment). We have identified a centromere associated protein called Mam1 which is essential for preventing bipolar attachment during meiosis I. We also show that the meiosis-specific cohesin, Rec8, is essential for maintaining cohesion between sister centromeres but not for monopolar attachment. We conclude that monopolar attachment of sister kinetochores during meiosis I requires a meiosis-specific protein and is independent of the process that protects sister centromere cohesion. Future work must address the mechanism by which Mam1 co-orients sister kinetochores and the mechanism that protects Rec8 in the vicinity of centromeres from separin at the first meiotic division.

S-12. PROTEIN KINASES REGULATING MITOSIS E. A. Nigg1, L. Arnaud2, P. Duncan1, O. Kelm1, V. Stucke1 and H. H. W. Sillje´ 1 1 Max-Planck-Institute for Biochemistry, Dept. of Cell Biology, Am Klopferspitz 18a, D-82152 Martinsried, Germany (E-mail: [email protected]); 2present address: Fred Hutchinson Cancer Research Center, Seattle, WA, U.S.A.

The principal purpose of mitosis is the faithful segregation of duplicated chromosomes to daughter cells. The error-free execution of this process is of fundamental importance to the viability of all cells and organisms, and it has long been recognized that mis-segregation of chromosomes (aneuploidy) is important for human diseases, including birth defects and cancer. Progression through mitosis is controlled by two posttranslational mechanisms, protein phosphorylation and protein degradation. The most prominent mitotic kinase is the

Cell Biology International, Vol. 25, No. 2, 2001 cyclin-dependent kinase 1 (Cdk1), the founding member of the Cdk family of cell cycle regulators. However, recent studies have brought to light additional mitotic kinases. These include members of the Polofamily (Plks), the Aurora family, the NIMA/Nek family, as well as kinases implicated in mitotic checkpoints. These kinases are thought to cooperate with Cdk1 in the regulation of mitosis and cytokinesis. Our laboratory presently focuses on the characterization of vertebrate Polo, Aurora, NIMA/Nek family members as well as spindle checkpoint kinases. Recent findings on the function and regulation of vertebrate Plks will be discussed. In addition, our laboratory also became interested in a novel kinase family, the Tousled-like kinases (Tlks). These enzymes are named after a kinase (Tousled) first described in Arabidopsis thaliana, where mutations in Tousled cause abnormal flower and leave development. Although animals do not make flowers, Tlks are highly conserved in multicellular organisms. To explore the molecular functions of these kinases, we have recently begun to characterize two Tousled-like kinases (Tlk1 and Tlk2) in human cells. Our recent results suggest that human Tlks positively regulate chromatin assembly during DNA replication, and they provide a plausible explanation for the pleiotropic developmental defects of plant Tousled mutants.

S-13. CONTROLLING S-PHASE IN THE CELL CYCLE Zoi Lygerou, Hideo Nishitani and Paul Nurse Cell Cycle Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K. (E-mail: [email protected])

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only upon exit from mitosis, ensuring that S-phase occurs once every cell cycle. This licensing control is thought to require Cdc6 (Cdc18 in fission yeast) dependent chromatin association of the MCM proteins. The control is over-ridden in fission yeast by over-expressing Cdc18 which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated and we have used a re-replication assay to identify Cdt1 as one such factor. Cdt1 co-operates with Cdc18 to promote DNA replication, physically interacts with Cdc18, is nuclear located, and peaks in level as cells exit mitosis and proceed to S-phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at mitotic exit and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants, suggesting that the co-operation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.

S-14. CELL CYCLE CONTROL, PROTEOLYSIS, AND GENETIC INSTABILITY Charles Spruck*, Heimo Strohmaier*, Susanna Ekholm†*, Kwang-Ai Won*, Anders Zetterberg† and Steven I. Reed* The Scripps Research Institute, La Jolla, CA, 92037 U.S.A. (E-mail: [email protected]); †Karolinska Institute, Stockholm, Sweden

Elevated cyclin E levels have been associated with a variety of human malignancies. Furthermore, a correlation between elevated cyclin E and aggressive disease has been established, suggesting a link to genetic instability. In order to investigate the molecular basis for these observations, we constructed cell lines capable of regulated cyclin E overexpression via the tetracycline-regulated transactivation system. In each experiment where cyclin E was expressed constitutively over a period of time, significant levels of aneuploidy were observed. Thus, it is likely that elevated expression of cyclin E promotes maligancy by causing cells to become aneuploid. Increases in chromosome loss are expected to accelerate the rate of loss of heterozygosity at tumor suppressor loci. In terms of mechanism, we have found that persistent cyclin E expression causes impairment of DNA replication. Using flow cytometry and deconvolution microscopy, it is clear that there is a severe disorganization of the replication machinery under these conditions, most likely leading to an increase in chromatid non-disjunction events. Elevated cyclin E in the context of malignancy results a failure to target cyclin E for ubiquitin mediated proteolysis. We have shown that targeting of cyclin E depends on phosphorylation followed by ubiquitination via a protein ubiquitin ligase known as SCF. In yeast,

A5 the relevant SCF isoform is SCFCdc4. We have identified a comparable activity in mammalian cells and are investigating its possible dysregulation in tumors where cyclin E is elevated. Another mechanism for hyperactivation of cyclin E associated kinase activity associated with aggressive malignant disease is downregulation of the Cdk inhibitor p27. p27 is targeted for ubiquitin mediated proteolysis by a protein ubiquitin ligase known as SCFSkp2. We have found that a small protein known as p9Cks1 is a rate-limiting factor in SCF-mediated ubiquitination of p27 and are currently determining whether this protein therefore has a role in oncogenesis.

S-15. CONTROL AND SENSITIVITY OF THE G2/M TRANSITION IN VERTEBRATES Conly L. Rieder and Richard W. Cole Div. of Molecular Medicine, Wadsworth Center, N.Y. State Dept. of Health, Albany, N.Y. 12201-0509 (E-mail: [email protected] or [email protected])

G2 ends when cells pass a ‘point of no return’ which commits them to the division process. In some cells this point occurs prior to visible signs of chromosome condensation but in others, especially those with large chromosomes (e.g., PtK1), it occurs well after condensation is evident (Rieder and Cole, 1998). As a result, irradiating these latter cells in the nucleus with a laser during early prophase induces the chromosomes to decondense and returns the cell to G2. In contrast, when similarly irradiated during late prophase (i.e., after the commitment point) progression into mitosis is not impeded even in the presence of broken chromosomes. The commitment to mitosis likely occurs when CDK1 is suddenly and rapidly activated in the nucleus (which happens in PtK1 during late prophase) (Hagting et al., 1999) after which the ability to downregulate CDK1 in response to DNA damage is lost. In PtK1 the early stages of chromosome condensation represent a visible cue that the cell is leaving G2, and the reversion of this process means that the cell cycle has been arrested. This ability to determine in vivo when a cell exiting g2, as well as when progress towards mitosis has been impeded, provides a unique approach for studying how the G2/M transition is regulated. We find that excessive illumination during video LM induces early prophase cells to revert in the absence of detectable DNA double strand breaks, and caffeine does not inhibit this reversion. Surprisingly when the illumination is reduced these cells remain arrested in G2 for days—while adjacent cells exposed to the same radiation enter and complete mitosis (Rieder and Cole, in press). This enhanced radiationsensitivity of cells in the terminal stage of G2 likely has important therapeutic implications. We have also found that when suddenly exposed to drugs (nocodazole, colcemid) that disrupt microtubules, early prophase cells return to G2 where they remain for 4–8 h prior to finally progressing into a C-mitosis (Rieder and Cole, 2000). This transient arrest at the G2/M boundary produces a characteristic ‘lag’ in the mitotic index which is not seen in response to drugs that stabilize microtubules or disrupt actin, and it is not eliminated by pre-treating cultures with caffeine. The recent demonstration that this nocodazole-induced lag is absent in many tumor cells, and requires the Chfr protein (Scolnick and Halazonetis, 2000) reveals that the G2/M transition in vertebrates is guarded by a non-DNA damage checkpoint. This pathway monitors the integrity of the cytoplasmic microtubule complex and, when triggered, arrests the cell cycle without using the ATM kinase. We are currently evaluating the hypothesis that the G2/M transition is inhibited by agents that disrupt DNA topoisomerase II without inducing DNA damage, and that this inhibition requires the ATM kinase (Downes et al., 1994).

References R CL, C RW, 1998. J Cell Biol 142: 1013–1022. H A, et al., 1999. Current Biology 9: 680–689. R CL, C RW, in press. CSH Symp Quant Biol 65 (in press). R CL, C RW, 2000. Current Biology 10: 1067–1070. S DM, H TD, 2000. Nature 406: 430–435. D, et al., 1994. Nature 372: 467–470.

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S-16. GROWTH CONTROL, CELL CYCLE CONTROL AND THE RESTRICTION POINT Anders Zetterberg, Susanna Ekholm, Fredrik Erlandsson and Peter Zickert Karolinska Institutet, Cancer Center Karolinska, R8:04, 171 76 Stockholm, Sweden (E-mail: [email protected])

Growth control (growth in cell size) on the one hand and cell cycle control on the other hand reflect two separate aspects of cell proliferation. Growth control represents the interaction between the cell and its environment, while cell cycle control reflects the coordination between cell size, DNA replication and cell division (checkpoint control). In multicellular organisms, growth is controlled both by mitogenic signals in the local microenvironment, such as components of the extracellular matrix or polypeptide growth factors, and by ‘long-range’ signals such as steroid or peptide hormones. These signals are transduced into the cell by a number of different interacting pathways, converging upon several key regulatory molecules that form a link between growth control and cell cycle control. The neoplastic process involves defects in both growth control and in cell cycle control. Defects in growth control lead to accumulation of cells through uncontrolled proliferation, whereas defects in checkpoint control lead to genetic instability seen in the tumor cells as aneuploidy, chromosomal rearrangements and, ultimately, tumor progression. One fundamental control point in the cell cycle is the restriction point, R, that operates stringently in normal cells but is defective in tumor cells. It is defined as the point in G1 in the cell cycle, after which the cell can complete a division cycle in the absence of growth factors. In cycling cells R divides G1 into two physiologically different intervals, a post-mitotic interval of G1 (G1 pm) of constant length (3–4 h) from which the cells rapidly exit to Go in the absence of growth factors, and a pre S-phase interval of G1 (G1 ps) of variable length (1–10 h) from which the cells can enter S in the absence of growth factors. The molecular mechanism underlying the control of passage through R is still unclear. We have studied physiological and molecular aspects of R in individual cells of unperturbed asynchronously growing populations. The cell cycle position was determined with time-lapse videomicroscopy analysis. Cell growth was measured as protein content per cell, determined cytophotometrically after staining with naphthol yellow S. Cell cycle specific proteins like cyclin E and cyclin A were determined semiquantitatively by immuno-cytochemistry. We found that passage through R is not followed by entry into S after a set time interval. Some cells enter S shortly (within 1 h) after passage through R, while other cells remain in G1 (in G1 ps) for many hours (up to 10 h) after passage through R before entering into S. Although progression through G1ps can occur in the absence of growth factors, this process is dependent on the attachment of the cell to the solid support throughout the whole of G1. Furthermore, passage through R seemed to be a prerequisite for growth in cell size. Finally it was found that G1 pm cells were negative for cyclin E. Only cells that had passed R (G1 ps cells) were found to express cyclin E, but at variable times after R (2–5 h before entry into S phase). These data show that passage through R is not dependent on the accumulation of cyclin E, but suggest that passage through R is a prerequisite for cyclin E accumulation. The relationship between R and Rb-phosphorylation will be discussed.

SECTION 2 (ORAL & POSTER PRESENTATIONS) 1. ROLE OF JUNB IN CELL CYCLE PROGRESSION S. Andrecht, A. Kolbus, P. Angel and M. Schorpp-Kistner Deutsches Krebsforschungszentrum (DKFZ), Div. Signal Transduction and Growth Control, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany (E-mail: [email protected])

The transcription factor AP-1, consisting of members of the Fos, Jun and ATF protein families, constitutes a major target for mitogen and

stress activated signal transduction pathways. AP-1 is critically involved in the regulation of cell proliferation, differentiation, transformation and apoptosis. To address the role of the AP-1 member JunB in vivo, we ablated JunB function in mice that resulted in embryonic lethality due to a placentation failure (Schorpp-Kistner et al., 1999). In order to assess the function of JunB in cell proliferation and transformation we isolated junB
/
fibroblasts from E9.5 mutant embryos (Szabowski et al., 2000). In contrast to c-jun
/
fibroblasts which exhibit a severe proliferation defect (Schreiber et al., 1999), both primary and immortalised junB
/
fibroblasts did not show any obvious alteration in cell growth. However, detailed analysis revealed substantial changes during cell cycle progression. Most strikingly, the number of S-phase cells is greatly increased whereas the typical G2/M population is diminished in junB
/
fibroblasts. Analysis of cyclins, relevant kinases and their inhibitors showed an accelerated transition of JunB-deficient fibroblasts from G1- to S-phase and a delay from S to G2/M. Deregulated cyclin D1-CDK4/ CDK6 kinase activity, decreased p16INK4 mRNA and protein levels and prolonged transcriptional activation of c-jun and its target genes could be identified and apparently cooperate to accelerate the transition from G1- to S-phase. The delay in the S- to G2/M-transition is caused by a deregulated cyclin A-CDK2 and cyclin B-CDC2 kinase activity. Detailed analysis revealed that the inducibility of cyclin A mRNA and protein expression is diminished and delayed in its kinetics. Evidence will be given that cyclin A promoter activity is potentiated by JunB. To causally relate the regulatory function of JunB to the phenotype of the junB
/
fibroblasts described above we reintroduced a posttranslationally inducible JunB-ER protein. Upon activation of this construct a normal cell cycle profile was restored since the changes on the molecular level could be reverted to a major extend. This ex vivo approach together with previous work (Passegue and Wagner, 2000) identifies a critical function for JunB in normal cell proliferation which cannot be complemented by other AP-1 dimeric complexes.

References S-K M, W Z-Q, A P, W EF, 1999. EMBO J 18: 934–948. S M, K A, P F, S A, MS U, T J, K M, A P, W EF, 1999. Genes Dev 13: 607–619. P E, W EF, 2000. EMBO J 19: 2969–2979. S A, M-S N, A S, K A, S-K M, F NE, A P, 2000. Cell 103, in press. 2. THE CELL CYCLE INHIBITOR p16/Ink4a SENSITIZES LYMPHOBLASTIC LEUKEMIA CELLS TO APOPTOSIS BY PHYSIOLOGIC GLUCOCORTICOID LEVELS Michael J. Ausserlechner1, Petra Obexer1, G. Jan Wiegers1, Bernd L. Hartmann1, Stephan Geley1 and Reinhard Kofler1,2 1 Institute for General and Experimental Pathology, Division of Molecular Pathophysiology, University of Innsbruck, Medical School, Austria; 2Tyrolean Cancer Research Institute, Innsbruck, Austria (E-mail: [email protected])

The cyclin-dependent kinase (CDK) inhibitor p16INK4A is frequently inactivated in childhood T-cell acute lymphoblastic leukemia (T-ALL). To investigate possible consequences of this genetic alteration for tumor development, we conditionally expressed p16INK4A in the T-ALL line, CCRF-CEM, which carries a homozygous deletion of this gene. In agreement with its reported function, p16INK4A expression was associated with hypophosphorylation of the retinoblastoma protein pRB and stable cell cycle arrest in G0/G1, documenting that the pRB/E2F pathway is functional in these cells. Unexpectedly, p16INK4A expression increased the sensitivity threshold for glucocorticoid (GC)induced apoptosis from therapeutic to physiologic levels. As a possible

Cell Biology International, Vol. 25, No. 2, 2001 explanation for this phenomenon, we found that p16INK4A-arrested cells had elevated GC receptor (GR) expression associated with enhanced GC-mediated transcriptional activity and increased responsiveness of the GC-regulated cyclin D3 gene. These data are supported by our previous findings that GR levels critically influence GC sensitivity, and imply that p16INK4A inactivation, in addition to allowing unrestricted proliferation, represents a mechanism by which lymphoid tumor cells might escape cell death triggered by endogenous GC. Supported by the Austrian Science Fund (F204).

3. REPLICATION TIMING AND HERITABLE GENE REGULATION V. Azuara, K. E. Brown, M. Merkenschlager and A. G. Fisher Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN, U.K. (E-mail: [email protected])

During embryogenesis, differentiated cells develop from omnipotent precursors through a series of intermediates with increasingly restricted lineage potentials. This progressive pathway is likely reflected by changes in gene expression. It is, however, not clear how patterns of gene expression that characterise differentiated cells are initially established nor how they are maintained through DNA replication and cell division. Several processes have been implicated in heritable gene expression patterns such as DNA/histone modification (by methylation or acetylation) or through the interaction of specific proteins to chromatin (e.g Polycomb and Trithorax group proteins). Other works suggest that the maintenance of cellular memory may involve the dynamic repositioning of repressed/active genes to distinct compartments within the nucleus of dividing cells. It has also been proposed that replication timing may serve as an epigenetic factor in heritable gene regulation based of the general correlation that expressed genes are replicated early in S-phase whereas silent genes are replicated late. We are currently studying the replication timing of a panel of tissue-and developmental stage specific genes in cells at distinct stages of haematopoietic development or earlier in development using ES cells. Replication timing has been evaluated by using either FISHbased ‘spot counting’ analysis or by adapting a PCR-based assay as previously described (Cell Vol. 73, 1403-1409, July 2, 1993). This latter approach directly examines replicated sequences that have been labelled with BrdU within a given stage of the cell cycle (i.e. G1, four windows of S-phase and G2/M). Our results suggest that several genes destined for heritable repression in mature B/T cells following differentiation (e.g. 5, Rag and TdT) are recruited to heterochromatic regions in the nucleus but do not undergo a change in their replication timing (from early to late) as scored by the PCR-based analysis. The FISH data on other hand may indicate that the repressed genes display retarded chromatidseparation during S-phase. Such apparently discordant data between the two different methods have previously been reported for some loci (e.g. early replication by PCR but late replication by FISH of the XIST locus in human). Such results may be significant for understanding how gene inactivity could be regulated and maintained.

4. MEASURING TRANSCRIPTIONAL REPRESSION OF CDK1 AND OTHER GENES WITH CDE AND CHR PROMOTER ELEMENTS DURING DNA DAMAGE-INDUCED G2/M ARREST IN HUMAN CELLS C. Badie1, J. E. Itzhaki2, M. J. Sullivan1, A. J. Carpenter1 and A. C. G. Porter1 1 Gene Targeting Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K. (E-mail: [email protected]); 2The Wellcome Trust, London NW1 2BE, U.K.

CDK1 is transcriptionally downregulated in response to DNA damage. This response, which is associated with G2 arrest, can be sustained

A7 for long periods (a week or more) and is p53-dependent. We have shown (Badie et al. (2000) Mol. Cell Biol. 20: 2358–2366) that this response is also dependent on CDE and CHR (Cell-cycle Dependent Element/Cell-cycle Homologous Region) sequences, cis-acting elements in the CDK1 promoter. Several other genes that, like CDK1, are maximally expressed in G2 and involved in the entry into, and progression through, mitosis (e.g. Topoisomerase II , CENPA, Cyclin B1, Cyclin A2) are also transcriptionally downregulated after DNA damage and also carry CDE/CHR element in their promoter regions. Although p53-independent mechanisms of G2 arrest are well known, it appears that transcriptional repression is required for sustained G2 arrest in order to avoid catastrophic mitosis and carcinogenesis. To date, such transcriptional repression has been measured mostly by RT-PCR in asynchronous cell populations. In order to better characterise and quantify the transcriptional repression of CDK1 and Topoisomerse II , we have linked their promoter sequences to a d2EGFP (enhanced green fluorescent protein with a reduced half-life) reporter. In this way flow cytometric measurements of EGFP and DNA content can be used to measure the amount of transcription at various stages of the cell cycle and how this changes after DNA damage and other stimuli or is affected by promoter (or other) mutations. We will describe our progress in developing and applying this new approach.

5. INDUCTION OF CYCLIN E-CDK2 KINASE ACTIVITY, E2F-DEPENDENT TRANSCRIPTION AND CELL GROWTH BY MYC ARE GENETICALLY SEPARABLE EVENTS Rudolf Beier, Andrea Bu¨ rgin, Astrid Kiermaier, Matthew Fero1, Holger Karsunky2, Rainer Saffrich3, Tarik Mo¨ ro¨ y2, Wilhelm Ansorge3, Jim Roberts1 and Martin Eilers Institute of Molecular Biology and Tumour Research, Emil-Mannkopff-Str. 2, 35033 Marburg, Germany (E-mail: [email protected], [email protected]); 1 Division of Basic Sciences, Howard Hughes Medical Institute, Fred Hutchison Cancer Research Center, Seattle, U.S.A.; 2Institute for Cell Biology, University of Essen, Virchowstrasse 173, 45122 Essen, Germany; 3Biological Instrumentation Programme, EMBL, Meyerhofstr. 169112 Heidelberg, Germany

The proto-oncogene c-Myc has been implicated in at least three distinct genetic pathways controlling the progression through the G1 phase of the cell cycle: induction of cyclin E/cdk2 kinase activity with sequestration of the cki p27kip1 from cdk2, activation of E2F-family transcription factors, and cell growth, i.e. increase in cell mass. These effector pathways were investigated by inducing c-Myc in mouse embryo fibroblasts (MEFs) with deregulated p27- (knockout) and E2F2- (constitutively) expression. When p27
/
MEFs and p27 +/+ wildtype MEFs were infected with c-Myc- or Myc-ER- expressing retroviruses, they did not show differences regarding colony formation, apoptosis, growth rate and S-phase progression. However, induction of cyclinE/cdk2 kinase activity showed to be dependent on p27 since it could not be induced by Myc in p27
/
cells. In these cells, after starvation in low serum, cyclinE/ cdk2 was found in complexes with the pocket protein p130, which had been shown to be able to act as kinase inhibitor. The p130/cdk2/ cyclinE complexes were not sensitive to the activation of c-Myc. In p27 +/+ cells those complexes were not detectable until very late after activation of Myc. When MycER was activated in p27
/
cells, cyclin D2 and E2F2 proteins, both direct targets of Myc, were still induced. Transcription of E2F2-target genes as assayed by RT-PCR and cell growth (assayed by FACS forward scatter profiles) was not inhibited compared with the wildtype cells. Deregulation of either cyclin D2 or E2F2 expression by retroviral infection of p27
/
MycER cells could only partially induce cell cycle progression. E2F2 clearly upregulated cyclin A-expression and increased the percentage of cells in S-phase about twofold. The ability of Myc to stimulate cell growth, however, was still unaffected by the deregulation of E2F2 and cyclin D2. Therefore the induction of cyclin E/cdk2 activity together with the sequestration of p27, the induction of

A8 E2F-dependent transcription and cell growth show to be three distinct effector pathways of Myc-signalling.

Reference B, et al., 2000. EMBO J 19: 5813–5823. 6. STRUCTURAL AND FUNCTIONAL ANALYSIS OF A NEWLY IDENTIFIED U5 SPLICEOSOME SUBUNIT P. Bello1, F. Simeoni1, A. Davy1, C. Gondeau1, K. Hopfner2, J. Tainer2 and G. Divita1,2 1 CRBM, CNRS UPR-1086, 1919 route de Mende, 34293 Montpellier, France (E-mail: [email protected]); 2 Scripps Research Institute, 10550 North Torrey Pines, La Jolla, California 92037, U.S.A.

The excision of introns from eukaryotic pre mRNAs to produce mature, translatable mRNA is governed principally by the spliceosome. This heteromeric complex comprises of at least 50 proteins and five small nuclear RNAs (U1, U2, U4/U6 and U5 snRNAs) that act in a co-ordinated fashion to recognize the intron-exon boundaries and to catalytically remove the intron. Aberrant splicing events have in some cases been implicated during the development of cancerous tumors, for example, breast and colon cancers and in Acute Lyphocytic Leukemia. Recently, a direct association between a cyclin/cdk and spliceosome subunit has been demonstrated although the physiological consequences and/or importance remains unknown. The U5 snRNP is the most complex of the snRNP subunits and its importance in both the human and yeast spliceosomes is well documented. We recently identified a novel human U5p15-like protein which we have designated U5p17 based on its strong homology with the former and with the Dim yeast orthologues. With the view of understanding the importance of this spliceosome component, we have (i) expressed and purified recombinant U5p17 to homogeneity and determined its crystal structure, (ii) identified potential interacting partners using the two hybrid system and (iii) have also performed in vivo studies on endogenous and exogenous expressed U5p17 (both wild type and mutant forms) for cellular expression, localization and effects.

7. CYTOPLASMIC ANCHORING OF TSP53 AND REGULATION OF GROWTH IN SUBCLONES OF THE C6 RAT GLIOMA CELL LINE O. Klotzsche, W. Deppert and W. Bohn Department of Tumor Virology, Heinrich-Pette-Institute for Experimental Virology and Immunology at Hamburg University, D-20251 Hamburg, Martinistr. 52, Germany (E-mail: [email protected])

Cytoplasmic sequestration of p53, a phenomenon found in differentiating tissue and in certain types of tumors, is considered as a mechanism of epigentic inactivation of the tumor suppressor protein. Subclones of the C6 rat glioma cell line were transfected with a temperature sensitive mutant p53 (tsp53val135) and used as an experimental system to determine the relationship between cytoplasmic anchoring of p53 and regulation of growth in the absence of genotoxic stress. Cytoplasmic sequestration of tsp53 in C6 cells was shown to depend on the presence of a proper intermediate filament scaffold composed of vimentin. While cells expressing the endogenous rat vimentin (vim+) anchored p53 to the cytoplasm at the non-permissive temperature, vimentin deficient cells (vim
) harboured p53 in the nucleus. Tsp53 localized to the cytoplasm again, when vim
cells had been restored to express a mouse vimentin protein. When shifted to the permissive temperature tsp53 adopted the wild type conformation, transiently accumulated in the nucleus in vimentin expressing cells, and subsequently relocated to the cytoplasm.Correspondingly, the cells shortly arrested in G1, but then resumed growth. In contrast, vim
cells, which were not able to sequester tsp53, stayed in this growth arrest and adopted a senescence like phenotype. Thus vimentin can function as a cytoplasmic anchor for p53. This anchoring of a functional p53 is a decisive step in blocking growth suppressive functions in unstressed cells, which otherwise would activate signalling pathways involved in lifespan regulation. Supported by Deutsche Krebshilfe.

Cell Biology International, Vol. 25, No. 2, 2001

8. CELL CYCLE POSITIONING FOR CELLS UNDERGOING APOPTOSIS BY MEANS OF DUAL PARAMETER FLOW CYTOMETRY E. Boiartchouk1 and V. Zenin2 1 Department of Cytology & Histology, Faculty of Biology & Soil Science, St.-Petersburg State University, St.-Petersburg 199034 Russia (E-mail: [email protected]); 2Institute of Cytology, Russia Academy of Science, St-Petersburg, 194064 Russia (E-mail: [email protected])

Flow cytometry remains a practically unique method allowing both cell cycle analysis and apoptosis detection. We used dual parameter flow cytometry (light scatter/ DNA content) for detection of apoptosis in cells pretreated with saponin and stained with propidium iodide (PI). Cell shrinkage resulting in decrease of forward light scatter signal was shown to be usual for apoptosis. Contrary to previous observations our specific pretreatment of cells elaborated in this work led to the increase both of side light scatter (SLS) and forward light scatter (FLS) signals in all studied cells (human lymphoblastoid GM130, 2151, 2139 and monoblastoid U937) and all kinds of inductors (etoposide (VP-16), staurosporine, nocodazole, campthtecin, or -irradiation). Morphological analysis and DNA fragmentation demonstrated by DNA-comet assay and gel electrophoresis proved that apoptosis do occur in these cells. Cell sorting of the FLS-increased subpopulation revealed cells with nuclear fragmentation. Suggested pretreatment of cell makes it possible to discriminate apoptotic cells simply using light scattering parameters without any need for staining with special dyes thus enabling the correct analysis of the DNA distribution in viable and apoptotic cells separately. This kind of analysis showed that the subG1-peak, which is one of the most prevailing characteristic of apoptotic changes, consists mostly of cells undergoing apoptosis in G1-phase. Cells undergoing apoptosis in other phases are located in the 2c-4c region of DNA histogram and disguised as viable cells. In standard cell cycle analysis these apoptotic cells used to be regarded as nonapoptotic ones that leads to erroneous calculations. The analysis of cell cycle distribution based on the overall and differential histograms gave quite diverse esteems percents of cells in different phases. VP-16-treated GM130 cells (0.5 ug/ml) showed the true G2/M arrest only after 24 h of incubation but longer treatment resulted in a decrease of the percent of G2/M cells and at the same time in a significant increase of the cell number with 4c DNA content in the apoptotic subpopulation. The increase of the S-phase cells percent observed with univariate analysis also was connected with the increase of the cell number with S-phase like DNA content whereas the percent of nonapoptotic cells progressively dropped down. Thus, the discrimination of apoptotic cells as an FLS-increased subpopulation can be used for correct and detailed quantitation using simple PI staining and makes it possible to avoid false assignment of cells in univariate DNA analysis.

9. EFFECTS OF FORMATE ON PROTEIN AND DNA DISTRIBUTIONS IN SACCHAROMYCES CEREVISIAE L. Brambilla, D. Porro and L. Alberghina Dip. di Biotecnologie e Bioscienze, Universita` degli Studi di Milano—Bicocca, p.zza della Scienza 2, 20126 Milano, Italy (E-mail: [email protected])

Control of both metabolism and cell cycle progression by the cellular environment has a key role in the regulation of growth and cell proliferation in all organisms. Saccharomyces cerevisiae is an eukaryotic microorganism widely used as model to study both control of metabolism and cell cycle progression. Formate is an energy source for S. cerevisiae. The addition of formate to glucose-limited continuous cultures of budding yeast allowed us to modulate the metabolic state of the cells, making cells to respire or to produce ethanol (i.e. respiro-fermentative metabolism), depending on the formate flux rate. In this respect, the control of the amount of energy source used from the yeast cells allows an easy modulation of the critical dilution rate value at which cells start to produce ethanol. We acquired samples from population continuously grown in many different conditions. The analysis by flow cytometry of the protein distribution of such cells showed that the average dimension of the

Cell Biology International, Vol. 25, No. 2, 2001 cells remains constant during the growth under oxidative conditions, while it sharply and continuously increases during the production/ accumulation of ethanol. We also developed a double staining flow cytometric technique, that yields biparametric distributions of protein and DNA content of a given yeast population. From the analysis of such distributions we could determine the values of Ps (i.e., the protein threshold required to enter the S phase) and Pd (i.e., the protein content of the cells at division) for each population. Alterations in the metabolic state of the cells, induced by formate addition, resulted in a strong modulation of these values. Finally, formate seems to control to a certain extent the partitioning of cells in the G1 and G2 +M phases. We observed small but significant variations in the DNA distributions of cells growing at a fixed specific growth rate, depending on the formate flux rate.

10. REGULATION OF AN RPD3-RELATED HDAC DURING THE CELL CYCLE OF PHYSARUM POLYCEPHALUM Eva-Maria Brandtner, Thomas Lechner, Alexandra Lusser and Peter Loidl Department of Microbiology, Medical School, University of Innsbruck; Fritz-Pregl Str. 3, A-6020 Innsbruck, Austria (E-mail: [email protected])

The dynamic state of acetylation of core histones, the basic protein components of eukaryotic chromatin, is maintained by histone acetyltransferases (HAT) and histone deacetylases (HDAC). HATs and HDACs exist in multiple distinct complexes and their activities, intracellular location and substrate specificities are regulated in a complex, cell cycle related manner. Macroplasmodia of the myxomycete Physarum polycephalum offer the advantage of a naturally synchronous cell cycle. We have previously shown that Physarum polycephalum has two HDAC activities (HD1, HD2) and five distinct HAT forms (HAT-B, HAT-A1–HAT-A4). All of them exhibited changing activity patterns dependent on the cell cycle stage. HD1- and HD2-activity fluctuated in a biphasic pattern with two maxima in early S-phase and mid G2-period, respectively. Here we report on the cloning of a Physarum HDAC (PpHDAC1) related to the Saccharomyces cerevisiae Rpd3 protein. The expression pattern of PpHDAC1 mRNA was analysed at different time points of the cell cycle. Furthermore, macroplasmodia were treated with the HDAC inhibitor trichostatin A at several cell cycle stages and the effects on cell cycle progression and expression of PpHDAC1 are described.

11. KINETICS OF TELOMERE LENGTH REGULATION IN SACCHAROMYCES CEREVISIAE Vanessa Brevet, Stephane Marcand* and Eric Gilson Ecole Normale Supe´ rieure de Lyon, CNRS/ENSL UMR 49, 69364 Lyon cedes 07, France; *Present address: CEA/ Saclay, 91191 Gif sur Yvette Cedex, France (E-mail: [email protected])

Telomeres are the extremity of eucaryotic chromosomes. They are responsible for genomic integrity and are necessary for the complete replication of DNA. In Saccharomyces cerevisiae, telomere length is maintained constant through a well-defined number of RAP1 molecules bound to the telomere. Telomeric repeats inserted in the vicinity of a telomere are taken into account by the length regulation mechanism. We took advantage of this to develop an experimental system to follow the elongation of a single yeast telomere. Two specific sites for the FLP1p recombinase were inserted between the internal and distal telomeric repeats and more internally downstream of a marker gene. Recombination by FLP1p deletes the internal telomeric repeats, leaving an abnormally short telomere of approximately 100 bp. This allowed us to follow the kinetics of elongation of a telomere in a population of yeast cells. We observed that telomere elongation is restricted to a few base pairs per generation and that this rate decreases progressively with increasing telomere length. By contrast, in absence of telomerase or in presence of an over-elongated telomere, the degradation rate due to the succession of generations appears to be constant, that is independent of telomere

A9 length. Together, these results indicate that telomerase is gradually inhibited at its site of action by the elongating telomere. We also followed telomere elongation at different stages of the cell cycle. We observed a lack of telomere elongation in stationary phase and when cells were blocked in G1 and M. In a synchronous population of cells, telomere elongation can be detected in cells progressing through S-phase. Furthermore, it appeared that telomere elongation was far more efficient when the telomere was on a replicating mini-chromosome, compared to a non-replicating one. The implications of this findings for the coupling between telomere elongation and DNA replication will be discussed. In addition, we are currently investigating the mechanisms of telomere length regulation occurring in the absence of tel1, a gene with a putative PI3-kinase domain which is homologous to the human gene ATM (mutated in Ataxia Telangectiasia). In the absence of tel1, yeast telomeres are maintained to a constant length—shorter than in wild type cells—thanks to a ‘cryptic’ mechanism, still unknown, that does not imply a RAP1 counting mechanism.

12. CLONING AND CHARACTERIZATION OF A NOVEL TOBACCO MAP KINASE KINASE FOR THE CELL CYCLE-REGULATED P43NTF6 MAP KINASE O. Calderini1, N. Glab2, C. Bergounioux2, E. Heberle-Bors1 and C. Wilson1 1 Institute of Microbiology and Genetics, University of Vienna, Dr Bohrgasse 9a 1030 Vienna (E-mail: [email protected]); 2Laboratoire Cycle Cellulaire et Recombinaison, Institut de Biotechnologie des Plantes, CNRS UMR 8618, Universite de Paris-Sud Bat. 630, Plateau du Moulon, F-91405 Orsay Cedex, France

Eukaryotic cells respond to a variety of extracellular stimuli via activation of specific MAP kinase cascades. MAPKs are usually phosporylated by upstream kinases (MEKs) which are in turn activated by a MEK kinase (MAPKKK). Signalling into this cascade can occur via tyrosine kinase receptors, G-protein linked receptors and protein kinase-C dependent pathways. once activated the MAP kinase mediates the signal to a cellular response by phosphorylating nuclear targets, as transcription factors, or cytosolic proteins, as cytoskeletal components, other protein kinases etc. a tobacco MAP kinase, p43Ntf6, with a possible role in cell plate formation and whose activity is peaking late in mitosis has been characterized in our lab. with the aim of identifying possible partners of p43Ntf6 a tobacco BY-2 cell suspension cDNA library was screened using this MAP kinasE AS BAIT AND A CDNA encoding a protein with features characteristic of a MAP kinase kinase (MAPKK) was isolated. Physical interaction between the MAPKK (NtMEK1) and the tobacco MAP kinase p43Ntf6 was confirmed by GST pull-down experiments and in vitro kinase assay showed that NtMEK1 is able to phosphorylate p43Ntf6. Northern analyses showed that the NtMEK1 and ntf6 genes have a similar pattern of expression both in plant tissues and following the induction of cell division in leaf pieces. These data suggest that NtMEK1 is a MAPKK for the p43Ntf6 MAP kinase.

13. REGULATION OF CYCLIN D3 EXPRESSION DURING SKELETAL MUSCLE DIFFERENTIATION F. De Santa, C. Cenciarelli, A. De Toni, A. Felsani and M. Caruso CNR, Istituto Biologia Cellulare, Viale Marx, 43 00137 Roma, Italy (E-mail: [email protected])

The expression of cyclin D3, which is normally thought as a proliferative factor, is greatly induced during the terminal differentiation of skeletal myoblasts. We showed that in differentiated C2 muscle cells cyclin D3 also becomes stabilised, and is found nearly totally complexed with unphosphorylated pRb. In addition, we found that cyclin D3 binds directly proliferative factors such as PCNA, cdk2 and cdk4, and mediates the interaction of these factors with pRb; these notions suggested that in differentiating myoblasts pRb needs cyclin D3 to sequester factors required for cell cycle progression and DNA replication (Cenciarelli et al., 1999). To verify this hypothesis, we sought to generate cyclin D3 mutants deficient in their ability to bind pRb, but

A10 with good stability even in the absence of pRb. Because cyclin D3 contains a LXCXE sequence motif, located near the N-terminus of the protein, we generated a number of 5 deletion mutants, as well as a mutant carrying two aa substitutions that destroy the LXCXE motif. We also introduced a mutation in the DNA sequence encoding a potential proline-directed phosphorylation site (Thr-283), located near the C-terminus of cyclin D3, in a position conserved with cyclin D1 (Thr-286 in cyclin D1). The interaction of cycD3 with pRb was tested by means of GST-pull down experiments using in vitro-translated, labelled cyclin D3 proteins (wt or mutated) and bacterially produced GST-Rb fusion proteins. It was found that wt cyclin D3 was able to bind efficiently the C-terminal region, but not the pocket region, of pRb. The cyclin D3 LXCXE mutants displayed the same pRb-binding properties as wt cyclin D3, which indicates that the LXCXE motif is not essential for the interaction with pRb. The analysis of cyclin D3 deletion mutants revealed that the region required for binding pRb is contained within the first 70 aa residues of cyclin D3. In order to verify whether the Thr residue 283 plays a role in the regulation of cyclin D3 stability, we generated a mutant in which Thr-283 was substituted by an alanine (T283A). The half-life of wt cyclin D3 was calculated to be 30 min, whereas that of the cyclin D3 (T283A) mutant exceeded three hours, indicating that Thr-283 positively regulates cyclin D3 degradation. Furthermore, we have been able to demonstrate that stabilisation of cyclin D3 (T283A) reflects its reduced ubiquitination relative to wt cyclin D3.

Reference C C, D S F, P PL, M E, R L, B F, F A, C M, 1999. Critical Role Played by Cyclin D3 in the MyoD-Mediated Arrest of Cell Cycle during Myoblast Differentiation. Mol Cell Biol 19: 5203– 5217. 14. CYCLIN D1 IS AN EARLY TARGET GENE IN THYROID HORMONE-INDUCED HEPATOCYTE PROLIFERATION M. Pibiri, C. Cossu, G. Simbula, D. Concas, G. M. Ledda-Columbano and A. Columbano Department of Toxicology, Oncology and Molecular Pathology Unit, University of Cagliari, Italy (E-mail: [email protected])

The thyroid hormone (T3), affects cell growth, differentiation and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have analyzed changes in activation of transcription factors, mRNA levels of immediate early genes and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-B and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun and c-myc that are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration following 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment, and by a 20-fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed following treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator activated receptor , PPAR) of the same superfamily of steroid/ thyroid receptors. T3 treatment also resulted in an increased expression of cyclin E, E2F and enhanced phosphorylation of pRb and p107, the ultimate substrates in the pathway leading to transition from G1 to S phase. The results demonstrate that cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors.

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15. CYCLIN D1 IS ELEVATED DURING HUMAN COLORECTAL TUMOR PROGRESSION AND IN A VITAMIN D RECEPTOR KNOCKOUT MOUSE COLON CANCER MODEL H. S. Cross, H. Hofer, P. Bareis, E. Bajna and M. Peterlik Department of Pathophysiology, University of Vienna Medical School, Waehringerguertel 18-20, A-1090 Vienna, Austria (E-mail: [email protected])

Cyclin D1 expression is elevated during transition from G1 to S phase of the cell cycle. We were able to demonstrate that during premalignity and in low grade cancer of the human colon cyclin D1 expression is upregulated. This occurs in parallel to increasing vitamin D receptor (VDR) levels. The VDR as a steroid hormone receptor and transcription factor mediates the genomic action of the active metabolite of vitamin D, 1,25-dihydroxycholecalciferol (1,25-D3). 1,25-D3 is well known to inhibit growth in many cell types by blocking G1 and/or G2/M phase of the cell cycle. In addition it promotes differentiation and apoptosis. Interestingly, the enzyme 25-D3-1alpha-hydroxylase (1-hydroxylase) which converts the precursor 25-D3 into the active metabolite, is not only present in kidney cells, but also in epithelial cells from other organs: we have demonstrated its mRNA, protein as well as enzymatic activity in colon cancer cells. Expression of the 1-hydroxylase is elevated during colon tumor progression in parallel to that of VDR. In late stage, high grade colorectal cancer however, VDR and 1-hydroxylase expression is diminished to almost nil. We therefore suggest that the vitamin D system, during incipient hyperproliferation as indicated by high cyclin D1 expression, is activated as a physiological autocrine/paracrine defense against advancing tumor progression—this defense however eventually fails in high grade cancer. In order to show the relevance respectively to elucidate possible mechanisms to therapeutically use the vitamin D system during tumor progression, we availed ourselves of a genetically altered mouse model: the VDR knockout (KO) mouse. Characterization of wildtype (WT) as opposed to heterozygote (HT) and KO genotypes provided evidence that reduced or eliminated expression of active VDR increases expression of PCNA (proliferating cell nuclear antigen) and of cyclin D1. In parallel, a marker for oxidative stress, 8-hydroxy-2 -deoxyguanosine, is elevated. These data provide evidence that a functional vitamin D system could indeed provide a check against elevated proliferation during premalignity. We are currently investigating regulatory aspects of this defense mechanism during tumor progression.

16. REGULATION OF THE CDK INHIBITOR P27KIP1 BY THE MAP KINASE PATHWAY C. Delmas1, S. Manenti1, A. Boudjelal1, C. Peyssonnaux2, A. Eyche`ne2 and J.-M. Darbon1 1 Laboratoire de Biologie Cellulaire et Mole´ culaire du Controˆ le de la Prolife´ ration, CNRS UMR 5088, Universite´ Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex, France (E-mail: [email protected]); 2CNRS UMR 146, Institut Curie, Centre universitaire, Laboratoire 110, 91405 Orsay Cedex, France

We investigated the effect of p42/p44 MAP kinase on the expression of the CDK inhibitor p27Kip1, a regulator of the G1 phase of the cell cycle. We demonstrated that chemical inhibition of MEK leads to p27Kip1 accumulation in NIH 3T3. Inhibition of MAP kinase by expression of a dominant negative MEK also increased p27Kip1 levels. Blocking the degradation of p27Kip1 with proteasome inhibitors impaired this accumulation, suggesting that MAP kinase did not act on p27Kip1 synthesis. We also demonstrated in transient co-transfection experiments that activation of MAP kinase with a constitutively active MEK mutant decreased the expression of ectopic p27Kip1, in the absence of growth factors or cell adhesion. To confirm these data, we performed in vitro degradation of recombinant p27Kip1 by cellular extracts from control cells or from cells kept in suspension. The robust p27Kip1 degradation observed in control cells was totally absent in suspended cells, suggesting that p27Kip1 degradation is adhesiondependent. When constitutively active MEK was overexpressed in suspended cells, the p27Kip1 degradation activity was restored, indicating that the inhibition of p27Kip1 degradation in these cells is due to

Cell Biology International, Vol. 25, No. 2, 2001 MAP kinase inhibition. Immunofluorescence experiments on cells expressing constitutively active MEK confirmed the down-regulation of endogenous p27Kip1 by MAP kinase, and suggested a MAP kinase-dependent cytoplasmic translocation of the CKI. These data demonstrate that MAP kinase regulates p27Kip1 in fibroblasts, downstream of growth factors and cell adhesion signalling, through a degradation mechanism possibly involving a cytoplasmic translocation of the protein.

17. CELL CYCLE-DEPENDENT LOCALIZATION OF FISSION YEAST CDK cdc2p AND CYCLIN B cdc13p A. Decottignies1 and P. Nurse1 1 Cell Cycle Lab, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, WC2A 3PX London, U.K. (E-mail: [email protected])

In eukaryotic cycling cells, from yeast to humans, cyclin dependent kinases control the onset of both S phase and M phase. In S phase, chromosomal DNA is replicated and cohesin molecules hold the sister chromatids together. The duplicated centrosomes, which act as microtubules organising centers, then separate and nucleate microtubules between them to form the mitotic spindle in M phase. The kinetochores of sister chromatid centromeres attach to the spindle and, after the cohesion between them has been lost at the onset of anaphase, sister chromatids move apart to form two nuclei. Cytokinesis then occurs to generate two cells. In our work, we have followed the localization of fission yeast cdc2p kinase and cdc13p cyclin B in live cells using YFP-tagged proteins. We found that cdc2p-YFP and cdc13p-YFP nuclear fluorescence are correlated throughout the cell cycle and are the lowest at mitotic exit. Unlike cdc13p, cdc2p-YFP fluorescence was detected in the cytoplasm throughout the cell cycle. cdc2-YFP and cdc13-YFP accumulate on the SPB of cycling G2 cells, or cells arrested at the G2/M transition, and both proteins were present on the SPBs and the mitotic spindle in mitosis. The MT-association of CDK did not require a functional dis1p. In anaphase, the fluorescence of both fusion proteins disappeared from the mitotic spindle at the same time, immediately prior to sister centromere separation. At this point, cdc13p-YFP accumulated in the nuclear periphery before cdc13p-YFP nuclear fluorescence disappeared completely. Using cyclins B-deleted strains, we showed that cdc13p, cig1p and cig2p are each able to drive back cdc2p-YFP into the nucleus during the G1 phase. In the absence of all three cyclins however, cdc2p-YFP did not re-enter the nucleus after mitosis. In a HU block, we found that cdc2p-YFP and cdc13p-YFP accumulate on the SPB, suggesting an early function of the kinase. If cdc13p is not recognized by the APC, both cdc2p and cdc13p remain on the spindle. Finally, we have followed cdc2p-YFP localization in meiosis. We found that cdc2-YFP associates with centromeres in mating cells, karyogamy and meiotic prophase.

18. ARABIDOPSIS E2F TRANSCRIPTION FACTORS ARE ENCODED BY A MULTIGENE FAMILY WITH DISTINCT ACTIVATION PROPERTIES S. M. de Jager1, M. Menges1, U.-M. Bauer2 and J. A. H. Murray1 1 Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, U.K. (E-mail: [email protected]); 2Wellcome/CRC Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, U.K.

Mammalian E2F transcription factors are composed of E2F and DP subunits, and with their negative regulators, Rb-related proteins, govern expression of cell division related genes. Six E2Fs and two DPs are present in mammals, but only single E2F genes are known from wheat, tobacco and carrot. Here we show that E2Fs are a multigene family in Arabidopsis, and report isolation of three E2F-like clones AtE2F1-3, with 45–62% identity to wheat, tobacco and carrot E2Fs. Sequence analysis reveals AtE2F1 and AtE2F3 are closely related to previously identified plant E2Fs, whereas AtE2F2 is related to human E2F6 and Drosophila dE2F2 which are unusual in lacking transcriptional activation potential. We show that AtE2F1 and AtE2F3

A11 activate transcription in yeast cells and bind a plant Rb protein, but AtE2F2 cannot activate transcription or bind Rb. Functional distinction between AtE2F1 and AtE2F3 is indicated by dimerisation of AtE2F3, but not AtE2F1, with an Arabidopsis DP protein AtDP1. Consensus E2F binding sites were identified in promoters of several cell cycle related genes, including the D-type cyclin CycD3 and the Arabidopsis homologue of the replication origin protein CDC6. Accumulation of AtE2F1-3 was observed in partially synchronised Arabidopsis cells re-entering the cell cycle, before induction of CycD3 and CDC6 expression in late G1. AtE2F1 complexes bound to consensus E2F sequences and to the AtCDC6 promoter in vitro. We conclude Arabidopsis contains a family of functionally distinct E2F genes, most probably involved in G1 to S phase progression.

19. STRUCTURE AND FUNCTION OF AN ESSENTIAL PHOSPHORYLATION-SITE DOMAIN OF HUMAN CDC25C WHICH INTERACTS WITH BOTH 14-3-3 AND CYCLINS M. C. Morris1, J. Mery2, A. Heitz3, F. Heitz2 and G. Divita1,2 1 The Scripps Research Institute, Department of Molecular Biology, La Jolla, U.S.A. (E-mail: [email protected]); 2 CRBM, CNRS-UPR 1086, 34293 Montpellier Cedex 5, France; 3CBS, Faculte de Pharmacie, 34060 Montpellier Cedex, France

Human cdc25C is a dual-specificity phosphatase involved in the regulation of cell cycle progression in both unperturbed cells and in cells subject to DNA-damage or replication checkpoints. In this study we describe the structure-function relationship of a 51 amino acid peptide designated MP51, derived from an essential domain of human cdc25C phosphatase which interacts with 14-3-3 proteins. In vivo, differential phosphorylation of this domain regulates both the induction of mitotic processes, and the checkpoint arrest of eukaryotic cells in response to DNA damage. By combining different spectroscopic and biochemical approaches we have investigated the mechanism of regulation of cdc25C by 14-3-3. We have demonstrated that this domain of cdc25C is a bi-functional motif which interacts with 14-3-3 on the one hand, and with human cyclins A and B on the other hand, with a similar high affinity, both in vitro and in vivo. As observed for full length cdc25C, the binding of this peptide to 14-3-3 is strongly increased when MP51 is phosphorylated by cds1 kinase. Using different truncated forms of the N-terminal domain of cdc25C, we have demonstrated that the interaction of MP51 with cyclins is primarily mediated through their P-box motif and is independent of MP51 phosphorylation. Characterization of the structural features of this domain by circular dichroism and NMR reveals it forms an elbow-shaped structure composed of two alpha helices interconnected by a loop carrying the 14-3-3 binding site. Moreover, we have shown that helical folding of MP51 is induced upon binding of 14-3-3, which suggests that the conformation of this domain of cdc25C may be regulated through interactions with partner proteins, notably 14-3-3 and cyclins. Combining our structural and biological data, we propose a detailed model of the molecular mechanism of cdc25C regulation by 14-3-3 and cyclins.

20. ACF 1, THE LARGEST SUBUNIT OF THE CHROMATIN ACCESSIBILITY COMPLEX MODULATES THE NUCLEOSOME SLIDING ACTIVITY OF ISWI A. Eberharter, S. Ferrari1, D. Corona2, G. La¨ ngst, Tobias Straub and P. B. Becker Adolf-Butenandt-Institut, Molekularbiologie, Schillerstrasse 44, D-80336 Mu¨ nchen, Germany (E-mail: [email protected]); 1 University of Brescia, Italy; 2University of California, Santa Cruz, 350 Sinsheimer Labs, Santa Cruz, CA 95064, U.S.A.

Nucleosome mobilization by energy-dependent remodelling ATPases emerges as a key principle involved in the establishment of dynamic chromatin structure. The nucleosome-stimulated ATPase ISWI has

A12 been found as the core subunit of various chromatin remodelling complexes in Drosophila (ACF, NURF, and CHRAC). We set out to characterise the subunit composition and mechanism of action of the CHromatin Accessibility Complex, CHRAC. CHRAC is composed of 4 integral subunits. Two small subunits of 14 and 16 kDa, CHRAC-14 and CHRAC-16, are developmentally regulated histone fold proteins. The largest subunit of CHRAC turned out to be Acf1, which had earlier been shown by the Kadonaga laboratory to associate with ISWI to form ACF. Acf1 contains several interesting motifs: two PhD fingers, a WAC domain, a WAKZ domain and a C-terminal Bromodomain. We currently investigate the functional interaction between of Acf1 and ISWI, through reconstitution of CHRAC and ACF form recombinant subunits Interestingly, we find that Acf1 modulates the nucleosomal sliding capacity of ISWI by changing the directionality of ISWI-induced nucleosome movement. We will report on our progress analyzing the role of various Acf1 domains in the interaction with ISWI as well as the nucleosomal substrate and in the process of nucleosome mobilization.

21. PATTERNS OF DNA SYNTHESIS IN PRIMARY RAT HEPATOCYTES I. Raffelsberger and P. Eckl Institute of Genetics and General Biology, University of Salzburg, A-5020 Salzburg, Hellbrunnerstr. 34, Austria (E-mail: [email protected])

Proliferation of primary rat hepatocytes cultured in MEM medium was analysed after applying different proliferation stimulating factors such as insulin, FCS and epidermal growth factor (EGF), and combinations of these. Cells were incubated for different periods of time in the presence of BrdU. the rate of DNA synthesis (labelling index) and the mitotic activity (mitotic index) were quantified by double-staining: immunocytochemical staining of BrdU followed by DAPI staining. under the above conditions supplementation with EGF and insulin revealed to be the most effective treatment for stimulation of DNA synthesis, leading to a steep increase of the labelling index as well as the mitotic activity. The highly differentiated primary hepatocytes are proliferatively quiescent (G0) and consist of di-, tetra- and octoploid mono- or binucleated cells. Therefore, it was expected that stimulation by EGF and insulin would yield solely labelled mitotic figures independent of the ploidy. In contrast, both labelled and unlabelled mitoses were observed, the latter up to a percentage of 0.35%, which equals approximately 17% of all mitotic figures. Besides this finding we also observed a pattern of labelling of mitotic figures which cannot be explained by the common understanding of the process of mitosis. In particular we observed the occurrence of interphase nuclei, half labelled half unlabelled; telophases, half labelled half unlabelled and the appearance of single unlabelled chromosomes in labelled meta- or anaphase stages. This observation was also confirmed by differential fluorescence plus Giemsa (FPG) staining of metaphase spreads. Since replication in vivo could be responsible for these observations rats were fed BrdU in the drinking water for 14 days according to an established protocol, and the isolated hepatocytes analysed for labelling. These experiments gave no detectable labelling indicating that the labelling patterns observed in primary culture cannot be attributed to replication prior to primary culture. These findings therefore may on the one hand indicate that (a) polyploid hepatocytes do not have to go through S-phase before entering mitosis, (b) the genomes of polyploid hepatocytes occupy particular territories within the nucleus and (c) both the separate genomes and single chromosomes are capable of replicating autonomously, which could be features of highly differentiated polyploid cells. On the other hand staining artefacts may be responsible for our observations.

22. ACCUMULATION OF CYCLIN E IS NOT A PREREQUISITE FOR PASSAGE THROUGH THE RESTRICTION POINT Susanna V. Ekholm1, Peter Zickert1, Steven I. Reed2 and Anders Zetterberg1 1 Department of Oncology-Pathology, Cellular and Molecular Tumorpathology, Karolinska Institute/Karolinska Hospital,

Cell Biology International, Vol. 25, No. 2, 2001

CCK R8: 04, S-171 76 Stockholm, Sweden (E-mail: [email protected]); 2Department of Molecular Biology, MB-7, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 U.S.A. (E-mail: [email protected])

The restriction point (R)* is defined as the point in G1 after which cells can complete a division cycle without growth factors and divides G1 into two physiologically different intervals in cycling cells, G1-pm (a post-mitotic interval of constant length, 3–4 h) and G1-ps (a pre-DNAsynthesis interval of variable length, 1–10 h). Cyclin E is a G1 regulatory protein whose accumulation has been suggested to be critical for passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, both with respect to passage through R and entry into S phase. We found that the post-mitotic G1-cells that had not yet reached R were negative for cyclin E accumulation. On the other hand, cells that had passed R were found to accumulate cyclin E at variable times (1–8 h) after passage through R and 2–5 h before entry into S. These kinetic data rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E, but suggest instead the converse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated within 1–2 h after entry into S.

23. THE JNK INTERACTING PROTEIN 1 (JIP-1) IS ACTIVATED UPON MITOGENIC STIMULATION OF QUIESCENT CELLS Wilhelm Engstro¨ m, Lisa Rohbe and Mia Larsson Department of Pathology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, PO Box 7028, S-750 07 Uppsala, Sweden (E-mail: [email protected])

JNK-interacting protein 1 (JIP-1) is a scaffolding protein in the JNK signalling pathway. Initially JIP-1 was described as a cytoplasmic protein that binds to JNK to prevent its translocation to the nucleus. Its expression pattern in different mouse tissues indicates that JIP-1 has a role in the regulation of different cellular events. By examining the JIP-1 expression in transgenic mice that were heterozygous for a functional IGF II gene we were able to show that abrogated IGF II expression was followed by a decreased transcription of the JIP-1 gene. To examine the potential growth regulatory effects of JIP-1 we stimulated quiescent Swiss 3T3 cells with either 10% serum and IGF II and examined the transcription of the JIP-1 gene. We found that the transcriptional rate of JIP-1 was increased within 1 hour which points at a potential role of this gene in the coordinated response during mitogenic stimulation.

24. EXPRESSION PATTERN OF CYCLIN A AND CYCLIN E IN NORMAL AND TRANSFORMED CELLS F. Erlandsson1, C. Wa¨ hlby2 and A. Zetterberg1 1 Department of Oncology-Pathology, Karolinska Hospital, CCK R8:04, 171 76 Stockholm, Sweden (E-mail: [email protected]); 2Centre for Image Analysis, Uppsala University, Lagerhyddsv. 17, 752 37 Uppsala, Sweden (E-mail: [email protected])

The amounts of cyclin E and cyclin A have been shown to be increased in tumors. It is however not clear whether this is simply a result of a higher degree of proliferation, or whether it reflects a disturbed cell cycle control in tumor cells. We therefore investigated how cyclin A and cyclin E are expressed over the cell cycle in 4 normal and 9 transformed cell lines. The cells were exposed to a short BrdU pulse, fixed, and then stained using a triple immunofluorescence staining protocol. The protocol stained for cyclin E using a mouse monoclonal antibody, cyclin A using a rabbit polyclonal antibody, BrdU using a sheep polyclonal antibody, and DNA by DAPI staining. Images of the cells were acquired with a cooled CCD camera, and then underwent digital image analysis. The amount of fluorescence emitted by the fluorophores in each nucleus could be measured, i.e. a measurement of the level of each of the antigens in every single investigated nucleus was

Cell Biology International, Vol. 25, No. 2, 2001 acquired. The simultaneous staining of DNA, BrdU, cyclin A, and cyclin E made it possible to determine the cell cycle position, as well as to measure the relative amounts of cyclin A and cyclin E in each of the analyzed cells. A method was developed to calculate an objective threshold for each stain defining the highest staining intensity found in the negative nuclei in the population. Using the thresholds, we could discriminate cells with minute amounts of staining from truly negative cells. Previously it has been unclear when cyclin A first appears in the cell nucleus, i.e. if it begins to accumulate during late G1- or early S-phase, and whether the expression pattern of cyclin A is altered in tumor cells. The acquired data clearly show that cyclin A appears in the nucleus within minutes from the onset of DNA replication. The levels of cyclin A then rise as the amount of DNA in the nucleus increases, and finally peaks in G2. No difference in the pattern of expression of cyclin A over the cell cycle in normal and tumor cells could be found. Cyclin E did on the other hand exhibit a striking difference in the expression pattern in normal and transformed cells. In normal cells, the highest amounts of cyclin E were present in cells around the G1/S border, and the amounts of cyclin E quickly fell upon entry into S-phase. In transformed cells, the highest levels of cyclin E were achieved during S-phase, high levels remained throughout S-phase, and in some cell lines cyclin E even remained in the nuclei during G2-phase. We are currently studying the expression pattern of cyclin E in samples from cervical carcinoma and carcinoma of the prostate in order to find out if this aberrant pattern of cyclin E expression observed in the tumor cell lines is a general property of tumor cells. Aberrant expression of cyclin E could also be restricted to a subgroup of tumor cells, such as the highly malignant tumors.

25. S PHASE-SPECIFIC ACTIVITY OF THE RICE CAK R2 Tanja Fabian and Margret Sauter Institut fu¨ r Allgemeine Botanik, Universita¨ t Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany (E-mail: [email protected]/E-mail: [email protected])

Progression through the eukaryotic cell division cycle is regulated through cyclin-dependent kinases (CDKs). Besides binding to a regulatory cyclin subunit, CDKs must be phosphorylated by a CDK activating kinase (CAK) for full activity. Known CAKs from human and yeast and their cyclin H binding subunit are regulated neither at the level of gene nor protein expression nor at the level of enzyme activity during the cell cycle. In rice, a CAK homolog, R2, has recently been identified. It is highly homologous to human CDK7 except for an 80 amino acid C-terminal extension not found in other CAKs. We showed that R2 is a nuclear protein. Both, at the gene and protein level R2 expression was elevated in S phase in synchronized suspension cells. Overexpression of R2 in rice suspension cells lead to an increased number of cells in G2 phase. Synchronization of R2 overexpressing cells resulted in an accelerated entry into and progression through S phase suggesting a regulatory role for R2 in DNA synthesis. The R2 binding and activating cyclin H subunit Os;cycH;1 was recently isolated from rice (Yamaguchi et al., 2000). During the cell cycle gene expression of Os;cycH;1 and R2 were coordinately regulated. Deepwater rice plants respond to submergence with increased cell division activity and internodal growth. Both, R2 and Os;cycH;1 gene expression were induced in the meristematic tissue upon submergence, whereas R2 protein abundance remained unaffected. Cyclin H protein has not yet been measured. R2 protein was not only expressed in the meristematic tissue of the internode but also in the elongation zone and at lower levels in the differentiation zone. Os;cycH;1 transcript levels, on the other hand, were specifically expressed in the meristem. Only low levels were found in elongating or in differentiated cells. Expression of R2 protein in elongating and differentiated cells support the idea that it might have functions other than cell cycle regulation. R2 was shown to phosphorylate not only rice cdc2Os-1 and human CDK2 but also the C-terminal domain of RNA polymerase II, suggesting a role in transcriptional control as is known from human CDK7 (Yamaguchi et al., 1998). In rice plants, CTD kinase activity was detected in growing tissues. Upon growth induction by

A13 submergence, CTD kinase activity increased in the meristem but not in non-dividing cells. In partially synchronized suspension cells CTD kinase activity of R2 did not fully correlate with R2 or Os;cycH;1 expression, suggesting a posttranslational regulation mechanism.

References Y M, U M, U H, 1998. A rice homolog of CDK7/MO15 phosphorylates both cyclin-dependent protein kinases and the carboxy-terminal domain of RNA poymerase II. Plant J 16(5): 613–619. Y M, F T, S M, B R, S J, S G, U M, U H, 2000. Activation of CDK-activating kinase is dependent on interaction with H-type cyclins in plants. Plant J 24(1): 11–20. 26. MUTANTS OF THE PROTEIN PHOSPHATASE 2A CATALYTIC SUBUNIT WITH ALTERED SUBSTRATE SPECIFICITY IN VIVO Thomas Fellner1, Claudia Juno1, Ingrid Mudrak1, Karin Kienbauer1 and Egon Ogris1 1 Institute of Medical Biochemistry, Department of Molecular Biology, University of Vienna, Vienna Biocenter, Vienna, Austria (E-mail: [email protected])

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase, appears to be involved in many cellular processes including growth and division. The core enzyme of PP2A is composed of a constant regulatory subunit (A subunit) and a catalytic subunit (C subunit). The association of the heterodimeric core with one of several regulatory B subunits determines substrate specificity and subcellular localization of the holoenzyme. The regulation of complex assembly and substrate specificity in vivo is not well understood yet. To define amino acid residues of the PP2A catalytic subunit that play a role in complex assembly and catalysis we performed a mutational analysis of the C subunit. The selection of the sites to be mutated was based on sequence alignments and crystallographic data of related serine/threonine phosphatases. Individual substitutions of amino acids in the putative okadaic acid (OA) binding region lead to the identification of a mutant which displayed striking effects in vivo. Expression of this mutant in mouse fibroblasts dramatically suppressed proliferation compared to cells expressing either wild type C subunit or containing only the control vector. In addition, these cells showed morphological defects including nuclear abnormalities and an increased cell size. Most interestingly, the introduction of an additional mutation (T304D) in the carboxy terminus of the OA-binding site mutant could neutralize these effects. The T304D C-terminal mutation is known to abolish the interaction with the regulatory B subunit. The loss of the substrate targeting subunit correlated with the loss of the in vivo effects indicating that these effects seemed to be mediated via the B subunit. Consistent with this result, expression of a mutant C subunit containing the OA binding site mutation and a C-terminal mutation, which increased the affinity for B subunit binding, resulted in enhanced in vivo effects. Therefore B subunit-targeted substrates appeared to be the prime target for the OA-binding site mutant. The consequences of the mutation in the OA-binding site on catalytic activity and complex assembly are currently investigated and the results will be presented.

27. ADENOVIRUS-MEDIATED OVEREXPRESSION OF P15INK4B INHIBITS HUMAN GLIOMA CELL GROWTH, INDUCES REPLICATIVE SENESCENCE AND INHIBITS TELOMERASE ACTIVITY SIMILARLY TO P16INK4A Jonas Fuxe1, Go¨ ran Akusja¨ rvi2, Helena M. Goike1, Go¨ ran Roos3, V. Peter Collins1* and Ralf F. Pettersson1 1 Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-17177 Stockholm, Sweden (E-mail: [email protected], [email protected]); 2Department of Medical Immunology and Microbiology, Uppsala University, Biomedical Center, Box 582, Uppsala, Sweden;

A14 3

Department of Pathology, Umea˚ University, Umea˚ , Sweden. *Present address: Department of Histopathology, Addenbrooke’s Hospital, Cambridge, U.K.

The genes encoding the cyclin-dependent kinase inhibitors p16INK4A (CDKN2A) and p15INK4B (CDKN2B) are frequently homozygously deleted in a variety of tumor cell lines and primary tumors, including glioblastomas in which 40–50% of primary tumors display homozygous deletions of these two loci. While the role of p16 as a tumor suppressor has been well documented, it has remained less well studied whether p15 plays a similar growth suppressing role. Here, we have used replication-defective recombinant adenoviruses to compare the effects of expressing wild-type p16 and p15 in glioma cell lines. Following infection, high levels of p16 and p15 were observed in two human glioma cell lines (U251MG and U373MG). Both inhibitors were found in complex with CDK4 and CDK6. Expression of p16 and p15 had indistinguishable effects on U251MG, which has homozygous deletion of CDKN2A and CDKN2B, but a wild-type retinoblastoma (RB) gene. Cells were growth arrested, showed no increased apoptosis, displayed a markedly altered cellular morphology, and repression of telomerase activity. Transduced cells became enlarged and flattened and expressed senescence-associated -galactosidase, thus fullfilling criteria for replicative senescence. In contrast, the growth and morphology of U373MG, which expresses p16 and p15 endogenously, but undetectable levels of pRB, were not affected by exogenous overexpression of either inhibitor. Thus, we conclude that overexpression of p15 has a similar ability to inhibit cell proliferation, to cause replicative senescence and inhibit telomerase activity as p16 in glioma cells with an intact pRB-pathway.

28. CELL-CELL COLLISION AS AN INSTRUCTIVE EVENT IN CELL GROWTH CONTROL IN VITRO R. Grigorian, N. Sarkissian, A. Sayadian and G. Gasparian Institute of Zoology of Armenian NAS, 7, Sevak St., Yerevan 37014, Armenia (E-mail: [email protected])

Liquid medium, solid substratum, and neighbor cells, constituents of in vitro cell microenvironment, were shown to be able to excite inside the cell signals controlling its viability and proliferation. These signals, in turn, can be integrated via the cross-talk between different intracellular signal transduction pathways. This integration is believed to suggest the interchangeability, at least partial, of mentioned factors regulating the cell functions. To verify this idea we have been applying two experimental cell systems. The first is a very sparse culture of normal embryo cells where cell-to-cell touching is a rare event. One can mimic the cell density rise and, therefore, intercellular collision frequency, by adding into the culture ethanol-fixed homologous cells. It is also possible to simulate medium enrichment changing the medium with conditioned one. This cell system allows one to identify separately effects of cell-cell contacts and diffusible factors of the medium excluding either of them. The second system is normal cells cultured in serum-free medium and, hence, deprived of exogenous cell growth and survival regulating molecules. Varying the cell population density enables the role of cell-to-cell contacts to be learned. Using the systems described we have been studying the contribution of intercellular contacts in the cell growth regulation. It was shown that they can both inhibit and stimulate the cell multiplication and for these reason the existence of two types of cell-to-cell contacts controlling the cell growth differently was proposed. Under unavailability of serum factors a cell survival-promoting influence of intercellular contacts was evidenced. The results suggest that insoluble molecules exposed on the cell surface can substitute for soluble serum factors controlling the cell behavior. In this case direct cell-to-cell interaction may be considered, like cell interaction with diffusible factors, as an event bearing instructive meaning in the integrated system of cellular signaling.

29. THE TIMING AND MECHANISM OF CYCLIN A DEGRADATION IN MITOSIS S. Geley1 and T. Hunt2 1 Institute for General and Experimental Pathology, University of Innsbruck, Fritz-Pregl -Str.3/IV, A-6020

Cell Biology International, Vol. 25, No. 2, 2001

Innsbruck, Austria (E-mail: [email protected]); Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, EN6 3LD, Herts, U.K. (E-mail: [email protected])

2

Cyclin A is degraded in mitosis before cyclin B1. To investigate the timing and mechanism of cyclin A degradation in mitosis we followed cyclin A-CFP and cyclin B1-YFP levels in cells going through mitosis. We found that cyclin A is destabilised shortly after nuclear envelope breakdown while cyclin B1 stays stable until metaphase. Both cyclins are degraded by an CDC20-APC/C-dependent mechanism suggesting that additional mechanisms must control the degradation of these cyclins. In contrast to cyclin B1, cyclin A is not stabilised in cells treated with nocodazole or taxol indicating that the degradation of cyclin A is not controlled by the spindle assembly checkpoint. Impairing this checkpoint by expressing dominant negative Bub1, on the other hand, accelerated the degradation of cyclin B1 in mitosis indicating that the spindle assembly checkpoint is responsible for the differential timing of cyclin A and B1. To understand these differences we analysed the destruction box of human cyclin A and found that it is longer than the destruction box of cyclin B1. Overexpression of wild-type cyclin A delayed the onset of anaphase up to several hours by a mechanism that required the presence of the destruction box but not cyclin A-associated kinase activity. Expression of non-degradable cyclin A, on the other hand, arrested cells in anaphase and delayed the degradation of cyclin B1. These data suggest that cyclin A has to be degraded early in mitosis to allow onset of anaphase, rapid degradation of cyclin B1 and exit from mitosis.

30. THREE-DIMENSIONAL STRUCTURE OF THE HUMAN ANAPHASE PROMOTING COMPLEX C. Gieffers1, P. Dube2, R. Harris3 and J.-M. Peters1 1 Research Institute of Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna, Austria (E-mail: gieff[email protected]); 2MPI for biophysical chemistry, Am Fassberg 11, D-37077 Goettingen, Germany (E-mail: [email protected]); 3University of Mainz, D-55099 Mainz, Germany (E-mail: [email protected])

The anaphase-promoting complex (APC) is a large multi-subunit complex that has cell cycle regulated ubiquitin-protein ligase (E3) activity. Together with an ubiquitin activating (E1) and an ubiquitinconjugating enzyme (E2) the APC catalyses the formation of polyubiquitin chains on substrate proteins such as securin and mitotic cyclins and thereby targets them for the degradation by the 26S proteasome. The human APC is composed of at least 11 subunits. We have recently shown that the smallest of these subunits, the 10 kDa RING-H2 protein APC11 alone is sufficent to ubiquitinate APC substrates in the presence of E1 and E2 enzymes. However, the function of the other 10 APC subunits is entirely unknown. To understand why the APC is composed of so many subunits we have begun to analyse the structure of the human APC. We have developed a method that allows the purification of human APC in a homogeneous soluble form suitable for electron microscopic analyses. EM analysis of negatively stained APC showed distinct particles of about 20 nm in diameter that often appeared triangular or V-shaped. To obtain a 3D model of the APC we used cryo electron microscopy. Based on the analysis of 10,000 molecular images we could generate a 3D model of the human APC with a resolution of 25 A r . On basis of this model we will discuss possible mechanisms by which the APC may ubiquitinate substrate proteins.

31. GALECTIN-8 INDUCES GROWTH ARREST AND APOPTOSIS OF TUMOR CELLS Rinat Arbel Goren, Yifat Levy, Denise Ronen and Yehiel Zick Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel (E-mail: [email protected])

Galectin-8, a secreted mammalian lectin is selectively overexpressed in human prostate and lung (1299) carcinomas. We have previously

Cell Biology International, Vol. 25, No. 2, 2001 shown that exogenously added recombinant-Galectin-8 (rGalectin-8) induces apoptosis in various cell lines. In addition, overexpression of Galectin-8 reduces colony formation in CHO and 1299 cells (Hadari, et al., 2000). In the present study, we aimed to characterize the effect of galectin-8 on cell growth and apoptosis. We could demonstrate that transient overexpression of GFP-Galectin-8 in CHO cells induced growth arrest as evident by reduced BrdU incorporation into the transfected cells. We could further demonstrate that rGalectin-8 (4 mM), exogenously added to 1299 cells in a serum-free medium, induced apoptosis, which was accompanied by cleavage of poly ADP ribose polymerase (PARP). Wortmannin (100 nM), an inhibitor of PI-3-kinase, significantly enhanced rGalectin-8-induced PARP cleavage. Similarly, inhibition of protein synthesis by cycloheximide (25 mg/ ml) potentiated rGalectin-8- induced PARP cleavage. In contrast, z-VAD-fmk (4 mM), a caspases inhibitor, decreased rGalectin-8induced PARP cleavage. Insulin (1 kM) which acts as an antiapoptotic agent, inhibited the apoptotic effects induced by rGalectin-8. However, addition of insulin (1 M) in the presence of cycloheximide (25 mg/ml) did not rescue the cells from PARP cleavage induced by Galectin-8. Taken together, these results indicate that overexpression of secreted Galectin-8, arrests the cell cycle and inhibits cellular growth, which might trigger an apoptotic process. Accordingly, exogenously added rGalectin-8, which presumably mimics the effects of the secreted galectin-8, activates ICE related proteases (caspases). This in turn contributes to PARP cleavage and apoptosis, while serum constituents such as insulin, acting as survival factor, overrides the apoptotic effects induced by rGalectin-8 through activation of the PI-3-kinase pathway. Since inhibition of protein synthesis enhances rGalectin-8-induced apoptosis, it suggests that apoptosis induced by rGalectin-8 is inhibited by proteins having a short half-life. Our findings implicate Galectin-8 as an inhibitor of cell growth and an inducer of apoptosis. Accordingly, tumor cells that overexpress this protein devised means to override the pro-apoptotic functions of Galectin-8.

Reference H YR, G AR, L Y, A A, A R, Z R, Z Y, 2000. J Cell Sci 113(13): 2385–2397. 32. MOLECULAR ANALYSIS OF Dd-Nek2, THE FIRST NON-VERTEBRATE HOMOLOGUE OF THE HUMAN CENTROSOMAL NIMA-RELATED KINASE NEK2 R. Gra¨ f Adolf-Butenandt-Institut/Zellbiologie, Schillerstr. 42, D-80336 Mu¨ nchen, Germany (E-mail: [email protected])

NIMA-related kinases (never in mitosis, Aspergillus nidulans) are a ubiquitous family of serin/threonine kinases promoting cell cycledependent events such as G2/M progression, chromosome condensation and centrosome splitting. Human Nek2 is unique among this kinase family since it is an integral component of the human centrosome (Fry et al. (1998) EMBO J. 17, 470). Recently, a cDNA sequence of a Dictyostelium member of this protein family was identified in the cDNA-project of the University of Tsukuba/Japan. Clone SLD805 contains a complete open reading frame encoding Dd-Nek2 which has 43% amino-acid identity to human Nek2. Within the catalytical domain (65% of the entire sequence) both sequences are even 54% identical. Moreover, both proteins have a similar domain structure and share appoximately the same length of 50 kDa. Thus, Dd-Nek2 is the first non-vertebrate homologue of human Nek2. Dd-Nek2 was expressed in E. coli as a fusion with maltose-binding protein (MBP). By contrast to human Nek2, bacterially expressed Dd-Nek2 is catalytically active, i.e. it exhibits autophosphorylation and uses /-casein as an artifical substrate. MBP-Dd-Nek2 was expressed in Dictyostelium as well. One-step isolation of MBP-Dd-Nek2 from cytosolic Dictyostelium extracts by affinity chromatography on amylose resin resulted in a highly pure catalytically active enzyme preparation. Thus, MBP is a well suited fusion-tag for expression and high purification of native proteins not only in E. coli but also in Dictyostelium.

A15 Immunofluorescence analyses with antibodies raised against bacterially expressed MBP-Dd-Nek2 and expression of a GFP-Dd-Nek2 fusion protein in Dictyostelium showed that Dd-Nek2 is localized at the centrosome during the entire cell cycle. The GFP-Dd-Nek2 mutants frequently exhibit supernumerary centrosomes indicating a role of Dd-Nek2 in centrosome dupliation. The centrosome duplication defect was not observed in the GFP-Dd-Nek2 316 mutant where the C-terminal 103 amino acids of GFP-Dd-Nek2 were deleted, although the truncated GFP-fusion protein still localized to centrosomes. Recently, a centrosomal 280-kDa substrate protein of human Nek2, called C-Nap1, could be identified (Fry et al. (1998) JCB 141, 1563). Interestingly, antibodies against human C-Nap1 specifically recognized a 280-kDa band in immunoblots of Dictyostelium centrosome preparations. Furthermore, immunofluorescence analysis revealed that anti-C-Nap1 antibodies bound to the Dictyostelium centrosome, as in human cells, only during interphase. Dissociation of C-Nap1 at the onset of mitosis, presumably triggered by its phosphorylation, is thought to facilitate centrosome splitting (Fry et al., 1998). The immunological evidence for a putative C-Nap1 homologue in Dictyostelium and the presence of Dd-Nek2 at the centrosome suggests that the important functional role of Nek2 in centrosome duplication is conserved from Dictyostelium to humans in a very similar fashion despite of considerable differences in morphology and duplication of human and Dictyostelium centrosomes. Supported by the Deutsche Forschungsgemeinschaft (SFB184). I am grateful to Thibault Mayor and Erich Nigg for their generous gift of anti-C-Nap1 antibodies.

33. PCTAIRE-1, A CDK-RELATED PROTEIN KINASE INVOLVED IN THE REGULATION OF NEURITE OUTGROWTH R. Graeser1,3, T. Dubois2, A. Aitken2 and T. Hunt3 1 KTB Tumorbiology Center, Breisacherstrasse 117, 79106 Freiburg, Germany (E-mail: [email protected]); 2University of Edinburgh, Department of Biomedical Sciences, Edinburgh, EH8 9XD, Scotland, U.K.; 3ICRF, Clare Hall Labs, South Mimms, Herts EN6 3LD, U.K.

PCTAIRE-1 is a cdk-related protein kinase found in terminally differentiated cells in brain and testis, and in many immortalised and transformed cell lines. Little is known about its way of activation and its cellular function. The bacterially expressed PCTAIRE-1 protein kinase is catalytically inactive. So we started by looking for activators in brain extracts. We did not find any interactors, but detected a strong phosphorylation of the N-terminus of PCTAIRE-1. Using nanospray massspectrometry we identified three sites, which carried the recognition sequences of protein kinase A (PKA). Phosphorylation by PKA of one site generates a 14-3-3 binding motif, which is functional in vitro as well as in vivo. Stimulation of PKA in cells using forskolin or dibutyro-cAMP decreases the activity of transfected PCTAIRE-1. Mutation of a second PKA site in the N-terminus of PCTAIRE-1 protects the kinase from inactivation. Moreover, it results in a 2–3 fold stimulation of PCTAIRE-1 kinase activity when compared to wild-type even if isolated from non-treated cells. There is some evidence that PCTAIRE-1 is not dependent on a partner subunit (cyclin) for kinase activity. Constructs encoding the activated PCTAIRE-1 kinase mutant generate active kinase when transfected into a variety of epithelial or fibroblast cell lines. This is in contrast to a neuronal cdk, cdk5, whose activity is strictly dependent upon its cyclin, p35nck, which is absent from non-neural cells. Moreover, gelfiltration analysis of brain extract, suggest that monomeric PCTAIRE-1 may be active. Finally, kinase-dead PCTAIRE-1 constructs transfected into neuroblastoma cell lines promote neurite outgrowth, whereas a double mutant, which lacks both above mentioned PKA sites in the N-terminus, prevents this process even in the presence of forskolin. This indicates a potential role for the PCTAIRE-1 gene product in the prevention of neurite outgrowth.

A16

34. ROLE AND CHARACTERIZATION OF PROTEIN KINASE C ISOFORMS IMPLICATED IN THE TRANSCRIPTIONAL ACTIVATION OF CYCLIN D1 BY TRANSFORMING HA-RAS H. H. Grunicke1, S. Kampfer1, M. Windegger1, F. Hochholdinger1, W. Schwaiger1, R. G. Pestell2, G. Baier3 and F. U } berall1 1 Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Austria (E-mail: [email protected]); 2 Albert Einstein College of Medicine, Bronx , NY 10461, U.S.A.; 3Institute of Medical Biology and Human Genetics, University of Innsbruck, Austria

HC11 mouse mammary epithelial cells express protein kinase C PKC) isoenzymes , ,  and . The implication of these PKC isotypes in the transcriptional regulation of cyclin D1 by transforming Ha-Ras was investigated by employing cyclin D1-luciferase reporter constructs. The studies revealed that cyclin D1 induction by transforming Ha-Ras is Rac-dependent and requires the PKC isotypes ,  and —not, however, cPKC-. The data indicate that in the signalling pathway from Ras to the cyclin D1 promoter aPKC- functions upstream of Rac whereas PKC isotypes  and act downstream of Rac. Accordingly, cyclin D1 induction by constitutively active V12 Rac was not inhibited by co-expressed dominant negative (DN) aPKC- whereas dominant negative (DN) versions of nPKC- or blocked transcriptional activation of cyclin D1 by V12 Rac. Deletion of the AP-1- and the CRE-sites of the cyclin D1 promoter abrogated the responsiveness to V12 Rac. Transforming Ha-Ras, however, was still able to activate transcription of the mutated cyclin D1 reporter construct. Transcriptional activation by Ras of the mutated cyclin D1 promoter lacking functional AP-1- and CRE-sites was inhibited by dominant negative (DN) nPKC- and but proved insensitive to kinase deficient, dominant negative (DN) aPKC-. Cyclin D1 induction by transforming Ha-Ras is depressed by PD98059, a selective inhibitor of the mitogen-activated kinase kinase MEK-1, demonstrating that Ha-Ras employs extra cellular signal regulated kinases (ERKs) for signal transmission to the cyclin D1 promoter. Evidence is presented that PKC isotypes  and —but not —are required for the Ras-mediated activation of ERK 1/2. Expression of dominant negative (DN) mutants of nPKC- or aPKC- inhibit ERK 1/2 activation by constitutively active (CA) Raf-1. Phosphorylation within the TEY motive and subsequent activation of ERK 1/2 by constitutively active (CA) MEK-1 were significantly inhibited by (DN) aPKC- —not, however, by (DN) nPKC- indicating that PKC- functions downstream of MEK-1. Cyclin D1 induction by transforming Ha-Ras requires the cooperative activity of PKC-isozymes ,  and . Evidence is presented that aPKC- acts upstream of Rac, i.e. between Ras and Rac whereas PKC isotypes  and are required for the activation of ERK 1/2.

35. REGULATION OF SP1 ACTIVITY BY A CYCLIN A/KINASE COMPLEX Eva Haidweger1, Michael Novy and Hans Rotheneder Institute of Medical Biochemistry, Division of Molecular Biology, Vienna Biocenter, University of Vienna, A-1030 Vienna, Dr.Bohr-G. 9, Austria (E-mail: [email protected])

Sp1, a ubiquitously expressed protein of 95–105 kDa, binds DNA through C-terminal zinc finger motifs and was one of the first cloned and characterized transcription factors. It stimulates transcription from promoters containing a G and C rich sequence, the GC-box, and is particularly important for the regulation of TATA-less genes that encode housekeeping proteins. Most growth factors and receptors are also encoded by such genes. Sp1 exhibits multiple domains which seem to be important for different aspects of its activity. It is able to act synergistically with itself, with other members of the family and with a variety of cellular and viral factors thought to confer promoter and cell-type specific activity. We have found recently that the C-terminal part of Sp1 interacts with both, the transcription factor E2F-1 and the negative regulator histone deacetylase 1 (HDAC1). Moreover coexpression of E2F-1 not only abolishes

Cell Biology International, Vol. 25, No. 2, 2001 HDAC1 mediated repression of SP1 but also physically displaces HDAC1 from Sp1. When studying the interactions between Sp1 and E2F-1 on one hand, and E2F-1 and cyclin A on the other hand, we found that there is an interaction between cyclin A and Sp1, too. The binding of cyclin A to Sp1 occurs in vivo (as tested by co-immunoprecipitations) as well as in vitro (in GST-pull down assays). Utilizing GST-cyclin A and purified Sp1, we could further show that the interaction is direct with no other proteins necessary. The interaction takes place within the region of Sp1 also mediating E2F-1 and HDAC1 binding. Furthermore we could show that immunoprecipitated cyclin A/kinase complexes are able to phosphorylate full length as well as truncated Sp1 comprising only the C-terminal domain. Cyclin A binds both, cdk2 and cdc2, giving two distinct cyclin A/kinase activities, one appearing in S-phase, the other in G2. We found that cyclin A is able to activate Sp1-dependent promoters, whereas inhibition of cdk2 results in repression of the very same promoters. This suggests that growth factor regulated phosphorylation of Sp1 may be a significant mechanism of Sp1 mediated gene expression.

36. ENDOREDUPLICATION IN ARABIDOPSIS ROOT DEVELOPMENT M.-T. Hauser, M. Axterer, A. Karsai and A. Mansfeld Center of Applied Genetics, University of Agricultural Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria (E-mail: [email protected])

Endoreduplication, the amplification of the genome without mitosis, is a common process in eukaryotes and most angiosperms develop tissues and cells with elevated ploidy levels. The increase in nuclear DNA content is implicated to be important for cell expansion, cell differentiation and metabolic activity. Whereas mitotic cell divisions in roots are restricted to the meristematic zone at the tip, the region where endocycles take place during root development is not yet defined. In Arabidopsis roots rapid cell elongation occurs in the cell expansion zone which follows the cell division zone. The cell expansion zone accommodates 6 to 10 tiers of cells and is about 500–900 m long. in this zone most of the root cells reaches their final shape and size. By measuring the cell volume of the three outer root tissues we revealed that cortex cells get 1.6 times larger than epidermis and 4.5 times larger than endodermis cells. As cell size is thought to correlate with ploidy levels we quantified the nuclear dna content. Unexpectedly, the ploidy levels are not dramatically different in the three root tissues. Furthermore, the amount of 2C, 4C, 8C and 16C nuclei changes similarly in the different tissues during root development. Most of the cells in the cell expansion zone exhibit a ploidy level of 4C. The nuclear DNA content increases to 8C in older differentiated parts of the root. Nuclei with 16C were only observed 5 mm distal of the quiescent center. These data implicate that endoreduplication occurs not only during cell expansion but also in later stages of root development. To identify genes, regulating endoreduplication we analyzed the DNA content of different cell expansion mutants and found that the number of 16C, 32C and 64C is dramatically increased in pom1 roots. The POM1 gene was isolated by a chromosomal walking approach and the deduced amino acid sequence is related to a gene family in Drosophila and mammals which interacts with a specific ubiquitinconjugating enzyme. Thus, we propose that POM1 is a new component which may be involved in the ubiquitylation and degradation of proteins regulating endomitosis. This work was funded by the EU grant PL 960217 and the FWF grant P14477-GEN.

37. MCM6 DISTRIBUTION IN PROLIFERATING CELLS DETECTED BY THE NOVEL MAB KI-MCM6 H. J. Heidebrecht1, F. Buck2, K. Andersen1, H. H. Wacker1 and R. Parwaresch1 1 Department of Hematopathology and Lymph Node Registry, University of Kiel, Michaelisstrasse 11, D-24105 Kiel, Germany (E-mail: [email protected]); 2 Institute of Cell Biochemistry and Clinical Neurobiology, University of Hamburg, Hamburg, Germany

Cell Biology International, Vol. 25, No. 2, 2001 Nuclear lysates of proliferating cells were used to immunize mice. One of the resulting new monoclonal antibodies (mabs) was specific for the human minichromosome maintenance protein 6 (Ki-Mcm6). Immunohistochemical stainings of cytospin preparations of L428 cells and mitogen stimulated peripheral blood lymphocytes (PBL) revealed that Ki-Mcm6 recognized an antigen that is expressed like a cell cycle associated antigen. Quiescent cells do not express the antigen whereas cells in G1, S, G2 and M phase do. In Western blot experiments Ki-Mcm6 recognized a protein of about 105 kDa in lysates of L428 cells. This 105 kDa protein was immunopurified and separated by SDS-PAGE. The Coomassie blue stained protein was excised and digested with LysC. Sequencing of the resulting peptides proved that the antigen detected by Ki-Mcm6 is the minichromosome maintenance protein 6. To our knowledge Ki-Mcm6 is the first mab specific for mcm6. Immunohistochemical stainings of cytospin preparations of mitogen stimulated peripheral blood lymphocytes revealed that the antigen is expressed in the cell nucleus during G1, S and G2 phase. During mitosis mcm6 is localized in the cytoplasm. In comparison to the pattern of the well-known cell cycle associated antigen Ki-67, the number of mcm6 expressing cells is slightly enhanced. These data indicate that mcm6 is already expressed in the early G1 phase which is not detected by mabs specific for the Ki-67 antigen. Immunostaining of sections of various human tissues and tumors confirmed the results. In all cases the number of mcm6 expressing cells was slightly enhanced compared with the number of Ki-67 antigen expressing cells. One remarkable observation was that human oocytes expressed mcm6 in contrast to other proliferation associated antigens (Ki-67 antigen; repp86). Confocal laser scanning microscopy revealed a dotlike distribution of mcm6 in the nucleus in mitogen stimulated PBL during G1, S and G2 phase. In mitotic cells mcm6 is distributed throughout the cytoplasm and, like the Ki-67 antigen, it is associated with the chromosomal scaffold. These preliminary results indicate that the new mab Ki-mcm6 will be a useful tool for the further characterisation of mcm6.

38. REGULATION OF p27KIP1 TRANSLATION Ulrich Go¨ pfert, Michael Kullmann and Ludger Hengst Max-Planck-Institut fu¨ r Biochemie, Munich, Am Klopferspitz 18a, D-82152 Martinsried, Germany (E-mail: [email protected])

The Cdk inhibitor p27Kip1 regulates cell proliferation by binding and inhibiting Cdk complexes in response to diverse antiproliferative signals. During G1 phase of the cell cycle, p27 is involved in the control of the restriction point passage by being present at high levels early in G1 and reduced levels as cells enter S-phase. Antiproliferative signals also induce increased p27 protein levels. Among other mechanisms translational control is responsible for the regulated oscillations of p27 protein levels during the cell cycle and the induction of p27 in growth arrest. To elucidate the contribution of translational control, we have isolated a complete p27 cDNA and determined the role of distinct regions of the p27 mRNA in its translation. The 5 UTR of the p27 mRNA contains a small uORF sequence that impairs overall translational efficiency for the downstream coding region for p27. Within this region in the 5 -end of the 5 UTR is an element responsible for the cell cycle phase dependent translation of p27. Specific proteins bind to this regulatory element in a sequence and cell cycle position dependent manner. We have purified and identified several of the proteins that bind to the p27 5 UTR. An internal ribosomal entry site (IRES) located downstream of the cell cycle responsive element in the 5 UTR enhances overall translational efficiency but does not interfere with the cell cycle regulated translational control of p27 expression. In contrast to the regulatory role of the 5 UTR in p27 translation, the 3 UTR reduces overall efficiency of p27 translation.

39. MUTATIONAL ANALYSIS OF DdCP224 DISCLOSES THE C-TERMINAL CENTROSOME-BINDING DOMAIN AND AN ENCRYPTED MICROTUBULE-BINDING AND BUNDLING ACTIVITY A. Hestermann, M. Schliwa and R. Gra¨ f

A17

Adolf-Butenandt-Institut/Zellbiologie, Universita¨ t Mu¨ nchen, Schillerstr. 42, 80336 Mu¨ nchen, Germany (E-mail: [email protected])

DdCP224 belongs to the TOGp/Stu2p family of microtubuleassociated proteins. It is the first member of this protein family which plays a role in centrosome duplication and is a permanent centrosomal resident (Gra¨ f et al. (2000) J. Cell Sci. 113, 1747–1758). The latter feature is so far shared only by yeast Stu2p. But as human, Xenopus and Drosophila homologues (TOGp, XMAP215, msps), DdCP224 is more than twice as long as yeast Stu2p which corresponds to the N-terminal half of DdCP224. Accordingly the
C deletion mutant of DdCP224 was constructed and fused to GFP, along with a
N-GFP construct covering the C-terminal half which is not homologous to Stu2p. GFP-fusions of the whole-length protein are localized to microtubules, kinetochores, and the centrosome. As Stu2p the
C-GFP-fusion was expected to localize to microtubules and the centrosome. However, it showed only an uniform distribution in the cytosol. In contrast, the
N-GFP-fusion protein localized to the centrosome and the kinetochore region, but did not bind to interphase-microtubules. For further mapping of the microtubule and centrosome-binding domains we constructed seven new deletion constructs of DdCP224 fused to GFP. The new C-terminal deletion mutants of
N-GFP surprisingly localized to microtubules. Microtubules frequently appeared bundled and much longer than usual. These long microtubules often curved along the plasma membrane, possibly due to an inability to interact with docking sites at the cell cortex. Therefore, the middle part of DdCP224 contains a microtubule binding and bundling activity which is hidden in the
N-GFP mutant. This microtubule binding site must be different from that mapped to the N-terminal half of the human and yeast homologues which is excluded in all of our
N-mutants. All new N-terminal deletion mutants of
N-GFP localized to the centrosome and not to interphase microtubules. Thus we could map the centrosome-binding site to the C-terminal 25% of the DdCP224 sequence. In the near future we plan to use the centrosome-binding domain as bait in a yeast two-hybrid screen in a search for the unknown centrosomal binding partners of DdCP224. Supported by the Deutsche Forschungsgemeinschaft (SFB184).

40. REGULATION OF B-MYB DURING THE CELL CYCLE S. Horstmann1, S. Charrasse2, I. Carena2, V. Brondani2, S. Ferrari2, K.-H. Klempnauer1 1 Institut of Biochemistry, University of Mu¨ nster, Wilhelm-Klemm-Str. 2, D-48149 Mu¨ nster, Germany (E-mail: [email protected]); 2Department of Oncology, Novartis Pharma AG, K-125.4.01, CH-4002 Basel, Switzerland (E-mail: [email protected])

Evidence obtained during recent years suggests that B-Myb, a highly conserved member of the Myb transcription factor family, plays a key role in cell proliferation. The expression of B-myb at the G1/S-phase boundary of the cell cycle is under control of the transcription factor E2F. Superimposed on the cell cycle dependent expression of B-myb mRNA are additional levels of post-translational regulatory mechanisms involving cyclins D1 and A. Cyclin D1 strongly inhibits the activity of B-Myb by formation of a specific complex with B-Myb without increasing the phosphorylation of B-Myb. In addition to this inhibitory effect the formation of the B-Myb/cyclin D1 complex increases the protein stability of B-Myb. In contrast to cyclin D1, cyclin A stimulates the activity of B-Myb by cyclin A/Cdk2dependent phosphorylation at the carboxyl-terminus of B-Myb. The phosphorylation, presumably, induces a change of the B-Myb conformation which exposes the transactivation domain of B-Myb for interaction with other proteins. We have identified a 131 amino acids spanning central domain of B-Myb as the principal transactivation domain of full-length B-Myb which interacts with the transcriptional coactivator p300. We have discovered a splice-variant of B-Myb lacking the transactivation domain and acting as a transcriptional repressor.

A18 Besides stimulating the transactivation potential of B-Myb, cyclin A dependent phosphorylation causes a pronounced reduction of B-Myb protein level. This increased degradation of the B-Myb protein is due to an interaction of B-Myb with p45Skp2, a component of the SCF ubiquitin E3 ligase that is associated with cyclin A/Cdk2 complexes. The coupling of activation- and degradation-signals for B-Myb leads to a short peak of B-Myb activity during the cell cycle which seems to be necessary for correct cell division. Current work is directed at understanding the molecular basis of fine-tuning of B-Myb activity by the antagonistic effects of cyclin D1 and cyclin a in more detail.

41. THE ROLE OF UBIQUITIN LIGASES IN REPLICATIVE SENESCENCE Mechthild Wagner, Eveline Huetter, Barbara Hampel, Wilhelm Krek* and Pidder Jansen-Du¨ rr Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, 6020 Innsbruck, Austria (E-mail: [email protected]); *Friedrich-Miescher-Institut, Basel, Switzerland

Normal human cells in primary culture have a limited proliferation capacity; after extended passaging such cells stop dividing and enter a quiescent state referred to as replicative senescence. While it is clear that senescent cells undergo cell cycle arrest in G1, the molecular mechanisms responsible for cell cycle arrest are to be defined. We have compared the expression of cell cycle regulatory genes in primary human fibroblasts and endothelial cells of different replicative age. Strikingly, we found a deregulation of genes controlling the ubiquitinmediated proteolytic degradation of cell cycle regulators in senescent human cells. These findings shed new light on the mechanisms of senescence-associated cell cycle arrest.

42. EFFECT OF p15INK4b EXPRESSION ON THE CELL CYCLE AND THE RELATIONSHIP BETWEEN p15INK4b AND PKC.MAPK SIGNAL IN HUMAN MELANOMA CELLS Liu Huitu, Liu Jun, Tong Yingkai, Zhang Wei and Gao Ping Key Laboratory of Cell Proliferation and Regulation Biology of Education Ministry, Beijing Normal University, Beijing, 100875, China (E-mail: [email protected])

Using the transfection technique, p15INK4b was introduced into melanoma A375 cells in which p15INK4b gene is deleted and a cell model MLIK6 overexpressing p15INK4b was constructed. Comparing with the control cells MLC2, MLIK6 cells in G1phase increased by 11%, but those in S phase decreased by 15%. The M and G1 Phase cells were obtained by using N2O-TdR blocking. The result of 3H-TdR incorporation indicated that the transition of G1/S of MLIK6 cells was delayed 2 h as compared with that of MLC2 cells and incorporation rate also decreased. The observation on expression of some G1/Srelated regulating genes showed that in MLIK6 cells the protein level of p27kip1 increased with the decreasing expression of Cyclin D1, Cyclin E and c-myc, especially Cyclin D1 in late G1phase. The expression of Cyclin E obviously decreased at G1/S transition, and c-myc was inhibited throughout all the process of G1-S phase. The effect of PKC inhibitor on G1-S progression in MILK6 cells was observed. When PKC activity was inhibited , the transition of G1/S could be ahead of 1–2 h and the G1 phase was shortened. At the same time, the PKC activity of MILK6 cells was obviously increased in the late G1 phase. At late G1 phase compared with MLC2 cells, the level of ERK1 and ERK2 almost have no change in MILK6 cells, but that of P-ERK1 and P-ERK2 (activated form) were decreased markedly. The results suggest the transition of G1/S was negatively regulated by PKC in melanoma cells overexpressing p15INK4b, and overexpressing p15INK4b can down-regulate the activity of ERK1 and ERK2.

43. THE CATENIN-RELATED APC-PATHWAY IS LINKED TO THE RB-PATHWAY VIA ACTIVE p27kip-1 MEDIATED CELL GROWTH CONTROL R. Huss and K. Thalmeier

Cell Biology International, Vol. 25, No. 2, 2001

Institute of Pathology, University of Munich, Thalkirchner Str. 36, D-80337 Munich, Germany (E-mail: [email protected])

Hematopoietic progenitors are derived from predominantly quiescent fibroblast-like cells, which rest in the site of hematopoiesis (e.g. as CD34-negative cells in the bone marrow) and eventually become activated. Activation occurs in a sequential manner. First, the growth factor receptor, e.g. a receptor tyrosine kinase (RTK) such as c-kit is expressed on the surface and later the signal transduction pathway becomes activated. Nevertheless, the majority of those cells are quiescent (>97%) and refractory to external signalling which is also mediated by the downregulation of MPF-associated factors (cdc2) and no STAT activation. Interleukin-6 (IL-6) is the major factor to induce proliferation and maintaining viability. But the stable transfection of quiescent cell clones with a differentiation antigen (DR) leads to a cellular transformation with the biological behaviour and phenotype of an immature myeloid leukemia (e.g. accelerated cell cycle progression and tumor growth in mice). The formerly quiescent cells now activate the IL-6-dependent JAK/STAT-pathway. The transformed cells also display an increased alternative kinase activity as shown in the constitutive activation of p38 MAPK, while non-proliferating cells activate ERK and MEK instead. Besides the increased expression and tyrosine-phosphorylation of the rasGAP-associated p62dok protein, cyclin D as well as the cyclin-dependent-kinase-inhibitors (CDKI) p21cip-1 and p27kip-1 are overexpressed, but apparently inactive, since Rb-phosphorylation remains unaltered. We could not find any mutation in p27 by SSCP (single strand conformation polymorphism). But phosphorylation of p27 can inactivate the protein and it becomes subject to the ubiquitin-associated degradation. To investigate the pivotal role of p27kip-1 in malignant transformation, we transfected the transformed clones with an additional wildtype p27 construct. The transgene expression of the active p27 protein reinstated the growth control of the CD34-negative cell clones in-vitro (e.g. contact inhibition and cell-cell-interaction) and the translocation of residual -catenin to the cell membrane, while -catenin was completely absent or located in the cytoplasm in the transformed cell clones. Transgene expression of p27 did not interfere with apoptosis or bcl-2/bax expression. In conclusion, the catenin-related APC-pathway is linked to the Rb-pathway via active p27kip-1 mediating cell growth control, while malignant cells bypass CDKI-growth control by an increased kinase activity. Transformed cells also utilize the p38 MAPK pathway rather than the conventional ERK and MEK activity.

44. PURIFICATION OF HISTONE MODIFYING COMPLEXES FROM DROSOPHILA EMBRYONAL EXTRACTS B. B. Hulsmann, B. Czermies, P. B. Becker and A. Imhof Adolf Butenandt Institut, University of Munich, Schillerstr. 44, D-80336 Munich (E-mail: [email protected])

Histone modifications play an essential role in setting up and maintaining stable patterns of geneexpression during differentiation and development. We have fractionated Drosophila nuclear extracts from 0–12 h larvae and assayed the fractions for histone acetyltransferase (HAT), histone kinase (HIK) and histone methyltransferase (HIM) activity. We were able to identify at least four different HATs which we termed HAT1-4, three different HIKs (HIK1-3) and five HIMs (HIMalpha-epsilon). A comparison of the specificities of these activities for different histones showed that the HIKs and the HIMs are all specifically modifying H3 whereas the different HATs clearly have distinct histone specificities. When using nucleosomes as substrates only three of the HATs could transfer 3H from Acetyl-CoA to the histones, suggesting that one of the identified HATs is the Drosophila orthologue of HAT1 which was isolated from various sources.Two of the HIMs and all of the HIKs were able to use nucleosomes as substrates with the same speficity as the free histones. One of the HIMs-HIMalpha -was able to methylate nucleosomes significantly better than free histones and this effect was even more pronounced after further purification.

Cell Biology International, Vol. 25, No. 2, 2001 HIM alpha activity is severely inhibited by ATP, some divalent cations such as Zn + + and phosphate. It consists of at least four polypeptides p120, p80 p60 and p42 and has a molecular weight of about 450 kDa on a superdex 200 column.

45. CELL VOLUME REGULATION IN THE CELL CYCLE OF NASOPHARYNGEAL CARINOMA CELLS L. Wang1,2, L. Chen1,2 and T. J. C. Jacob1 1 School of Biosciences, Cardiff University, Biomedical Building, Museum Avenue, Cardiff CF10 3US, U.K. (E-mail: jacob@cardiff.ac.uk); 2Department of Physiology, Guangdong Medical College, Zhanjiang, Guangdong 524023, China (E-mail: [email protected])

Cells increase their volume before division during the cell cycle and do not pass the restriction point (the critical checkpoint between G1 and S) until they reach a threshold size. To increase cell volume, a cell activates its regulatory volume increase (RVI) mechanisms and inhibits its regulatory volume decrease (RVD) mechanisms by readjusting its set point to a higher level. However, there are no reports to date on the role of RVD in the cell cycle. In this study, the RVD of synchronised, poorly differentiated nasopharyngeal carcinoma cells, CNE-2Z cells, was investigated. Cell volume was calculated from the cell diameter taken from cell images. The results showed that cell volume increased throughout interphase and reached the peak during G2 phase of the cycle. Although the cells were capable of regulating their volume by RVD when exposed to hypotonic challenge, the RVD was incomplete. When challenged with hypotonic solution the cells could only achieve a partial recovery in volume. RVD capacity changed periodically as the cells passed through the cell cycle. The cells increased their RVD capacity gradually, reaching a peak during G1 (78.0%6.4%, n=22, P<0.01). Then, RVD capacity dropped when the cells entered S phase, reaching a minimum at the end of S phase (36.7%8.5%, n=15, P<0.01), and then increased gradually again in G2 and M phases until the end of the next G1 phase. The RVD in CNE-2Z cells was associated with the activity of chloride channels. The RVD could be inhibited by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), tamoxifen, and ATP. Of the three inhibitors, ATP was the most effective. 10 mM of ATP blocked the RVD by 91.2% (n=5, P<0.01). NPPB (100 M) and tamoxifen (20 M) inhibited a part of the RVD (by 74.0% and 48.7% respectively; n=10 and 19, P<0.01). The expression of ClC-3, one of the candidates for the volumeactivated chloride channel, in the CNE-2Z cell was also monitored during the cell cycle by immunofluorescence techniques. ClC-3 was expressed predominantly inside the cells, probably inside the nucleus, but not in the cell membrane. ClC-3 expression was restricted to the nuclear region during G1, and then spread out gradually when the cells progressed through S and G2 phases. However, ClC-3 immunofluorescence was again restricted to the nuclear region following cell division. The changes in ClC-3 levels were directly opposite to the changes of RVD capacity: the cells expressed low levels of ClC-3 in G1 when RVD capacity peaked, whereas ClC-3 expression increased greatly during S phase when RVD capacity declined to a minimum. The results suggest that RVD may play an important role in controlling the cell cycle. RVD in the CNE-2Z cell is associated with chloride channels. ClC-3 may not function as a chloride channel associated with RVD in the CNE-2Z cell. Rather, ClC-3 may work as an important controlling factor in the progress of the cell cycle. Supported by the Wellcome Trust and MRC.

46. GENE REGULATION IN SENESCENT FIBROBLASTS: THE ROLE OF TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATORS Pidder Jansen-Du¨ rr Institute for Biomedical Ageing Research, Austrian Academy of Sciences, Rennweg 10, 6020 Innsbruck, Austria (E-mail: [email protected])

As most primary mammalian cells, diploid human fibroblasts actively proliferate when explanted into tissue culture until the cells reach a quiescent state referred to as replicative senescence. Senescent fibroblast show a specific cell cycle defect in that they arrest in the G1-phase of the cell cycle. G1-arrest of senescent fibroblasts has been associated

A19 with changes in the expression of a variety of growth regulatory genes, in particular, genes coding for inhibitors of cyclin-dependent kinases. While it is clear that some of these inhibitors accumulate in senescent cells, mechanisms underlying their senescence-specific accumulation remain unclear. We have addressed the role of chromatin-modifying enzymes in replicative senescence and found senescence-specific changes in histone deacetylases. The role these changes play in senescence-specific gene expression is currently under investigation. We have also addressed the possibility that differential metabolic stabilization of cdk inhibitors contributes to the accumulation of these proteins in senescent fibroblasts. To this end, we have determined the translation rate and metabolic stability of p16, p21, p27, and a few additional cell cycle regulators. The results suggest that there is no general decrease of proteasome activity in senescent fibroblasts; however, it appears that there are specific defects in ubiquitination occurring in senescent fibroblasts. Current studies address the role of type E3 ubiquitin ligases in replicative senescence.

47. S-PHASE AND MITOSIS IN STEM CELLS OF BASAL FLATWORMS P. Ladurner, K. Nimeth, R. Gschwentner, M. Mahlknecht, M. Hrouda, A. Bode, W. Salvenmoser and R. Rieger Institute of Zoology and Limnolgy, University of Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria (E-mail: [email protected])

Pluripotent stem cells are found in many metazoan taxa. Stem cells are not differentiated, they generate more stem cells (self renewal) and give rise to a variety of differentiated cell types. The mechanisms by which stem cells differentiate are extensively studied in certain model systems (hematopoietic cells, neural crest cells, mouse and human embryonic stem cells or cnidarian interstitial cells). Free-living Platyhelminthes (‘Turbellaria’) possess a unique totipotent stem cell system. In ‘Turbellaria’, stem cells (neoblasts) are the only cells that can divide and supply all cells for development, growth and regeneration. We have applied 5-Bromo-2 -deoxy-uridine (BrdU) and phosphorylated Histone H3 (phos H3) labeling to demonstrate the distribution of S-phase and mitotic cells and in the flatworm Macrostomum sp. After 30 days of starvation, the number of mitoses decreased from 26.88.5 (n=22) to 0.70.9 (n=23). Dramatic mitotic response was observed after feeding. The number of mitoses increased after 30 min feeding to 4.22.8 (n=10) mitotic cells and to 17.85.9 (n=12) mitoses after a feeding period of 2 h. A first maximum of mitotic cells was reached after 6 h (41.08.6; n=9) which was followed by a decline to 26.37.5 (n=10) after 12 h feeding period. The initial rapid increase of mitoses up to 6 h after feeding is probably due to neoblasts arrested in G2 phase. After depletion of this G2 pool of neoblasts, the number of mitoses decreased (minimum at 12 h) and rose again to 57.119.5 (n=11) probably because neoblasts have gone through S-phase and G2 reached mitosis. BrdU/phos. H3 double labeling reveald additional information on cell cycle duration of different neoblast populations. Ultrastructural analyses have shown three stages of neoblast differentiation based on nuclear morphology and heterochromatin patterning. In order to identify individual discernible neoblasts with a short cell cylce we focus to ‘pair-forming’ neoblasts at the level short behind the eyes. Those pairs appeared to occur in a constant pattern in 4 h pulse chase experiments indicating a recent division. We expect those stem cells to be localized in a specific ‘niche’. After asymmetric division the stem cell remains in the niche while the daughter cells migrates torwards the rostrum (pairs after 4 h chase time). Additional experiments using pulse chase experiments with different chase times, treatment of animals with cytochalasin B or hydroxyurea, microinjection of fluorescent tracers or labeled neoblasts, and molecular markers (e.g. vasa-like genes) as well as immunogold labeling will lead to a better understanding of the cell cycle of stem cells in ‘Turbellaria’. FWF-13060-BIO.

48. HISTONE DEACETYLASE 1 IS ESSENTIAL FOR EMBRYONIC DEVELOPMENT IN MICE AND UNRESTRICTED PROLIFERATION OF EMBRYONIC STEM CELLS G. Lagger1, D. O’Carroll2, M. Rembold1, H. Kier1, G. Weitzer3, T. Jenuwein2 and C. Seiser1

A20 1

Institute of Medical Biochemistry, Division of Molecular Biology, University of Vienna, Dr.Bohrgasse 9/2, 1030 Vienna (E-mail: [email protected]); 2Institute of Molecular Pathology, Dr.Bohrgasse 7, A-1030 Vienna; 3 Institute of Medical Biochemistry, Div. Biochemistry, University of Vienna, Dr.Bohrgasse 9/2, A-1030 Vienna

An important step in the regulation of eukaryotic gene expression is the posttranslational modification of nucleosomal histones, which allows specific chromosomal regions to become transcriptionally active or silenced. Over all, reversible acetylation of conserved lysine residues within the N-terminal tails of the core histones has been studied extensively during the last years. Various acetyltransferases (HATs) and histone deacetylases (HDACs) have been cloned to date and they seem to play an important role for transcription, chromatin remodelling and differentiation. Histone deacetylases often act as transcriptional corepressors, present in huge multiprotein complexes , and a possible role in human cancer seems to get more and more feasible as suggested by several recent publications (for review see 1). We cloned murine histone deacetylase 1 (mHDAC1) with a differential mRNA approach and investigated the role of HDAC1 for growth of the T cell line B6.1 (2). Since little is known about the role of HDACs in development, we generated a gene disruption of HDAC1 in mice using conventional knock out technologies. While mice heterozygous for the mutation are not distinguishable from wildtype animals, null mice die around day 9.5 of gestation. They exhibit extreme developmental abnormalities, visible from 7.5 days post coitum (d.p.c.) on. To get insight in the molecular mechanism of this effects, we performed blastocyst outgrowth experiments. We were able to isolate several embryonic stem cell lines, which were wildtype, heterozygous and null for the HDAC1 allele. All cell lines are stable over more than twenty passages. In vitro differentiation experiments demonstrated a reduced proliferation capacity of cell lines homozygous for the HDAC1 mutation, although differentiation per se seems not to be affected. The reduced proliferation rates of HDAC1 null cell lines are not due to increased apoptosis levels. We are currently investigating the expression patterns of different proteins, important for cell cycle regulation and have some candidates which might be responsible for the cell cycle prolongation of homozygous HDAC1 cells. Although HDAC1 belongs to a protein family with several homologous enzymes, often present in the same complexes, we can show with this work, that HDAC1 is indispensable for mouse development and unrestricted proliferation of embryonic stem cells.

References C WD, S E, 2000. Journal of Cellular Physiology 184: 1–16. B S, T J, L G, K H, K K, S C, 1996. Mol Cell Biol 17(9): 5033–5543. 49. Cdc25A PHOSPHATASE INTERACTS WITH AKT/PKB AND SUPPRESSES APOPTOSIS INDUCED BY SERUM DEPRIVATION C. Leisser1, G. Fuhrmann1, G. Rosenberger1, M. Grusch1, T. Halama2, I. Mosberger1, C. Cerni3 and G. Krupitza1 1 Institute of Clinical Pathology, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria (E-mail: [email protected]); 2Department of General Dermatology, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria; 3Institute of Tumor Biology and Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria

The phosphatase Cdc25A was shown to be a target of the transcription factor c-Myc. Myc-induced apoptosis entirely depended on Cdc25A expression and Cdc25A over-expression could substitute for Myctriggered apoptosis. These findings suggested that an universal downstream component of Myc-mediated apoptosis was identified. However, we recently reported that during TNF-induced apoptosis,

Cell Biology International, Vol. 25, No. 2, 2001 which required c-Myc function, Cdc25A was down-regulated in human ovarian carcinoma N.1 cells. Here we provide evidence that Cdc25A rendered immortalised rat embryonic 423 cells refractory to apoptosis which was induced by serum deprivation and in absence of detectable c-myc levels. The specificity of the survival-promoting activity of Cdc25A was evidenced by the reversion of the anti-apoptotic cdc25A-phenotype by a conditional, full length cdc25A antisense construct. Akt Coimmunoprecipitated with Cdc25A and inhibition of apoptosis of cdc25A- and akt/PKB- over-expressing pooled clones with SU4984, NF023 and Rapamycin placed cdc25A and akt function downstream of FGF.R, PDGF.R, G-proteins and PP2A. Moreover, control cells, cdc25A and akt over-expressing clones exhibited similar apoptotic patterns upon treatment with LY-294002, thus, neither akt nor cdc25A depended on PI.3 kinase function in rat 423 cells. Cdk2, which is the only known target of Cdc25A, became activated by this phosphatase and Cdc25A limitation inhibited Cdk2 activity. Cdk2 over-expression, which was expected to substitute for Cdc25A, did not mimic the anti-apoptotic effect of Cdc25A. This suggests that the survival effect of Cdc25A involved targets different from Cdk2. Survival-promoting target(s) of Cdc25A seem to be more common substrates and not entirely specific to Cdc25A because cdc25B and cdc25C over-expression also inhibited apoptosis of rat 423 cells.

50. ENZYMES INVOLVED IN HISTONE ACETYLATION DURING THE CELL CYCLE: LESSIONS FROM MAIZE AND PHYSARUM Peter Loidl Department of Microbiology, University of Innsbruck, Medical School; Fritz-Pregl Str. 3/II, A-6020 Innsbruck, Austria (E-mail: [email protected])

Posttranslational acetylation of core histones is involved in the regulation of various cellular processes, like transcription, silencing, replication, repair and recombination. Histone acetylation is established and maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs); these enzymes exert their function in multiprotein complexes. A significant contribution to our knowledge on HATs and in particular on HDACs stems from studies of plants and fungi. It is well known that histone acetylation is regulated in a cell cycle dependent manner and the involved enzymes also fluctuate during the cell cycle. We have demonstrated this during the naturally synchronous cell cycle of macroplasmodia of the myxomycete Physarum polycephalum and during the partially synchronous cell cycle of meristematic root tip cells in germinating maize embryos. As in vertebrate cells, both enzyme activities exist in multiple enzyme forms that belong to distinct HAT- and HDAC-families. In addition, plants contain numerous forms of a nucleolar HDAC (HD2) which has not been described in vertebrates so far. This contribution provides a summary of the various HAT- and HDAC-forms in maize, arabidopsis and physarum as well as a synopsis of the biochemical properties, subcellular localization, substrate and site specificity and expression levels during the cell cycle. current data suggest that distinct enzyme forms are predominantly associated with certain nuclear processes.

51. NF-Y MEDIATES THE TRANSCRIPTIONAL INHIBITION OF THE CYCLIN B1, CYCLIN B2, AND CDC25C PROMOTERS UPON INDUCED G2 ARREST Isabella Manni1,2, Giuseppina Mazzaro1, Aymone Gurtner1, Roberto Mantovani, Ulrike Haugwitz4, Karen Krause4, Kurt Engeland4, Ada Sacchi1, Silvia Soddu1 and Giulia Piaggio1,2 1 Laboratorio Oncogenesi Molecolare, Istituto Regina Elena, CRS; Rome, Italy (E-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]); 3Dipartimento di Biologia Animale Universita` di Modena e Reggio, Italy (E-mail: [email protected]); 4Medizinische Klinik II, Max-Burger-Forschungszentrum, Universitat Leipzig, Leipzig, Germany ([email protected])

During normal cell cycles, the function of mitotic cyclin/cdk1 complexes, as well as of cdc25C phosphatase, is required for the G2 phase

Cell Biology International, Vol. 25, No. 2, 2001 progression. Accordingly, the G2 arrest induced by DNA-damage is associated with a down-regulation of mitotic cyclins, cdk1, and cdc25C phosphatase expression. We found that the promoter activity of these genes is repressed in the g2 arrest induced by DNA-damage. We asked whether the CCAAT-binding factor NF-Y modulates mitotic cyclins, cdk1, and cdc25C gene transcription during this type of G2 arrest. In our experimental conditions, the integrity of the CCAAT boxes of cyclin B1, cyclin B2, and cdc25C promoters, as well as the presence of a functional NF-Y complex are strictly required for the transcriptional inhibition of these promoters. Furthermore, a dominant-negative p53 protein, impairing doxorubicin-induced G2 arrest, prevents transcriptional down-regulation of the mitotic cyclins, cdk1, and cdc25C genes. We conclude that, as already demonstrated for cdk1, NF-Y mediates the transcriptional inhibition of the mitotic cyclins and the cdc25C genes during the p53-dependent G2 arrest induced by DNA-damage. These data suggest a transcriptional regulatory role of NF-Y in the G2 checkpoint after DNA-damage.

52. CELL CYCLE-DEPENDENT CHANGES OF NEGATIVE REGULATORS (p53, p16 AND p21) IN NON-TRANSFORMED HUMAN FIBROBLAST CULTURES Zoltan L. Marcsek and Zsuzsanna Kocsis Department of Experimental Pathology and Mutagenesis, National Institute of Chemical Safety, ‘Jozsef Fodor’ National Center for Public Health, H-1097 Budapest, Gyali t 2-6, Hungary (E-mail: [email protected])

It is widely accepted that cell proliferation is regulated by the cyclindependent kinases (cdk-s) around the restriction point in the G1 phase of the cell cycle. The mechanism was clarified regarding the proliferating populations but much less is known about the regulatory processes active in resting/G0 cell population, maintain the resting state. We are studying the behavior of the cdk related regulatory pathways in non-transformed human cell systems (WI-38 and MRC5 cell lines). Both cell lines grow in monolayer, exhibit contact inhibition and their proliferation is serum dependent. Moreover, all studied proliferation regulatory pathways are wild-type (p53, cdk-s, cyclins, RB, cdk inhibitors). Using a proliferation panel composed of cultures in different (exponentially growing, serum starved and contact inhibited) proliferation states we examined p53, p21(WAF1), p15, p16 expression at both the mRNA and protein level in parallel cultures. Our results clearly shows that the regulatory mechanisms of proliferating cell are different from the control processes active in cell cycle arrest and/or in resting cell population. In our experimental system it seems, that the p53 protein levels do not activate the transcription of p21 mRNA in silent, non-proliferating cells, and the p53 protein level does not correlate with the cellular p53 mRNA content. On the basis of our results we propose a p53-independent control mechanism which keep cells in non-proliferating state in fibroblast-originated non/ transformed models.

53. ANALYSIS OF CYCLIN D1 AND CYCLIN D3 IN CORRELATION WITH THE RESTRICTION POINT IN SINGLE MAMMALIAN CELLS Hanna-Stina Martinsson, Anders Zetterberg and Maria Starborg Department of Oncology-Pathology, Karolinska Institutet, CCK R8:04, 171 76 Stockholm, Sweden (E-mail: [email protected])

The importance of D-type cyclins for progression through G1 has been shown in numerous investigations. Most studies have been performed on cell populations that have been starved and allowed to reenter the cell cycle or synchronized by drugs. The aim of this study is to investigate the importance of D-type cyclins for G1 progression in single cells under normal culture conditions using time-lapse video-microscopy in combination with immunofluorescence. Time-lapse video-microscopy allows determination of cell age after mitosis of individual cells. This method has been used to show that the restriction point in Swiss-3T3 cells occurs 180–240 minutes after mitosis. The age determined cells can later be identified after immuno-

A21 fluorescence staining. Normally growing Swiss-3T3 cells were time lapse recorded and labeled against cyclin D1 and cyclin D3. Both of these proteins were present in the nucleus immediately after mitosis. Levels of cyclin D1 increased around 200 minutes after mitosis, coinciding with the restriction point, and decreased around 300–350 minutes when the cells entered S-phase. A second increase was observed around 500–600 minutes after mitosis indicating higher cyclin D1 levels in G2. Cyclin D3 follows a similar pattern, but with a delayed decrease in S-phase. Investigation of the levels of cyclin D1 and cyclin D3 compared to DNA content showed high levels of both cyclins in G1. The levels dropped in S-phase as the DNA-content of the cells increased, but cyclin D3 levels remained high further into the S-phase than cyclin D1 levels. A peak in G2 was also observed for both cyclins. Presence of cyclin D1 and cyclin D3 in the nucleus during the entire G1 phase with a rise in levels at the restriction point indicates that cyclin D1 and cyclin D3 could play an important role in regulation of the passage through the restriction point. Higher levels of cyclin D1 and cyclin D3 in G2 suggests that the two cyclins are expressed in G2. To test this Swiss-3T3 cells were double labeled for one D-type cyclin and cyclin A. About 10% of the cells that expressed cyclin D1 also had cyclin A expression, and about 30% of the cells that expressed cyclin D3 had cyclin A expression. Since cyclin A only is expressed during S-phase and G2, this indicates that the two D-type cyclins are expressed sometime during S or G2. The higher number observed for cyclin D3 is consistent with the observation that cyclin D3 levels remained higher for a longer period of the S-phase than the cyclin D1 levels. Determining if the overlapping expression of the D-type cyclins and cyclin A occurs in S or G2 is now under further investigation.

54. G1 CYCLINS AND CDKS IN THE EARLY DEVELOPMENT OF SEA URCHINS Jennifer C. Moore1,2, Jan L. Sumerel1,2, Jason A. Nichols1,3, Bradley J. Schnackenberg1,4, Albert J. Poutska5, Gary Wessel6 and William F. Marzluff1–4* 1 Program in Molecular Biology and Biotechnology, 2 Department of Biochemistry and Biophysics, 3Department of Biology, 4Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill (E-mail: [email protected]), and 5Max Planck Institute for Molecular Genetics, Berlin, 6Department of Molecular and Cellular Biology, Brown University

Unlike most organisms in which oocyte development is arrested in meiosis, sea urchins store their gametes as haploid eggs. The eggs are arrested in a pseudo-G0 state and proceed into S phase after remodeling of the sperm chromatin and fusion of the male and female pronuclei. The developing embryos then undergo several rapid rounds of DNA synthesis followed by cell division. These early cell cycles do not contain G1 or G2 phases. We have cloned the G1 cyclins E and D and their partners cdk2 and cdk4/6 from sea urchins. In Xenopus it has been shown that the embryo transcribes no RNA before the midblastula transition. One of the major cell cycle regulatory events that occurs at the frog MBT is the down regulation of cyclin E protein and associated kinase activity and the up-regulation of p27xic1, which presumably plays a role in the introduction of gap phases. It has previously been shown that unlike frogs, sea urchins undergo some RNA synthesis in the unfertilized egg and critical genes are transcribed in the early embryo. However like frogs, there is a time when the cell cycle regulation changes from alternating S and M phases to cycles containing gap phases. We have termed this the ‘early blastula transition’. Although cdk4 protein and mRNA levels are relatively constant during the first 22 h of embryogenesis, cyclin D mRNA is first found in significant amounts at the morula stage (10 to 12 h) and remains present during later embryogenesis. When the levels of cyclin D mRNA increase, there is a substantial decrease in cyclin E activity and protein levels. Ectopic expression of cyclin D early in embryogenesis results in embryo death, possibly due to misregulation of the cell cycle that disrupts development. A sea urchin pRb homologue has recently been identified and its role in this process is currently being investigated. These results suggest that sea urchins undergo a transition at the start of the blastula stage. Cyclin D may be involved in

A22 establishing gap phases and down regulating cyclin E, completing the switching of cell cycle regulation in the embryo. Supported by NIH Grant GM58921.

55. REGULATION OF GLYCOLYSIS DURING MAMMALIAN CELL CYCLE Dieter Morandell1,2, Erich Eigenbrodt3, Pidder Jansen-Du¨ rr1,2 and Werner Zwerschke1,2 1 Tyrolean Cancer Research Institute, at the University of Innsbruck, Austria (E-mail: [email protected]); 2Austrian Academy of Sciences, Institute for Biomedical Aging Research, Innsbruck, Austria (E-mail: [email protected]); 3Institute for Biochemistry & Endocrinology, University of Giessen, Germany

Although our knowledge of the protein machinery regulating cell proliferation has increased tremendously over the recent years, we are still at the beginning to understand how nutrients contribute to proliferation control in multicellular organisms. There is, however, evidence that phosphometabolites derived from both glycolysis and the pentose phosphate pathway provide some of the signals linking metabolic conditions to cell proliferation. The glycolytic phosphometabolites, which are necessary for the biosynthesis of nucleic acids, phospholipids and complex carbohydrates, are up-regulated in the G1 phase of the cell cycle, and constant high levels of phosphometabolites have been detected in rapidly proliferating tumor cells. The key enzymes regulating the glycolytic phosphometabolite pools and glycolytic flux rate are hexokinase, 6-phosphofructo 1-kinase, and pyruvate kinase. Special isotypes of this enzymes are expressed in proliferating cells, and the expression is cell cycle regulated. We have analyzed the expression and regulation of glycolytic key enzymes, especially pyruvate kinase, during the cell cycle in human fibroblasts.

56. CKS1 IS REQUIRED FOR THE TIMELY REGULATION OF CDC28 PHOSPHORYLATION/ DEPHOSPHORYLATION IN THE MORPHOGENESIS CHECKPOINT OF S. CEREVISIAE M. C. Morris1 and S. I. Reed1 1 The Scripps Research Institute, Department of Molecular Biology, 10550 North Torrey Pines Rd, La Jolla, CA-92037, U.S.A. (E-mail: [email protected])

Timely progression through the cell cycle of eucaryotic cells is regulated by activation of cdk-cyclin complexes, by reversible phosphorylation of three residues, T161 by CAK kinase, and T14 and Y15 by homologs of the Wee1/Mik1 and Myt1 kinases and members of the Cdc25 phosphatase family. In Saccharomyces cerevisiae in contrast, regulation of the unperturbed cell cycle is altogether independent of phosphorylation of Cdc28, the homolog of Cdc2, by Swe1, the homolog of Wee1, mitotic progression being primarily regulated by the periodic accumulation and proteolysis of mitotic cyclins and regulators of the APC. Perturbation of the actin cytoskeleton however leads to activation of a surveillance mechanism termed the morphogenesis checkpoint, which delays nuclear division, by increasing the levels of Cdc28 tyrosine phosphorylation by Swe1 kinase. Small Cks proteins are essential for viability and cell cycle progression through direct interaction and regulation of cyclin-dependent kinases. Several studies have suggested they are involved in promoting entry into mitosis, by modulating the phosphorylation status of cdks, presumably by targeting Cdc25 phosphatase to cdk-cyclin complexes. We have investigated the role of Cks1 in the regulation of Cdc28 by Swe1 and Mih1, the homolog of Cdc25, in S.cerevisiae. Our data reveal that Cks1 plays an essential role in regulating the onset of nuclear division in the morphogenesis checkpoint of S.cerevisiae. Cks1 is indeed involved in regulating formation, periodic tyrosine phosphorylation and activation of mitotic Cdc28-Clb2 complexes. Upon activation of the morphogenesis checkpoint, cells lacking functional Cks1 were unable to undergo nuclear division due to transcriptional downregulation of Clb2, downregulation of Cdc28-Clb2 complex formation, constitutive expression of Swe1 and consequent constitutive tyrosine phosphorylation of Cdc28. In addition, deletion of Swe1 partially suppresses the phenotype of a temperature-sensitive Cks

Cell Biology International, Vol. 25, No. 2, 2001 strain, and the inviability of Cks1 deletion, revealing a genetic and functional interaction between these two gene products. Finally, we show that Cks1 only binds Cdc28-Clb2 complexes which are phosphorylated by Swe1 on Y19, suggesting that Swe1 phosphorylation may induce a conformational change which increases the affinity of Cks1 for Cdc28-Clb2 complexes. From these data, we propose that in the morphogenesis checkpoint, Cks1 plays a central role in coupling the phosphorylation status of Cdc28 to pathways which regulate entry into mitosis, and may act as a switch to promote either dissociation of Swe1 from Cdc28 and/or targeting of Mih1 to phosphorylated Cdc28-Clb2 for subsequent dephosphorylation.

57. THE HUMAN PAPILLOMAVIRUS E7 ONCOPROTEIN BINDS TO THE NUCLEAR LOCALIZATION SITE OF IGFBP-3 Barbara Moser1,2, Sue M. Firth3, Boris Mannhardt1,2, Robert C. Baxter3, Pidder Jansen-Du¨ rr1,2 and Werner Zwerschke1,2 1 Tyrolean Cancer Research Institute at the University of Innsbruck, Innrain 66, 6020 Innsbruck/Austria (E-mail: [email protected]); 2Institute for Biomedical Aging Research of the Austrian Academy of Sciences, Rennweg 10, 6020 Innsbruck/Austria; 3Growth Research Laboratory, Kolling Institute of Medical Research, St Leonards, NSW 2065, Sydney/Australia

Human papillomaviruses of the high-risk group cause cancers in humans and this requires the activity of the viral proteins E6 and E7. Expression of both, E6 and E7, is sufficient to overcome the cellular senescence program in primary human keratinocytes. Although it is generally assumed that this property of E7 depends on its ability to overcome the inhibitory activity of cell cycle regulatory proteins, such as the members of the retinoblastoma protein family or the cyclindependent kinase inhibitors p27Kip1 and p21Waf1, it is clear from many other studies that additional activities of E7 are necessary for cellular immortalization. We found that E7 targets insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), the product of a p53-inducible gene that is highly expressed in senescent cells. IGFBP-3 is a member of a protein family that regulates the mitogenic activity of IGF-I and mediates antiproliferative and proapoptotic activity in several cell types. IGFBP-3 induced apoptosis is blocked by E7, which triggers the proteolytic cleavage of IGFBP-3. An IGF-I-independent function of IGFBP-3 was assumed, which involves the uptake of extracellular IGFBP-3 and subsequent localization in the nucleus. Recently, it was shown that nuclear import of IGFBP-3 depends on the Importin / complex. We mapped the E7-binding domain on IGFBP-3 to the conserved carboxyterminal domain. A point mutation in the nuclear localization site (NLS) of IGFBP-3, which is essential for IGFBP-3/Importin interaction, completely abolished binding of E7. These data suggest that E7 may interfere with the nuclear uptake process of IGFBP-3.

58. A COMMON COMPONENT OF EUKARYOTIC CELL-CYCLE REGULATION IS REQUIRED FOR ORGAN SPECIFIC CYTOKINESIS IN ARABIDOPSIS THALIANA Sabine Mu¨ ller and Marie-Theres Hauser Centre of Applied Genetics, University of Agricultural Sciences Vienna, Austria (E-mail: [email protected])

Although the mechanism of cell division is rather conserved throughout evolution, plant cells seem to have adopted a distinct way to undergo cytokinesis, a process that physically separates the cytoplasm of daughter cells after nuclear division. Mutation in the Arabidopsis thaliana PLEIADE gene results in a root specific phenotype, depicting several features that are characteristic for cytokinesis defects in plants as multinucleate cells, cell wall stubs and synchronised cell division in incompletely separated cells. The observation, that root morphology is dramatically altered, while the aerial parts of the plant are not affected by the mutation, might provide new insights in the regulation of organ specific cytokinesis. The significant sequence similarity of PLEIADE

Cell Biology International, Vol. 25, No. 2, 2001 to a CDK substrate, which upon inactivation blocks cellular cleavage, but not nuclear division in human cells, support PLEIADE’s requirement in cytokinesis. We thus suggest that PLEIADE belongs to a new class of proteins required for late steps of cytokinesis in plants and animals. This project was funded by the EU grant PL960217 FORMA.

59. CYCLIN D3 IS CRITICAL FOR GLUCOCORTICOID-INDUCED CELL CYCLE ARREST BUT NOT FOR APOPTOSIS IN LYMPHOBLASTIC LEUKEMIA CELLS Michael J. Ausserlechner1, Petra Obexer1 and Reinhard Kofler1,2 1 Institute for General and Experimental Pathology, University of Innsbruck, Medical School, Austria (E-mail: [email protected]); 2Tyrolean Cancer Research Institute, Innsbruck, Austria

Glucocorticoids (GC) induce G1 cell cycle arrest and apoptosis in lymphoblastic leukemia cells, but the molecular basis for this therapeutically exploited phenomenon is not well understood. We investigated the effect of GC on cell cycle regulatory genes in bcl-2 overexpressing human CCRF-CEM acute lymphoblastic leukemia cells, in which ectopic bcl-2 delays GC-induced cell death but does not effect cell cycle arrest. GC-treatment was associated with complete repression of cyclin D3, reduction of CDK4, hypophosphorylation of pRB and repression of E2F-dependent genes as well as of c-myc and its targets, suggesting that these two pathways were shut down by GC. To investigate whether cyclin D3 repression might be critical for GCinduced cell cycle arrest and/or apoptosis we generated CCRF-CEM sublines with conditional overexpression of cyclin D3. Conditional overexpression of cyclin D3 per se increased proliferation in these leukemia cells but did not alter their apoptotic response to GC treatment making the analysis of cyclin D3 effects upon GC-induced inhibition of proliferation difficult. To study the role of cyclin D3 in GC-mediated cell cycle arrest in the absence of obscuring apoptotic effects, we infected the above cell lines with a recombinant retrovirus that constitutively expressed bcl-2, thereby preventing apoptosis. We found that cyclin D3 overexpression almost completely reconstituted proliferation under GC-treatment. Repression of cyclin D3 and activation of the pRB/E2F pathway seems therefore to be critical for GC-mediated cell cycle arrest but not for apoptosis induction. Supported by the Austrian Science Fund (F204).

60. CYPLASIN KILLS PREFERENTIALLY AUTONOMOUS CYCLING CELLS C. Petzelt1 and D. Werner2 1 Experimental Anesthesiology, Dept. of Anesthesiology and Intensive Care, University Hospital Charite´ , FH31, Spandauer Damm 130, D-14050 Berlin, Germany (E-mail: [email protected]); 2Dept. of Cellular Biochemistry, German Cancer Research Center, INF 280, D-60120 Heidelberg, Germany (E-mail: [email protected])

Cyplasin is a cytotoxic protein isolated from the secreted mucus of glands from the European sea hare Aplysia punctata. It has a MW of 46 kd and is heavily glycosylated. Its cytotoxic properties are intriguing insofar as it affects in nanomolar concentrations only cells which are, or have become, capable of autonomous growth, a property characteristic for many if not all tumor cells. In order to test such a hypothesis, numerous human and non-human established cell lines as well as primary cultures were treated with cyplasin. In all cases, the cytotoxicity predicted was obtained. On the other hand, injection of cyplasin in mikromolar concentrations is well tolerated in all animals studied (humans included). To compare the response of resting versus cycling cells, two systems were chosen. (A) Human endothelial cells of the vein of the umbilical cord, which within the wall of the vein behave as differentiated cells with all their characteristics of endothelial cells but do not divide. Within minutes after their isolation, they enter into the cell cycle and start dividing. If the endothelial cells still within the vein are incubated with micromolar

A23 concentrations of cyplasin, they are not affected at all; when the cyplasin is removed and the cells isolated, a normally growing culture ensues. If, however, the cells are first isolated and then treated with cyplasin, they become sensitive to it (at nanomolar concentrations) within a few minutes. (B) The second system studied has similar but inverse properties: Dopaminergic phaeochromocytoma cells, PC-12, grow in vitro as a normal cell line. Upon addition of nerve growth factor they differentiate within a few days into neuron-like cells and stop completely and irreversibly their proliferation. Consequently, proliferating undifferentiated PC-12 cells are susceptible to cyplasin in the nanomolar range; the more the cells differentiate (visible by the formation of dendritelike structures), the less sensitive they are to cyplasin, until finally, after four days, even micromolar concentrations of cyplasin have become ineffective. Recently it was established that cyplasin actually belongs to a family of closely related proteins of various sizes having long common sequences together with some microheterogeneities. Not all of those cyplasins are cytotoxic. We have now produced a recombinant cyplasin, about a third longer than the original one, which has very similar cytotoxic properties as the original native cyplasin, thus allowing further studies on its cycle-dependent toxicity. Supported in part by Marine Therapeutics, Ltd.

61. CYCLIN B1 GENE IS ACTIVELY TRANSCRIBED DURING MITOSIS IN HELA CELLS Selvaggia Sciortino*†, Aymone Gurtner*, Anup Dey§, Ada Sacchi*, Keiko Ozato§ and Giulia Piaggio* *Laboratorio di Oncogenesi Molecolare, CRS-IRE, Rome, Italy (E-mail: [email protected]; [email protected]; [email protected]); §Laboratory of Molecular Growth Regulation, NICHD, NIH, Bethesda, Maryland 20892 (E-mail: [email protected]; [email protected]; [email protected])

In mammalian cells, the expression level of cyclin B1 gene plays a critical role in the progression through mitosis. Here we demonstrate that the transcriptional activity of the human cyclin B1 promoter as well as the rate of gene transcription is high during mitosis. Indeed, the cyclin B1 promoter maintains an open chromatin configuration at the mitotic stage. Consistent with this, we show, by genomic footprinting and chromatin immunoprecipitation experiments, that cyclin B1 promoter is occupied and bound to NF-Y during mitosis in vivo. Our results provide the first example of selective RNA polymerase II dependent transcription during mitosis in mammalian cells.

62. ANALYSIS OF CELL CYCLE PROTEINS DURING MOUSE HEPATOCYTE PROLIFERATION INDUCED BY THE MITOGEN TCPOBOP M. Pibiri, C. Cossu, G. Simbula, D. Concas, A. Columbano and G. M. Ledda-Columbano Department of Toxicology, Unit of Oncology and Molecular Pathology, University of Cagliari, Italy (E-mail: [email protected])

It was previosuly shown that hepatocyte proliferation induced by the mitogen TCPOBOP is independent of changes in cytokines, immediate early genes and transcription factors considered to be necessary for liver regeneration after partial hepatectomy (PH). To further investigate the differences between mitogen-induced mouse hepatocyte proliferation and liver regeneration after PH, in the present study we have measured the expression of cyclin D1, cyclin E, cyclin A, and of cyclin-dependent kinases CDK4 and CDK6. The involvement of the cyclin-dependent kinase inhibitors p21 and p27 and of the oncosuppressor gene p53 was also examined at different times after stimulation of hepatocyte proliferation. Results showed that a single administration of TCPOBOP caused a very rapid increase in the levels of cyclin D1 when compared to 2/3 PH (8 h versus 30 h). The rapid increase in cyclin D1 protein levels was associated with a faster increase in the expression of the S phase-associated cyclin A (24 h versus 36 h of PH mice). Accordingly, measurement of BrdU incorporation revealed that while approximately 8% hepatocytes were

A24 BrdU-positive as early as 24 h after TCPOBOP, no significant changes in BrdU incorporation were observed at the same time point after 2/3 PH. A different pattern of expression in the two models of hepatocyte proliferation, was also observed in other proteins involved in cell cycle control such as CDK2, CDK4 and CDK6. The results indicate that cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by the primary mitogen TCPOBOP and suggest that a direct effect on this cyclin may be responsible for the rapid onset of DNA synthesis. Supported by AIRC and MURST ex-40%, Italy.

63. HCF-1 INTERACTS WITH THE MYC-INTERACTING Zn-FINGER PROTEIN MIZ-1 D. Piluso, H. Wong and J. P. Capone Department of Biochemistry, McMaster University, 1200 Main St. W. Hamilton, Ontario, Canada, L8N 3Z5 (E-mail: [email protected])

Host cell factor (HCF-1) is a large and highly unusual cellular polypeptide which was originally identified as an accessory factor required for transactivation of herpes simplex virus genes by the viral transactivator VP16. Increasing evidence indicates that HCF is a multifunctional protein that is interwoven into complex combinatorial networks of cellular factors that variously serve to regulate cellular and viral gene expression, cell proliferation, and transformation. HCF is essential for cell cycle progression since cells that harbor a temperature-sensitive point mutation in HCF reversibly arrest at G0/G1 at the non-permissive temperature. The cellular roles and mechanisms of action of HCF remain to be determined. To begin to elucidate the cellular functions of HCF, we used two hybrid interaction cloning strategies in order to identify novel HCFinteracting proteins. Through this approach, we have identified the Zn-finger transcription factor Miz-1 as a novel cellular factor that binds to HCF. Miz-1 was previously identified as a factor that binds to the cellular oncogene c-myc. Moreover, Miz-1 was shown to arrest cell cycle progression and to activate transcription of certain promoters that are involved in cell cycle regulation in a manner attenuated by Myc. We show here that Miz-1 binds directly to HCF and does so via determinants that are required for cell-cycle progression by HCF. Conversely, HCF independently targets two separate regions of Miz-1; the amino-terminal POZ domain, an element that is known to mediate repression of transcription, and a region near the C terminus, the latter which is also required for Myc interaction. Interestingly, the C terminal region serves as a potent transactivation domain when tethered to DNA, indicating that HCF directly targets the transactivation function of Miz-1. Our findings suggest that there is a functional interplay between Miz-1 and HCF in modulating cell cycle control, and perhaps in the regulation of viral and cellular genes.

64. MOLECULAR DISSECTION OF NPM/ALK ONCOGENICITY Doris Polgar1, Sandra Fassl1, Ingrid Simonitsch1, Peter Duchek1, Andrea Lamprecht1 Christa Cerni2 and Georg Krupitza1 1 Institute of Clinical Pathology, University of Vienna, Waehringer Guertel 18-20, A-1090 Wien, Austria (E-mail: [email protected]); 2Institute of Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Wien, Austria

Anaplastic large cell lymphomas (ALCL) constitute approximately 5% of non-Hodgkin’s lymphomas and are highly associated with a chromosomal translocation t(2;5). This results in the production of a chimeric protein NPM/ALK, in which the shuttle protein nucleophosmin is fused to the catalytic domain of the tyrosine receptor kinase ALK. Since the oncogenicity of NPM/ALK is not well understood, we analyzed the effects of NPM/ALK and the specific contribution of each fusion partner in a standardized cell culture system using primary rat embryo cells (REC) as cellular targets. We demonstrate by several biological parameters that NPM/ALK is an immortalizing oncogene which provides unlimited, yet normal growth potential to REC and, upon cooperation with a c-Ha-ras oncogene, induces cellular transfor-

Cell Biology International, Vol. 25, No. 2, 2001 mation. Targeting NPM/ALK to the nucleus by means of a nuclear localization signal diminishes its oncogenicity indicating that the fusion protein exerts its action mainly in the cytoplasm. Expression of the mere catalytic domain of ALK, which is unable to oligomerize, is insufficient with regard to immortalization and transformation. However, re-establishing the potential of ALK to homo-dimerize by fusing the bacterial dimerization domain of the tetracycline repressor to ALK re-inforces the immortalizing function and again allows powerful cooperation with c-Ha-ras to transform REC. Moreover, homodimerization was evidenced by immunoprecipitation of tagged TetR/ ALK proteins. We conclude that dimerization of overexpressed ALK is not only necessary, but indeed sufficient for the oncogenic potential of NPM/ALK protein.

65. GENOMIC ORGANIZATION OF ROPP120: A WD REPEAT PROTEIN OVEREXPRESSED DURING MITOSIS M. Pollmann1, F. Buck2, M. Szczepanowski1, R. Parwaresch1 and H. J. Heidebrecht1 1 Department of Hematopathology and Lymph Node Registry, University of Kiel, Michaelisstrasse 11, D-24105 Kiel, Germany (E-mail: [email protected]); 2 Institute of Cell Biochemistry and Clinical Neurobiology, University of Hamburg, Hamburg, Germany

Human ropp120 (restrictedly overexpressed proliferation-associated protein 120) is an as yet unknown cytoplasmic WD repeat protein of about 120 kDa that is overexpressed in mitotic cells. Ropp120 was immunoaffinity purified from cytoplasmic lysate of the human Hodgkin cell line L428 using the monoclonal antibody Ki-Mit. Following LysC digestion of the ropp120 protein the obtained peptide sequence data were used for the design of degenerated oligonucleotides. PCR screening of a L428 cDNA library yielded a ropp120 cDNA fragment which overlapped with several human EST clones at both its 5 and 3 end. Combination of these sequences resulted in a cDNA sequence of 3419 bp including the complete coding frame of ropp120 (981 amino acids). Fluorescence in situ hybridization (FISH) mapped the gene of ropp120 to the short arm of chromosome 2 (2p21-p22). Screening of a human chromosome 2 PAC library with different cDNA probes yielded two genomic ropp120 clones which were subsequently sequenced. One of these clones (LLNLP708B052Q3) contained the genomic 3 fragment of ropp 120 (including cDNA nucleotides 1391-3419), the other clone (LLNLP708E103Q3) contained the 5 end with the putative promoter region. Computer analysis of this region predicted a GC-rich but TATA-less promoter. Combination of the PAC sequences with sequences of human genomic databases revealed the entire gene structure of ropp120 consisting of 23 exons with lengths ranging from 25 to 772 nucleotides. Taking the putative promoter sequence as basis we performed PCR experiments with L428 cDNA as template in order to get information on the startpoint of the ropp120 transcript. The results enabled us to define a short sequence from -54 to -40 (refering to the first nucleotide of the known cDNA sequence) as the putative transcription start region of ropp120. Further studies on the ropp120 promoter region will yield essential information on its regulatory potential with regard to the expression of ropp120 and its possible modulation by cell cycle specific transcription factors.

66. RESCUE OF A CONDITIONAL CDC2 MUTANT HUMAN CELL LINE WITH A CDC2 KINASE THAT CANNOT BE PHOSPHORYLATED AT THR14 AND TYR15 M. Gupta, J. E. Itzhaki, A. J. Carpenter and A. C. G. Porter MRC Clinical Sciences Centre, Imperial College School of Medicine, Du Cane Road, London W12 0NN, U.K. (E-mail: [email protected])

The prototypic cyclin dependent kinase CDC2 controls entry into mitosis and its kinase activity can be attenuated by phosphorylation of residues Thr 14 (mammalian cells) and Tyr 15 (yeast and mammalian

Cell Biology International, Vol. 25, No. 2, 2001 cells). Numerous experiments in yeast and mammalian systems suggest that phosphoryation of these residues prevents premature, and potentially harmful, entry into mitosis both during the normal cell cycle and after activation of DNA damage and DNA replication checkpoint pathways. HT2-19 is a human fibrosarcoma cell line in which endogenous CDC2 gene expression is dependent on an inducer (IPTG). In the absence of IPTG, while DNA synthesis continues and nuclei become polyploid, cell division ceases and cells undergo programmed cell death (Itzhaki et al. [1997] Nat Genet 15: 258–265). A similar phenotype is observed when parental HT1080 cells are treated with agents that cause double stranded-DNA breaks, treatments known to cause the transcriptional downregulation CDC2 and many other genes required for mitosis (Badie et al. [2000] Mol Cell Biol 20: 2358–2366). Viable IPTG-independent HT2-19 derivatives can be recovered after transfection of a gene (CDC2-TY-HA) coding for a hemagglutinintagged CDC2, and this is true even for a mutant gene (CDC2-AF-HA) encoding a CDC2 that cannot be phosphorylated at positions Thr14 and Tyr15. Western analyses confirm that neither CDC2-TY-HA- nor CDC2-AF-HA-rescued clones have upregulated endogenous CDC2 expression, but express similar, physiological quantities of HA-tagged CDC2 represented by three bands in the former clones and a single, low mobility band in the latter. We have compared doubling times and plating efficiencies of CDC2-TY-HA- and CDC2-AF-HA-rescued clones, as well as their responses do DNA damage and inhibition of DNA synthesis, but so far have we have not detected any clear differences. More detailed comparisons are in progress. Thus, in this tumour line at least, and under the conditions tested to date, mechanisms other than the phosphorylation of CDC2 at residues 14 and 15 appear to be sufficient for preventing premature entry into mitosis both before and after activation of the DNA damage or DNA replication checkpoint pathways.

67. THE ROLE OF PP1 IN KINETOCHOREMICROTUBULE INTERACTION N. Rachidi1, T. Hyman2 and M. J. R. Stark1 1 School of life sciences, WTB/MSI, University of Dundee, Dow street, DD1 5HN, Dundee, Scotland, U.K. (E-mail: [email protected]); 2European Molecular Biology Laboratory, 69012 Heidelberg Germany (E-mail: [email protected])

Chromosome segregation depends upon kinetochores, which mediate the binding of sister chromatids to mitotic spindle microtubules. The catalytic subunit of Type 1 Protein Phosphatase (PP1) is encoded by the single, essential gene GLC7 in the budding yeast Saccharomyces cerevisiae. We have examined the critical functions of PP1 in yeast by characterisation of temperature-sensitive (Ts
) glc7 alleles. Two of our alleles, glc7-10 and glc7-12, arrest cells in mitosis. The arrest phenotype of glc7-10 is dependent on the spindle checkpoint, which blocks cells prior to the metaphase to anaphase transition when spindle formation is perturbed. glc7-10 mutants cells show an elevated frequency of chromosomal loss in vivo and a defect in the attachment of kinetochores to microtubules in vitro, demonstrating that PP1 plays a critical role in kinetochore function. Ndc10p, an essential kinetochore component, is thought to be the target of PP1, which specifically regulates the attachment of kinetochores to microtubules via Ndc10p dephosphorylation. Ndc10p seems also a target for Ipl1p, a protein kinase also required for chromosome segregation. ipl1 temperaturesensitive mutants cause a severe chromosome segregation defect and many cells exhibit a DNA content greater than 2N at the restrictive temperature. It is likely that in each single mutant, the defect is due to a change in the balance between Ipl1p and PP1. The kinetochoremicrotubule interaction may therefore be regulated by a balance between Ipl1p and PP1. We have investigated the role of PP1 and ipl1p in the regulation of the kinetochore-microtubule interactions by generating ipl1 glc7-10 double mutants. Ours results show that glc7-10 ipl1 double mutants are able to grow at the non-permissive temperature (37C), as a consequence of better viability at this temperature. furthermore, according to cell cycle analysis, especially FACS profiles, the double mutants do not arrest at the mitotic checkpoint like glc7-10, a large proportion of cells go through mitosis but without showing abnormal

A25 DNA content like ipl1 mutants. During this period of time, the cells double in number and exhibit a more normal DNA content. The phenotype of each single mutation is therefore at least partially suppressed when they are combined together. This result suggests that ipl1p and PP1 act in opposition to regulate the kinetochoremicrotubule interactions.

68. Dd-EB1, A NOVEL MEMBER OF THE EB1 FAMILY OF MICROTUBULE-ASSOCIATED PROTEINS, IS A PERMANENT CENTROSOMAL RESIDENT IN DICTYOSTELIUM M. Rehberg and R. Gra¨ f Adolf-Butenandt-Institut/Zellbiologie, Universita¨ t Mu¨ nchen, Schillerstr. 42, D-80336 Mu¨ nchen, Germany (E-mail: [email protected])

EB1, was originally identified as a binding partner of the adenomatous polyposis coli (APC) tumor suppressor protein. EB1 proteins are microtubule-associated proteins which usually have a size of 35– 38 kDa and were found in every organism so far. They are involved in microtubule search and capture at both kinetochores and the cell cortex and regulate microtubule dynamics, cell polarity and chromosome stability (reviewed by Tirnauer and Bierer (2000) JCB 149, 761). Consequently, EB1 proteins are weakly localized along microtubules but are concentrated at their plus ends. We have cloned the complete cDNA of Dictyostelium discoideum EB1 (Dd-EB1) by screening of a size-fractionated cDNA library using a 400 bp genomic sequence as a probe which was identified in the Dictyostelium genome project by its high similarity to human EB1. Dd-EB1 exhibits its highest amino acid identity to the human EB1 (44%) followed by budding yeast Bim1p (39%) and fission yeast Mal3 (37%). Compared to its homologues Dd-EB1 contains three sequence insertions in its central part and therefore Dd-EB1 is the largest known member of this protein family with a molecular mass of 57 kDa. Polyclonal antibodies raised against bacterially expressed Dd-EB1 revealed that Dd-EB1 is localized to astral microtubules and centrosomes and, during mitosis, to the spindle, spindle poles and kinetochores. Compared to yeast Bim1p, Dd-EB1 localization along interphase microtubules was unexpectedly strong and it was not biased to their plus ends. During metaphase we also observed a weak staining around the nuclear envelope. Most importantly, anti-Dd-EB1 antibodies also strongly bound to isolated centrosomes which are free of microtubules (Gra¨ f et al. (1998) Eur J Cell Biol 76: 167). Furthermore, centrosomal Dd-EB1 localization was unaffected by treatment of cells with the microtubule-depolymerizing drug thiabendazole. Thus, DdEB1 appears to be the first member of this protein family which is a genuine centrosomal component. Overexpression of a Dd-EB1-GFP fusion protein in Dictyostelium essentially confirmed the localization pattern described above. Interestingly, Dd-EB1-GFP cells were unable to grow in shaking culture and cell division was dependent on cell adhesion to a solid surface. Further studies of these mutant cells might explain this growth defect in the future. Furthermore, we will try to identify centrosomal Dd-EB1 binding partners with a two-hybrid approach and by isolation of possible binding partners copurifying with maltose-binding protein-tagged Dd-EB1. One promising candidate could be DdCP224 (Gra¨ f et al. (2000) JCS 113: 1747–1758), the homologue of yeast Stu2p which has been shown to interact with Bim1p (Kosco et al. (1999) MBC 10 Suppl, 256a). Supported by the Deutsche Forschungsgemeinschaft (SFB184).

69. INTERACTION OF TRANSCRIPTION FACTOR SP1 WITH THE MULTIFUNCTIONAL PROTEIN NUCLEOLIN Michael Novy, Eva Haidweger, Tina Weiland and Hans Rotheneder Institute of Medical Biochemistry, Division of Molecular Biology, Vienna Biocenter University of Vienna, Dr. Bohr-Gasse 9 A-1030 Wien (E-mail: [email protected])

Sp1, a ubiquitously expressed protein of 95-105 kDa that binds DNA through C-terminal zinc finger motifs stimulates transcription from promoters containing a G+C rich sequence, the GC box. It is

A26 particularly important for the regulation of TATA less genes. Previously it has been discovered that Sp1 belongs to a family of four closely related factors named Sp1, Sp2, Sp3 and Sp4 and several more distantly related proteins. Sp1 is able to act synergistically with itself, with other members of the family and with a variety of cellular and viral factors thought to confer promoter and cell-type specific activity. To get more insight into the function of Sp1 we used the yeast two hybrid system to look for novel Sp1-interacting proteins. By screening a human cDNA-libary, nucleolin, a multifunctional phosphoprotein, was detected. Nucleolin is present in both nucleoplasm and more abundantly in the nucleolus and shows helicase and chromatin decondensing activities. Nucleolin also functions as a cell surface receptor, where it acts as a shuttling protein between cytoplasm and nucleus, and thus can even provide a mechanism for extracellular regulation of nuclear events. As an antibody against nucleolin was made available to us, we were able to confirm the interaction with Sp1 by co-immunoprecipitation in mammalian cells. GST-pulldown assays with truncated Sp1 constructs assign the region necessary for binding of nucleolin to the zinc-finger motif in the C-terminal part of Sp1. Transient co-transfection of U2OS cells with Sp1 dependent promoterreporter constructs and increasing amounts of HA-tagged nucleolin results in a remarkable decrease of promoter-activity. This findings suggest that nucleolin acts as a negative regulator of Sp1 activity.

70. S. POMBE RAD17 FORMS A 5-MOLECULE PROTEIN COMPLEX WITH REPLICATION FACTOR Cs Shigeaki Saitoh, Hayes MacDonald, John Yates III and Paul Russell The Scripps Research Institute, La Jolla, CA 92037, U.S.A. (E-mail: [email protected])

When DNA replication is disturbed or genomic DNA is damaged in eukaryote, cell cycle progression is blocked. This cell cycle block is called ‘checkpoint’, mediated by Chk1 and Cds1 protein kinases that can deactivate Cdc2. So far, several genes including rad17 are reported to be essential for activation of these kinases. However, the molecular mechanism regulating the checkpoint is still not clear. In order to seek for proteins involving this control mechanism, we tried to purify S. pombe Rad17 protein complex under a native condition. After two affinity purification steps, Rad17 complex became almost pure. By silver staining we found four proteins besides Rad17, which ranged from 30 Kd to 40 Kd, in the complex. We applied peptidase digestion and mass spectrometry analysis to the whole purified complex and found that the complex included Rfc2, Rfc3, Rfc4, Rfc5 and Rad17. No other significant proteins were detected. The interaction between Rad17 and RFCs was not affected by treating cells with hydroxyurea or MMS. Thus, it seems that these five proteins form a stable equi-molar complex throughout cell cycle.

71. MAPK/SAPK SIGNALING IN APOPTOSIS AND FGF-2 MEDIATED SURVIVAL C. Schamberger and C. Cerni Institute of Cancer Research, University of Vienna, A-1090 Vienna, Borschkegasse 8A (E-mail: Chantal–[email protected])

MAPK/SAPK signaling cascades play an important role in proliferation, differentiation, cytokine production and cytoskeletal organization, depending on stimuli and cell type. These pathways are also involved in other important cellular processes such as apoptosis and survival, although these mechansims are less understood. Apoptosis, a form of programmed cell death, is characterized by morphological changes like cell shrinkage, condensation of chromatin, membrane blebbing and finally fragmentation of cell content into vesicles. The embryonic rat cell line 423 undergoes rapid apoptosis upon serum withdrawal. Morphological changes were already observed 15 min after serum reduction. Detachment of cells started after 3 h and, by 8 h, approximately 60% of the cells were apoptotic, as determined by microscopy, CASY analysis, DNA laddering and immunodetection of relevant proteins. In the presence of Fibroblast Growth Factor-2 (FGF-2) apoptosis was prevented in 423 cells. Since c-Ha-rassupertransfected 423 cell clones resisted serum reduction, we reasoned

Cell Biology International, Vol. 25, No. 2, 2001 that FGF-2 might activate the Ras/Raf/MAPK/MEK pathway. However, two different MEK1 inhibitors, U0126 and PD98059, which efficiently prevented phosphorylation of ERK1 and 2 in 423 cells, did not alter neither the kinetics of apoptosis, nor FGF-2mediated survival. We concluded that this pathway was not involved in either cellular reaction. Because of a transient but strong phosphorylation of p38/SAPK2 3 h after apotosis induction, we studied the influence of various p38/SAPK2 inhibitors. Surprisingly, addition of PD169513 prevented apoptosis in a concentration dependent manner, while two other p38/SAPK2 inhibitors, SB202180 and SB 203590, exhibited little or no effects on apoptosis and survival. This might suggest that the inhibitors affect different isoforms of p38. We are currently studying which of them is relevant for apoptotic signaling by immunoprecipitation and kinase assays of flag-tagged-p38 isoforms. Since FGF-2 induced survival irrespective of MEK1-and p38- inhibition, we assume that FGF-2 acts either further downstream in these cascades or by an independent route.

72. DIFFERENTIAL EXPRESSION OF CELL-CELL ADHESION PROTEINS, CYCLIN-D AND P27KIP1 IN MEK1-TRANSFORMED MDCK CELLS I. Marschitz, J. Lechner, I. Mosser, M. Dander and H. Schramek Institute of Physiology, University of Innsbruck, A-6010 Innsbruck, Austria (E-mail: [email protected])

Overexpression of a constitutively active mutant of the MAP kinase kinase MEK1 (caMEK1) in epithelial MDCK-C7 cells disrupts morphogenesis, induces an invasive phenotype and is associated with a reduced rate of cell proliferation. The role of cell-cell adhesion molecules and cell cycle proteins in these processes, however, has not been investigated yet. We report loss of E-cadherin expression as well as a marked reduction of -catenin and -catenin expression in transdifferentiated MDCK-C7 cells stably expressing caMEK1 (C7caMEK1) when compared with epithelial mock-transfected MDCK-C7 (C7Mock1) cells. These results were confirmed by standard and confocal immunofluorescence studies. While uniform, continuous -catenin staining at lateral cell borders was obtained in epithelial C7Mock1 cells, -catenin expression was restricted to single patches along C7caMEK1 cell borders. No increase in nuclear betasign-catenin expression was detected in C7caMEK1 cells. At least part of the remaining -catenin was co-immunoprecipitated with -catenin, while no E-cadherin was detected in -catenin immunoprecipitates. In both cell types, the proteasome-specific protease inhibitors ALLN (25 M) and lactacystin (10 m) led to a time-dependent accumulation of -catenin, including the appearance of high molecular weight -catenin species. These post-translational -catenin modifications occurred to a comparable extent in C7Mock1 cells and C7caMEK1 cells. While cyclin-A was equally expressed in both cell types, quiescent as well as serumstimulated C7caMEK1 cells showed a higher cyclin-D but lower p27Kip1 expression than epithelial C7Mock1 cells. Incubation of C7caMEK1 cells in the presence of 10 M of the MEK-inhibitor U0126 inhibited ERK phosphorylation, cyclin-D expression and almost abolished the already reduced cell proliferation rate. Ee conclude that the transdifferentiated and invasive phenotype of C7caMEK1 cells is associated with a diminished expression of proteins involved in cell-cell adhesion. Although -catenin expression is reduced, C7caMEK1 cells show a higher expression of U0126-sensitive cyclin-D protein.

73. EXAMINATION OF PROTEINS INVOLVED IN LOSS OF SISTER CHROMATID COHESION Klarissa Schroettner1, Tim Skern1 and Kim Nasmyth2 1 Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, Austria (E-mail: [email protected]); 2 Research Institute of Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna, Austria (E-mail: [email protected]) In eukaryotic cells, sister DNA molecules remain physically connected from their production at S-phase until their separation during anaphase. This sister chromatid cohesion is essential for the alignment

Cell Biology International, Vol. 25, No. 2, 2001 of chromosomes on the metaphase plate and for their equal separation into daughter cells. Cohesion between sister chromatids depends on a multiprotein complex called cohesin. Loss of sister chromatid cohesion is triggered through proteolytical cleavage by a recently discovered protein called separin or Esp1. Separin is responsible for removing the cohesion subunit Scc1 from the chromosomes which leads to sister chromatid separation. Little data about the separin protein is available. Esp1 resembles the CD class of cysteine proteinases, which include, for example, caspases and clostripains. So far no determination of the biochemical structure of the separin protein has been done. It is known that the catalytic dyad contains a conserved histidine and cysteine residue, which is surrounded by characteristic small residues (glycine or serine) and each is predicted to be flanked by hydrophobic beta sheets. We are trying to determine the crystal structure of the proteinase separin from different organisms by X-ray crystallography. To this end, we are expressing in E. coli the C-terminal domains, which are highly conserved among different organisms and which are responsible for the proteolytic activity of Esp1. We wish to compare the biochemical properties of the proteolytically active domains of separin from different organisms, such as S. cerevisiae, S. pombe, D. melanogaster and H. sapiens in order to clarify structural and functional differences.

74. RSC2, A COMPONENT OF THE RSC COMPLEX IS REQUIRED FOR 2
PLASMID MAINTENANCE IN SACCHAROMYCES CEREVISIAE Michael C. V. L. Wong2, Suzanna Scott-Drew1, Matthew J. Hayes and James A. H. Murray1 1 Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, U.K. (E-mail: [email protected]); 2Taman Universiti Indah, 43300 Seri Kembangan, Selangor, Malaysia

Replicating plasmids are highly unstable in yeast, because they are retained in the mother cell at mitosis, by a process known as Maternal Inheritance Bias (MIB). The 2  circle plasmid overcomes this MIB by using a partitioning system that uses both plasmid and host encoded proteins. We have previously shown that the plasmid encoded Rep1p and Rep2p proteins form discrete foci within the nucleus. We have now used GFP fused to the lac repressor protein to target plasmids containing the lac repressor DNA sequence (Straight et al. 1997). These plasmid/protein foci are tightly associated with the chromatin throughout the cell cycle and are efficiently segregated to the daughter cell during cell division. However, the plasmid/protein foci are distinct from both the nucleolus and the telomeres. RSC2, a subunit of the RSC (Remodels the Structure of Chromatin) complex, has been identified as a host gene essential for 2 m plasmid maintenance. The instability of 2  plasmid in rsc2 mutant strains is coincident with an alteration of the chromatin structure of a cis-acting DNA sequence STB, which is essential for efficient plasmid segregation. RSC1, another component of the RSC complex, is not essential for 2  plasmid stability. However, both RSC1 and RSC2 are required for the stable maintenance of centromeric plasmids. We suggest that chromatin remodelling of the STB locus plays a role in the segregation of 2  plasmid during mitosis.

75. APOPTOSIS DURING DEVELOPMENT AND INVOLUTION OF BOVINE MAMMARY GLAND F. Sinowatz1, S. Ko¨ lle1 and D. Schams2 1 Institute of Veterinar Anatomy, University of Munich, Germany (E-mail: [email protected]); 2Institute of Physiology, Technical University of Munich, Germany

The mammary gland is a fascinating model for the study of apoptosis. Its development is characterised by a proliferative phase during mammogenesis and lactation, followed by a phase of involution, when extensive restructuring of the tissue occurs which includes the elimination of many secretory epithelial cells. The molecular biological events involved in this processes are only poorly understood. Therefore, we studied apoptosis in the bovine mammary gland on distinct days of mammogenesis, lactation and involution using the TUNEL method

A27 and compared it with the immunohistochemical expression of apoptosis regulating genes bcl2, bax and p53. Apoptosis was confirmed by demonstrating ultrastructural changes in the chromatin texture of secretory cell nuclei. Additionally the expression of growth hormone receptors (GHR) was studied at the RNA (RT-PCR, in situ hybridisation) and protein level (immunohistochemistry, western blots), as we could show previously that growth hormone may have an antiapoptotic effect. Our results demonstrate that the highest apoptotic index is seen at the end of lactation, when the bovine mammary gland still displays high secretory activity. The number of TUNEL-labelled nuclei significantly decreases during the early stages of involution and remains on a low level during most part of the involution. The expression of GHR on the other hand is low during lactation and increases during involution. We conclude that apoptosis in the bovine mammary gland is most pronounced at the end of lactation. Already in the early involution phase, on the other hand, regenerative phenomena, like increasing proliferative activity in the glandular epithelium and increasing GHR-expression occur, which contributes to the restructuring of the mammary gland observed during this phase.

76. ACTIVATION OF DEOXYCYTIDINE KINASE BY DEOXYADENOSINE AND BY OTHER DEOXYNUCLEOSIDE-ANALOGUES, IN DIFFERENT CELLS Maria Staub*, Gergely Keszler*, Zsolt Csapo,* Tatjana Spasokoukotskaja*, Maria Sasvari-Szekely* and Zygmunt Kazimierczuk† *Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University of Medicine, H-1444 Budapest, P.O.B. 260, (E-mail:[email protected]); †Department of Chemistry, Agricultural University, Rakowieczka str. 26-30, 02-528 Warsaw, Poland

Deoxycytidine kinase (dCK) is the main salvage enzyme of deoxynucleosides, it can phosphorylate deoxycytidine, deoxyadenosine and deoxyguanosine. The dCK is constitutively expressed mainly in normal and transformed lymphoid cells. The sensitivity of tumour cells againts nucleoside analogue drugs, is dependent on the level of dCK activity. The aim of our work was to increase the sensitivity of cells to chemotherapeutic drugs. The activity of deoxycytidine kinase (dCK) could be potentiated by treating normal lymphocytes with the antileukaemic drug, Cladribine (2-chloro-2 -deoxyadenosine, CdA) previously shown (Sasvari-Szekely et al., 1998, Biochem Pharmacol 56: 1175–1179). Here we present evidences that the natural substrat, deoxyadenosine itself, is also a potent stimulator of dCK, if its deamination was inhibited by deoxycoformycine. Moreover, the deoxycytidine kinase activity was also increased (130%), by the new derivative, tri-fluoro-deoxyadenosine and by Gemcitabine (difluorodeoxycytidine) (200%), without influencing the thymidine kinase activities. Under same conditions CdA was the more effective stimulator (300%). When different lymphocyte populations were compared, it was shown, that mouse thymocytes respond to CdA treatment to a lesser extent (130%), than spleen cells (200%). Enhancement of dCK was higher in resting lymphocytes (300% in human PBMC and 280% in G0/G1 phase lymphnode cells), than in S-phase lymphocytes (170%). However, 2-Cl-ribo-adenosine was ineffective in all cell types investigated. There was no inrease of the dCK-protein and dCK-mRNA content in the drug treated cells, thus a secondary modification of the enzym was suggested. The increased activity of dCK might increase the sensitivity and efficacy of all drugs to be activated by dCK during therapy.

77. Sic1 MUTANTS IN CK2 CONSENSUS SITE SHOW AN ALTERATION IN CELL CYCLE PROGRESSION IN S. CEREVISIAE F. A. Sternieri, R. Rossi, P. Coccetti, D. Porro and L. Alberghina

A28

Department of Biotechnology and Bioscience, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano (E-mail: [email protected])

Sic1p, a key cell cycle regulator both in G1/S transition and in mitotic exit, has a highly hydrophilic aminoacidic stretch around Ser201 that very well matches a Ck2 consensus site. This observation, according to the fact that Ck2 physiological role and its targets are still widely unexplored, has induced us to replace the Ser201 with a residue of Ala or Glu, using site directed mutagenesis. We observed that although the doubling time was not modified in exponentially growing cells, mutant Sic1Ser201/Glu resulted in a decrease in the average protein content; on the contrary the mutant Sic1Ser201/ Ala exhibited an increase in the doubling time and an increase in the average protein content. Using cells synchronised in G1 with -factor we noticed that mutant Sic1Ser201/Glu showed delay in the G1/S transition as compared with the control colture, while the mutant Sic1Ser201/Ala did not. We have also preliminary indications that mitotic events could be influence too, but further analyses are required. After a shift in a medium with limiting carbon source the mutant Sic1Ser201/Glu was able to divide more quickly than the wild type, while the mutants Sic1Ser201/Ala accumulated cells of smaller size later than the control colture. Taken together these data show that mutants exhibit an alteration in cell cycle progression, suggesting that critical size(s) for cell cycle transition has been modified and that physiological role of Ck2 consensus site could be relevant. These data complement an analysis from our laboratory of a Ser to Ala mutant in Cdc28 Ck2 consensus site (see Biochem J, 2000, 351: 143–150).

78. IDENTIFICATION OF NOVEL CLEAVAGE SUBSTRATES OF THE YEAST SEPARASE ESP1 M. Sullivan and F. Uhlmann Chromosome Segregation Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, United Kingdom, WC2A 3PX (E-mail: [email protected])

Separase is the protease responsible for cleavage of cohesin complexes at the transition from metaphase to anaphase, thus allowing DNA separation. This occurs by the degradation of its inhibitor securin via the APC, thus freeing separase to begin proteolysis of its cellular targets. Separase has precise cellular targets, which it appears to recognise through a specific amino-acid sequence. To date in the Saccharomyces cerevisiae proteome, two targets (Scc1 and Rec8) have been identified both of which are subunits of cohesin complexes. It has been proposed that separase will have further mitotic (and/or meiotic) cleavage targets and we set out to identify these targets by the construction of a consensus cleavage sequence. Four cleavage sites for separase have been found (two sites in the mitotic cohesin subunit Scc1 and two in its meiotic homolog Rec8) and we used this information to generate potential models of the separase cleavage site. These models were used to screen the Saccharomyces cerevisiae proteome for other proteins with separase cleavage sites and a number of potential new targets were identified. These proteins were examined for cleavage fragments resulting from proteolysis by separase, and from this analysis we identify Slk19 as a novel target of separase. Slk19 is a non-essential mitotic, but essential meiotic, protein in the yeast kinetochore and is implicated to have a function in microtubule dynamics. We have confirmed both in vitro and in vivo that Slk19 is a cleavage target of separase. Like Scc1, Slk19 is cleaved at the metaphase to anaphase transititon and is thus a mitotic (and presumably meiotic) target. We are investigating the effect of mutations in the separase cleavage sequence to elucidate the cellular function of Slk19 cleavage. Like Scc1, Slk19 cleavage might be regulated by mitosis-specific phosphorylation. Cleavage of Scc1 by separase is vital for the destruction of the cohesive bridges linking sister chromatids in mitosis, and the C-terminal cleavage products of Scc1 are rapidly degraded by the N-end rule pathway. However unlike Scc1, the C-terminal cleavage product of Slk19 is stable and virtually full-length. Thus we are investigating whether the C-terminal cleavage product of Slk19 has a key cellular role, and what the effect of destabilising it might be.

Cell Biology International, Vol. 25, No. 2, 2001

79. THE DROSOPHILA TFIIH COMPONENT XPD/ERCC2 DOWNREGULATES CDK7’S CAK ACTIVTY AND PROGRESSION THROUGH MITOSIS Jian Chen, Ste´ phane Larochelle1 and Beat Suter Department of Biology, McGill University, Montre´ al, QC, Canada H3A 1B1 (E-mail: [email protected]); 1 present address: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, U.S.A.

Cdk7 is the catalytic subunit of the metazoan Cdk-activating-kinase (Cak) that phosphorylates and activates other Cyclin-dependentkinases (Cdks) through phosphorylation of a highly conserved threonine residue in the T-loop. In addition to its Cak function, Cdk7 also serves as the kinase subunit in TFIIH, the general transcription/DNA repair factor IIH. As part of TFIIH, Cdk7 phosphorylates the CTD (C-terminal domain) of the largest subunit of RNApolII. This step allows promotor clearance and transcription elongation to take place. We identified xpd/Ercc2 (Xeroderma Pigmentosum disorder group D/ Excision repair complementing group C) as a strong dominant suppressor of the Drosophila cdk7ts1 mutant, an allele that shows only a mitotic but no transcriptional defects. Elimination of one genomic copy of xpd does, however, not suppress cdk7ts2, an allele that does not show cell cycle defects. These results indicate that Xpd acts as a repressor of the cell cycle activity of cdk7. We further show that over-expression of Xpd, a helicase subunit of the TFIIH complex, results in the reduction of cellular Cak activity. During cycle 14, Xpd over-expressing embryos arrest their cell cycle. In the syncytial cycles that lack checkpoint control, Xpd over-expression causes a reduction in cell divisions instead of cell cycle arrest. These results lead us to propose that Xpd recruits the Cdk7 complex that provides the Cak activity into a larger complex that lacks Cak activity. Because mitosis and transcription are two mutually exclusive functions that both involve cdk7 activity, the uncovered mechanism can contribute to a co-ordinated regulation of these two activities.

80. GENOMIC STRUCTURE OF repp86—A CELL CYCLE ASSOCIATED PROTEIN M. Szczepanowski1, M. Pollmann1, F. Buck2, H. H. Wacker1, R. Parwaresch1 and H. J. Heidebrecht1 1 Department of Hematopathology and Lymph Node Registry, University of Kiel, D-24105 Kiel, Michaelisstr. 11, Germany (Email: [email protected]); 2 Institute of Cell Biochemistry and Clinical Neurobiology, University of Hamburg, D-22609 Hamburg, Germany

Repp86 (=restrictedly expressed proliferation associated protein 86) is a nuclear protein expressed from the transit G1/S until the end of cytokinesis. The antigen is recognized by the monoclonal antibody KiS2 (IgG1). Sequencing of the immunoaffinity purified protein yielded peptides, which enabled us to establish degenerated oligonucleotides. With different oligonucleotide combinations, we screened the L428 cDNA library (NCBI). We obtained a cDNA sequence of 3151 bp, which contained the complete coding frame of repp86. cDNA sequence comparison with the databank (NCBI) revealed the availability of a BAC clone containing the complete genomic sequence of repp 86 (RP11-243J16; Sanger Centre, United Kingdom). The repp 86 gene has a TATA-less promoter and consists of 18 exons. We found five CpG islands in the upstream gene region. The translation start is localized at the third exon. Since the primer extension assay failed, we approximated the transcription start (data not shown) by means of the PCR method. Currently this restricted region encloses 70 bp, and is localized 30 bp in front of the known cDNA sequence. By means of confocal LASER microscopy, we detected a colocalization between repp86 and ropp120, a proliferation associated protein containing WD repeats, which is overexpressed during mitosis. With the yeast two hybrid method, we verified the possible interaction between repp86 and ropp120. Repp86 cDNA was cloned into pGADT7 (Clontech). In this vector, repp86 is expressed as a fusion protein with the GAL 4 activating domain, whereas ropp120 cDNA, cloned in pGBKT7, is expressed as a fusion protein with the GAL 4 DNA binding domain. Both plasmids were transformed into the yeast

Cell Biology International, Vol. 25, No. 2, 2001 strain AH109, which carries point mutations in adenine, histidine, leucine, and tryptophan metabolism enzymes. The auxotrophy is usually compensated by reporter genes encoded in the vectors we used and by the putative protein interaction. Our results showed that repp86 does not interact with ropp120 in spite of the strict colocalization. In further experiments, we will investigate the interaction between repp 86 and other proteins encoded in the HeLa cDNA library obtained from Clontech.

81. THREONINE-11, PHOSPHORYLATED BY THE RAD3-RELATED KINASE ATM IN VITRO, IS REQUIRED FOR ACTIVATION AND FUNCTION OF FISSION YEAST CHECKPOINT KINASE CDS1 Katsunori Tanaka, Xiao-Bo Chen, Michael N. Boddy, Clare H. McGowan and Paul Russell Department of Molecular Biology, MB3, The Scripps Research Institute, La Jolla, CA 92037, U.S.A. (E-mail: [email protected])

Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also regulates replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia-mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, human ATM phosphorylates the N-terminal domain of Cds1 at the motif T11Q12. Substitution of threonine-11 with alanine (T11A) abolished in vivo phosphorylation and activation of Cds1 that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1
null mutant. Cds1T11A was unable to enforce the S-M checkpoint. These results strongly suggest that phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.

82. THE P53-INDUCIBLE GENE PC3 INDUCES ARREST OF G1-S PROGRESSION ACTING AS INHIBITOR OF CYCLIN D1 TRANSCRIPTION Daniele Guardavaccaro1, Giuseppina Corrente1, Francesca Covone1, Laura Micheli1, Igea D’Agnano2, Giuseppe Starace3, Maurizia Caruso4 and Felice Tirone1 1 Istituti di Neurobiologia, 2Tecnologie Biomediche, 3 Medicina Sperimentale, 4Biologia Cellulare, Consiglio Nazionale delle Ricerche Viale Carlo Marx 15, 00137, Rome, Italy (E-mail: [email protected])

The p53-inducible gene PC3 (TIS21, BTG2) is the founding member of a family of negative regulators of cell cycle, and had been originally isolated as an immediate-early gene induced by nerve growth factor in the PC12 cell line, at the onset of neuronal differentiation (Bradbury et al., 1991). We and others have demonstrated that PC3 inhibits the cell cycle progression (Montagnoli et al., 1996; Rouault et al., 1996). Several novel antiproliferative genes with partial homology to PC3 have also been isolated, e.g., BTG1 and Tob. Evidence has been presented for a role of PC3/TIS21/BTG2 in the growth arrest of the neuroblast preceding its differentiation in post-mitotic neuron (Iacopetti et al., 1994), and in the p53-dependent cell survival after genotoxic damage (Rouault et al., 1996). Thus, given that the growth arrest induced by PC3 can be responsible for important processes such as cellular differentiation and DNA repair, we sought to clarify the molecular mechanisms by which PC3 inhibits cell cycle. Here we report that expression of PC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb, leading to G1 arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that the PC3-mediated G1 arrest was Rb-dependent. Furthermore, (i) the arrest of G1-S transition exerted by PC3 was completely rescued by co-expression of cyclin D1, but not by cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells,

A29 accompanied by inhibition of CDK4 activity in vivo; (iii) the removal from the PC3 molecule of residues 50–68, a conserved domain of the PC3/BTG/Tob gene family, that we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 down-regulation as the main factor responsible for the growth inhibition by PC3. Such effect was associated to a decrease of cyclin D1 transcript and of cyclin D1 promoter activity, whereas no effect of PC3 was observed on cyclin D1 protein stability. Taken together, these findings indicate that PC3 impairs G1-S transition by inhibiting pRb function in consequence of a reduction of cyclin D1 levels, and that PC3 acts, either directly or indirectly, as a transcriptional regulator of cyclin D1 (Guardavaccaro et al., 2000).

References B A, P R, S EM, T F, 1991. PNAS USA 88: 3353. M A, G D, S G, T F, 1996. Cell Growth Differ 7: 1327–1336. R J-P, et al., 1996. Nat Genet 14: 482–486. I P, B G, T F, M L, C F, 1994. Mech Devel 47: 127–137. G D, C G, C F, M L, D’A I, S G, C M, T F, 2000. Mol Cell Biol, in press. 83. DEVELOPMENTAL CONTROL OF THE CELL CYCLE IN ANTIRRHINUM MAJUS Matthew Towers, Patricia A. Lunness, Valerie Gaudin, John H Doonan John Innes Centre, Norwich NR4 7UH, U.K. (Email: [email protected])

Developmental patterning and cell cycle control appear to be coordinated throughout plant growth. We are studying how cell cycle control genes such as D-cyclins are regulated during development. In both animals and plants, the D-cyclins are implicated in the cell’s response to extracellular growth factors and are believed to determine the balance between cell proliferation and differentiation. Using Arabidopsis genes as probes, we have isolated three D-type cyclins from a snapdragon inflorescence library. Two genes were found to belong to the D3 group and were accordingly named CycD3a and CycD3b. These two D3 cyclins were subsequently shown to display distinct and different RNA expression patterns in apical meristems. CycD3a is expressed exclusively in the peripheral zone of the meristem, particularly in the organ primordia. CycD3b expression completely overlaps the domain of D3a and is also present throughout the central zone. Furthermore, CycD3b is repressed in the aborted staminoid through the action of the CYCLOIDEA gene product (Gaudin et al., Apr 2000 Plant Physiol 122). These results suggest that theD3 class of cyclins to play a role in co-ordinating the interface between development and cell cycle control. Presently, further work is being undertaken to understand the functions of the D3 cyclins. Results of gene knockout screens to find endogenous Antirrhinum transposon insertions in or around the cyclin gene loci will be presented. A gain of function approach, using epitope and GFP tagged D3 cyclins under the control of an inducible promoter in Arabidosis is also in progress. Northern and In situ data indicate that CycD3a expression is modulated by the PHANTASTICA gene product. PHANTASTICA is a myb transcription factor involved in primordia growth and development. These experiments should give further insights to show how the cell cycle and growth are inter-linked.

84. DIFFERENT E2 ENZYMES MEDIATE CYCLIN A AND CYCLIN B DESTRUCTION IN A XENOPUS CELL FREE SYSTEM Chizuko Tsurumi and Tim Hunt ICRF Clare Hall Laboratories, South Mimms, Herts EN6 3LD, U.K. (E-mail: [email protected])

Xenopus early embryonic cell cycles consist of S and M phases only. Extracts made from activated eggs reproduce these events faithfully,

A30 and are therefore a good model system to study cell cycle regulation. We are interested in the degradation of mitotic cyclins. It has been known for a while that cyclin A is degraded shortly before cyclin B, in pro-metaphase—but both cyclins are destroyed by the APC/ proteasome system in a destruction box dependent manner. So how does the degradation machinery distinguish between the two cyclins? There seem to be subtle differences in the destruction of A and B-type cyclins: (1) the cyclin A-destruction box can not readily be grafted onto other proteins, and, (2) A-type cyclins, but not B1 cyclin, require the binding to a cdk-subunit in order to be degraded To investigate this further, we used antibodies and mutants interfering with various steps in the cyclin degradation machinery. First, we tested the effect of a dominant negative (DN) UbcH10 enzyme, the E2 ubiquitine conjugating enzyme for cyclin B. Addition of UbcH10 DN into an extract at the time of entry into mitosis (measured by H1 kinase activity and phosphorylation of the APC (anaphase-promoting complex) subunit cdc27), arrested the extract in M-phase with stable cyclin B. Cyclin A, however, was unstable. Using an interfering Fizzy antibody, we then tested whether this destruction was still APC-dependent. Addition of this antibody at a similar timepoint as the UbcH10 DN protein again resulted in an M-phase block. This time, however, both cyclins were stabilized, indicating that the degradation was still APC-dependent. Possible explanations for this observation are that either the APC uses two different kinds of E2 enzymes, or there are two different species of APC. At present, I am checking the effect of other dominant-negative E2 enzymes.

85. TGF-1 INDUCED GROWTH ARREST IN MALIGNANT B CELLS D. Tvrdik, R. Djaborkhel and J. Mu¨ ller Laboratory of Gene Expression, 3rd Faculty of Medicine, Charles University and Department of Cell Biology, Institute of Experimental Medicine, Czech Academy of Sciences, Albertov 4, Prague 2, Czech Republic (E-mail: [email protected])

Acquired resistance to inhibitory action of transforming growth factor beta1 (TGFbeta1) is commonly associated with the tumorigenesis. However, the B cell line derived from non-Hodgkin’s malignant lymphoma of follicular subtype (FL) retains the sensitivity to TGFbeta1 in vitro, similarly to the normal B lymphocytes. The presence of TGFbeta1 notably reduced the number of FL cells that entered S phase after 48 h of treatment (16%), compared to control unstimulated cells (44%). TGFbeta1 treatment led to notable increase in cdkinhibitor p21WAF1 expression, while no increase of p15INK4B, p16INK4A or p27KIP1 expression was observed due to the treatment. The level of early/mid G1 cyclins, cyclin D2, cyclin D3 and cyclin E or the level of cyclin-dependent kinases (cdk), cdk4, cdk6 and cdk2 was not significantly modulated by the treatment. However, TGFbeta1-induced growth arrest was associated with a profound reduction of S phase cyclin A expression, both at protein and RNA level. The expression of cyclin A is presumably related to the activity of transcription factors CREB/ATF family. TGFbeta1 treatment resulted in notable dephosphorylation of CREB-1 and in the significant reduction of ATF-2 expression. The profound effect on CREB/ATF transcription factors family indicates the complexity of TGFbeta1 action on FL B malignant cells.

86. ORCHESTRATING ANAPHASE IN YEAST Matthew Sullivan and Frank Uhlmann Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K. (E-mail: [email protected])

In metaphase, the sister chromatids of each chromosome are aligned pair-wise on the mitotic spindle, held together by the chromosomal cohesin complex. Sister kinetochores are under the tension of microtubules extending to opposite spindle poles, thereby establishing the bipolar orientation necessary for segregation of sister chromatids into opposite directions in anaphase. At anaphase onset, separase, an evolutionarily conserved protease, cleaves cohesin. This cut separates the sister chromatids. Segregation of the sisters is then driven by both the shortening of the kinetochore to spindle pole microtubules (ana-

Cell Biology International, Vol. 25, No. 2, 2001 phase A) and the lengthening of the spindle pole to pole distance (anaphase B). In budding yeast, cohesin cleavage in metaphase by an ectopic protease different from separase is sufficient to trigger sister chromatid separation and segregation to opposite cell poles. However, unlike in separase-triggered anaphase the spindle is weak and unstable. This implies an additional function of separase in stabilisation of the mitotic spindle. We determine the importance of spindle stabilisation by separase for high fidelity chromosome segregation, and we address how separase might stabilise the mitotic spindle during anaphase. We find that a protein implicated in spindle stabilisation, Slk19, is cleaved by separase in anaphase, and we are analysing the effect of Slk19 cleavage on spindle stability. The separase’s specific proteolytic activity at anaphase might be utilised for cleavage of additional target proteins to orchestrate high fidelity chromosome segregation.

87. POLYAMINES AND CELL CYCLE REGULATION IN THE UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII J. Voigt, C. Theiss and P. Bohley Institute for Physiological Chemistry, University of Tu¨ bingen, Hoppe-Seyler-Str. 4, D-72076 Tu¨ bingen, Germany

Polyamines play an important role in the control of cell growth and cell division. As in animal cells, biosynthesis of the commonly occurring polyamines (putrescine and spermidine) in the unicellular green alga Chlamydomonas reinhardtii is dependent on the activity of ornithine decarboxylase (ODC) catalyzing the formation of putrescine which is the precursor of spermidine (Voigt et al., 2000). When dark-adapted C. reinhardtii cultures were transferred to the light, a rapid 10-fold increase in ODC activity was observed without a measurable lag-phase which could be prevented by inhibition of photosynthesis or protein biosynthesis, but not by inhibition of RNA synthesis (Voigt et al., 2000). These findings indicate that this lightinduced increase in ODC activity is due to translation of pre-existing ODC-mRNA. In C. reinhardtii cultures synchronized by light-dark cycling, a further 3-fold increase in ODC activity was observed during transition to the cell division phase (Voigt and Bohley, 2000). This second up-regulation of ODC activity was not due to an increased ODC-mRNA level as revealed by Northern-blot analyses, but correlated with an increased half-life of this enzyme activity (from 1.1 to 3.2 h). When cell division was prevented by raising the temperature, no increase, but a decrease in ODC activity was measured indicating that an increase of the intracellular polyamine level is essential for cell division as assumed for animal cells. The evidence for a direct effect of polyamines on division of animal cells (Koza and Herbst, 1992), however, is not convincing: Since spermidine is required for the activation of the initiation factor eIF-5A (Jakus et al., 1993) and, therefore, for protein biosynthesis, the observation that inhibition of polyamine synthesis affects division of animal cells (Koza and Herbst, 1992) is presumably due to a decreased growth rate. C. reinhardtii, however, is a suitable model system to study direct effects of polyamines on cell division because this particular organism does not immediately divide upon doubling its cell mass, but is able to multiply its cell mass under optimal growth conditions before entering the division phase. In the subsequent dark period, cells divide several times without a further G1-phase. Therefore, direct effects of inhibitors of polyamine synthesis on cell division can be studied when applied to the cultures after the cells have doubled their cell mass (=8–9 h after beginning of the light period). Under these conditions, inhibition of cell division could be achieved by addition of spermine which caused a decrease in ODC activity by a decreased rate of ODC synthesis. This spermine induced cell-cycle arrest could be overcome by a subsequent addition of spermidine and, to a lesser extend, also by addition of putrescine indicating that spermidine is required for cell division.

References V J, D B, B P, 2000. Physiol Plant 108: 353–360. V J, B P, 2000. Physiol Plant (in press). K RA, H EH, 1992. Biochem J 281: 87–93. J J, W EC, P MH, F EJ, 1993. J Biol Chem 268: 13151–13159.

Cell Biology International, Vol. 25, No. 2, 2001

88. TWO DISTINCT PATHWAYS REMOVE MAMMALIAN COHESIN FROM CHROMOSOME ARMS IN PROPHASE AND FROM CENTROMERES IN ANAPHASE Irene C. Waizenegger1, Silke Hauf1, Andreas Meinke2 and Jan-Michael Peters1 1 Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria (E-mail: [email protected]); 2InterCell GmbH, Dr. Bohr-Gasse 7b, A-1030 Vienna, Austria

The equal segregation of sister chromatids in anaphase depends on cohesion, a mechanism that holds sisters together from S-phase until the onset of anaphase. Sister chromatid cohesion depends on a complex of chromosomal proteins, called cohesins. In yeast, cleavage of the cohesin subunit Scc1/Mcd1p and subsequent dissociation of the cohesin complex from chromosomes is required for the initiation of anaphase. This process is initiated by activation of the anaphasepromoting complex/cyclosome (APC) which ubiquitinates the securin Pds1p. Securin proteolysis activates the separase Esp1p which then mediates cohesin cleavage. In vertebrates, APC activation and securin proteolysis are also required for anaphase but cohesins dissociate from chromosomes already in prophase, i.e. long before sisters separate and the APC is thought to be activated (Losada et al., 1998; Sumara et al., 2000). Here, we show that in vertebrate cells residual amounts of cohesin SCC1 remains associated with human centromeres until the onset of anaphase when a similarly small amount of SCC1 is cleaved. In Xenopus extracts, SCC1 cleavage depends on the APC and on separase. Separase immunoprecipitates are sufficient to cleave SCC1, indicating that separase is associated with a protease activity. Separase activation coincides with securin destruction and partial separase cleavage, suggesting that several mechanisms regulate separase activity. Based on these results we propose that in vertebrates a cleavage-independent pathway removes cohesin from chromosome arms during prophase, whereas a separase-dependent pathway cleaves centromeric cohesin at the metaphase to anaphase transition.

89. ISOLATION OF INHIBITORS OF MIZ-1 FUNCTION BY RETROVIRAL CDNA EXPRESSION LIBRARY SCREENING Michael Wanzel and Martin Eilers Institute of Molecular Biology and Tumour Research, Emil-Mannkopff-Str. 2, D-35033 Marburg, Germany (E-mail: [email protected], [email protected])

The Myc associated transcription factor, Miz-1 (Peukert et al., 1997) induces G1-arrest and senescence in a pocket protein-dependent manner and inhibits cdk4 kinase activity. Miz-1 binds to the initiator element of the p15ink4b promoter and upregulates the expression of p15ink4b. Myc prevents Miz-1 induced G1-arrest by forming a complex with Miz-1 at the p15 initiator. Alleles of Myc that are unable to bind to Miz-1 fail to inhibit accumulation of 15ink4b mRNA (Staller et al., submitted). We describe a genetic screen to identify additional genes that, like Myc, inhibit cell cycle arrest by Miz-1. Rat1 fibroblasts were infected with a high complex retroviral human cDNA library and subsequently superinfected with viruses expressing Miz-1 and a hygromycin selection marker. After selection no clones grew in control plates, demonstrating that Miz-1 indeed completely blocks colony formation. In library infected plates, a total of 12 clones were identified that grew despite expression of Miz-1. A further rescue step, superinfection of the clones with MoMULV-viruses, revealed that 4 out of the 12 clones really express both wildtype Miz-1 and an inhibitor of Miz-1 function. Two clones were excluded from further analysis because they expressed a non-functional Miz-1 mutant form. PCR analysis from genomic DNA out of the 4 isolated clones showed that each clone contained a high number of retroviral infectants. Cloning and sequencing of some of these retroviral inserts revealed that nearly all cloned genes originated from the human cDNA library.

A31 Rat1 fibroblasts were infected with such identified genes and then superinfected with viruses expressing Miz-1. Using these conditions, we isolated one candidate gene which inhibits Miz-1 function. We are currently characterizing this candidate and its connection to Miz-1 function. Since our cloned gene is not Myc it seems that Miz-1 is not exclusively repressed by Myc.

References P, et al., 1997. EMBO J 16: 5672–5686. S, et al., submitted. 90. VISUALIZATION OF MICROTUBULE MOVEMENTS IN LIVING MITOTIC PLANT CELLS EXPRESSING CDC2-GFP Magdalena Weingartnera, Alois Schweighofera, Irute Meskienea, Erwin Heberle-Borsa, John Doonanb and La´ szlo´ Bo¨ grec a Institute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria (E-mail: [email protected]); bJohn Innes Research Centre, Cell Biology Department, Colney, Norwich NR4 7UH, U.K.; cSchool of Biological Sciences, Royal Holloway, University of London, Surrey, U.K.

To determine the dynamic localization of plant Cdc2 protein, we generated a fusion of the green fluorescent protein with the alfalfa Cdc2MsB kinase (Cdc2-GFP) and expressed it in tobacco cells. In vitro kinase assays demonstrate that the ectopically expressed fusion protein is following the same pattern of activation as the endogenous Cdc2-kinase during the cell cycle. Furthermore Cdc2-GFP is able to complement a yeast Cdc2 mutation. Thus the Cdc2-GFP fusion protein is functional and since its ectopic expression does not interfere with normal cell cycle progression, it is useful to study the in vivo localization of a plant Cdc2 during mitosis. During interphase Cdc2-GFP is found both in the cytoplasm and in the nucleus. However, these localizations are specific, because Cdc2GFP is retained in the nucleus and on cytoskeleton structures in detergent-extracted cells, while the GFP expressed by itself is washed away in these conditions. In pre-mitotic cells Cdc2-GFP signal appears along the preprophase band, a microtubule ring on the cell cortex predicting the future division site in plant cells. In metaphase and anaphase, Cdc2-GFP is associated with the spindle apparatus. It is still localized to the phragmoplast in telophase, suggesting a role for plant Cdc2 in cytokinesis. Because of its tight association to the cytoskeleton Cdc2-GFP allowed us to visualize the dynamic re-organization of the mitotic apparatus in living cells. Recording time-lapse images we could follow how from a diffuse fluorescence the Cdc2-GFP became localized to the metaphase spindle and the transition of the interzone spindle from anaphase 1 to anaphase 2. In telophase we observed Cdc2-GFP migrating with the phragmoplast to the margins of the division plane. In later time points during telophase to G1 transition the Cdc2-GFP reassociated with the chromatin. Our in vivo observed co-localizations of Cdc2 with microtubules are confirmed by cosedimentation assays showing interaction of an endogenous Cdc2kinase and Cdc2-GFP with in vitro polymerized microtubules from plant extracts. The association of its active form with microtubules at all mitotic stages suggests a role for a plant Cdc2 kinase in regulating microtubule behavior during mitosis.

91. ARGININE DEPRIVATION AND REDUCED RESTRICTION (‘R’) POINT STRINGENCY: KILLING CANCER CELLS IN VITRO AND IN VIVO Denys N. Wheatley1, Linda Scott2, Justin Lamb3 and Slobodan Tepic4 1 Department of Cell Pathology, University of Aberdeen, MacRobert Building, 581 King Street, Aberdeen AB24 5UA, U.K. ([email protected]); 2Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, U.K.;

A32 3

Department of Adult Oncology, Dana Farber Cancer Insrtitute, Binney Street, Boston MA 02165 U.S.A.; 4 Department of Surgery, School of Veterinary Medicine, University of Zu¨ rich, CH-8005 Zu¨ rich, Switzerland

Cells with reduced checkpoint (R-point) stringency in G1 continue to reinitiate S-phase when adverse conditions (e.g. amino acid deprivation) are experienced. Normal cells respond by entering quiescence (G0). The very obvious lack of response in anaplastic and fast-growing malignant cells leads to cell death for reasons which have yet to be fully elucidated, and are not necessarily apoptotic in character. Amino acid deprivation, either by omission from the medium or catabolic removal in situ has proved to be particularly devastating. Of all the amino acids explored, arginine deletion has given the greatest effects. Work which consolidates these findings for at least 26 tumour cell lines will be reviewed and summarised (Scott et al., 2000), and the latest data on several different enzymes causing the destruction of arginine in situ will be compared. Very few tumour cell types show ‘resistance’ in this respect, and those which do retain elements of control of cycle progression which allow them to enter a ‘cell cycle freeze’. The second aspect of these investigations will be concerned with the extremely difficult and complex problem of translating the in vitro findings of the selective destruction of tumour cells to practical approaches whereby this result can be achieved in vivo. Reduction of arginine in the whole body has serious impacts on numerous vital metabolic pathways, and consequently disturbs many physiological processes, all of which need to be brought under control. After nearly 6 years, we have now succeeded in achieving adequately low levels of arginine availability in the body, and the protocol is now being developed for the treatment of human cancer. The nature of the intervention requires windows of treatment which take cognisance of the cycle dynamics and recovery of normal cells relative to malignant cells. This systemic method of treatment should be applicable to late cancer-bearing cases with or without widespread metastatic disease.

References S L, L J, S S, W DN, 2000. Brit J Cancer 83: 800–810. 92. HIGHLY ACCURATE GENE EXPRESSION PROFILING Roland Wicki1 and Peter Ho¨ sle2 1 Applied Biosystems, Grundstrasse 10, CH-6343 Rotkreuz, Switzerland (E-mail: roland–[email protected]); 2Applied Biosystems, Brunner Strasse 59, A-1235 Wien, Austria (E-mail: peter–ho¨ [email protected])

Many cellular decisions concerning survival, growth and differentiation are reflected in altered patterns of gene expression. The ability to quantitate transcription levels of specific genes has therefore always been important to any research into gene function (Zamorano et al., 1996). RT-PCR is the most sensitive and the most flexible of all mRNA quantification methods. The application of fluorescence techniques to the RT-PCR, together with ABI PRISM Sequence Detection Systems capable of combining amplification, detection and quantification, has led to the development of kinetic or real-time RT-PCR methodologies that are revolutionising the possibilities for quantitation of nucleic acids. The TaqMan assay utilising the 5 nuclease activity of Taq Polymerase to cleave a target-specific probe during the PCR process is used to detect amplification of target-specific products. The development of fluorogenic probes made it possible to eliminate any post-PCR steps (Livak et al., 1995). The entire process, starting at the reverse transcription and ending with full relative or absolute quantification is now automated. The process of creating quantitative assays is streamlined because the construction and characterisation of an in-tube standard is no longer required in kinetic or real-time PCR. Many traditional variables, such as magnesium chloride concentration and the thermal cycling protocol itself, have been standardised using our latest guide-

Cell Biology International, Vol. 25, No. 2, 2001 lines (Applied Biosystems, 1999), greatly reducing assay development time. We will here present our latest guidelines for universal TaqMan PCR, today’s and future applications such as gene expression profiling in a 96 well microcard format, gene duplication/deletion determination and automated snp analysis.

References Z PL, M VB, B DW, 1996. Quantitative RT-PCR for neuroendocrine studies. A minireview. Neuroendocrinology 63: 367–407. L KJ, F SJA, M J, G W, D K, 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR products and nucleic acid hybridization. PCR Methods and Applications 4: 357–362. A B, 1999. Sequence Detection Systems Assay Design and Optimisation. 93. REGULATION OF CDC2/B-TYPE CYCLINS DURING G1IN FISSION YEAST Satoko Yamaguchi1,2, Hiroto Okayama2 and Paul Nurse1 1 Cell Cycle Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K. (E-mail: [email protected]); 2Department of Molecular Biology, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0033, Japan

Downregulation of Cdc2/B-type cyclin complexes is important not only during cell cycle progression but also in G1 arrested cells undergoing differentiation. We have studied Srw1, a member of the highly conserved Fizzy-related protein family in fission yeast. Srw1 is required for G1 arrest and conjugation, which is considered as differentiation in fission yeast. We have found that Srw1 is required for the degradation of mitotic cyclin B only during G1 arrest, but not at mitotic exit. Srw1 is phosphorylated by Cdc2/mitotic cyclin B throughout the cell cycle and only becomes dephosphorylated during G1 arrest. This phosphorylation targets Srw1 for proteolysis and inhibits its activity. These results demonstrate that Srw1 is involved in a G1-specific system of mitotic cyclin B degradation. To better understand G1 regulation of Cdc2/B-type cyclins, screenings for other factors involved in the regulation are in progress.

94. CDC18 ESTABLISHES REPLICATION COMPETENCE IN FISSION YEAST Stephanie K. Yanow1, Zoi Lygerou2 and Paul Nurse1 1 Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, U.K. (Email: [email protected]); 2Laboratory of General Biology, Medical School, University of Patras, Greece

In all eukaryotes, it is critical that DNA replication occurs once, and only once, per cell division cycle. Current models suggest that after mitosis, a complex of proteins called the pre-replicative complex (pre-RC) assembles onto replication origins rendering them competent for replication. Once S phase is complete this complex disassembles, precluding origins from being activated until the following G1. In fission yeast, overproduction of one of the proteins in this complex, Cdc18, causes cells to persist in S phase and re-replicate their DNA without going through mitosis. In cycling cells, Cdc18p is normally degraded during S phase and this is thought to contribute to the block over re-replication that exists in G2. We questioned whether overexpression of Cdc18 in G2 cells would be sufficient to override this control and allow origins to be re-licensed for replication. To test this, we overexpressed Cdc18 in cdc25-22 cells arrested in G2 and found that high levels of Cdc18 induce these G2 cells to re-replicate. Using neutral/neutral 2D gel electrophoresis as an assay for origin firing, we have shown that the origin ars3001 re-fires inappropriately when Cdc18p accumulates in these cells.

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To determine whether the mechanism of replication initiation in G2 is similar to that which is operative in a G1/S cell, we tested the requirement for several components of the pre-RC genetically, as well as their association with chromatin when Cdc18 is overexpressed. Our results suggest that the mechanism is similar in that Orp1, Cdc21, and Cdc2 are all required for re-replication. However, there is a decreased requirement for Cdt1 and Cdc21 association with chromatin which we propose may only be sufficient to allow inefficient re-firing of origins in the presence of high levels of Cdc18.

95. CORTICOTROPIN-RELEASING HORMONE AFFECTS CELL CYCLE OF HUMAN HaCaT KERATINOCYTES. BłazJej Zbytek, Piotr Trzonkowski, Andrzej Mys´liwski Department of Histology and Immunology, Medical University of Gdansk, ul. Deb¸ inki 1, 80-210 Gdan´ sk, Poland (E-mail: [email protected])

Corticotropin-releasing hormone, secreted by hypothalamus, coordinates systemic response to stress. It is expressed locally in the extracranial tissues including skin. Human HaCaT keratinocytes possess functional CRH receptors. We present original data indicating that CRH affects the cell cycle of HaCaT cells. In nanomolar concentrations it inhibits proliferation and tritiated thymidine incorporation. CRH-related peptides (sauvagin, urocortin, urotensin) also have this effect, however, to a lesser degree. Flow cytometric cell cycle analysis with the use of iodium propide staining of cell nuclei shows decrease of the cell population in the S phase and increase in the G1 phase. Flow cytometric analysis of ICAM-1 expression on the cells shows significant increase thereof. CRH, functioning as an element of a local epidermal regulatory circuit, may change keratinocyte phenotype from proliferative to inflammatory state.{/113}

INDEX: (N.B. By presenter’s names only) SECTION 1. INVITED SPEAKERS Bartek Bell Diffley Edgar Hunt Ju¨ rgens Krek La Thangue Lehner Mitchison Nasmyth Nigg Nurse Reed Rieder Zetterberg

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16

SECTION 2. ORAL AND POSTER PRESENTATIONS Andrecht Ausserlechner Azuara

1 2 3

Badie Beier Bello Bohn Boiartchouk Brambill Brandtner Brevet Calderini Caruso Columbano Cross Darbon Decottignies de Jager Divita Eberharter Eckl Ekholm Engstro¨ m Erlandsson Fabian Fellner Fuxe Gasparian Geley Gieffers Goren Graef Graeser Grunicke Haidweger Hauser Heidebrecht Hengst Hesterman Horstman Heutter Huitu Huss Imhof Jacob Jansen-Du¨ rr Ladurner Lagger Leisser Loidl Manni Marecsek Martinsson Moore Morandell Morris Moser Mueller

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

A34

Obexer Petzelt Piaggio Pibiri Piluso Polgar Pollmann Porter Rachidi Rehberg Rotheneder Saitoh Schamberger Schramek Schroettner Scott-Drew Sinowatz Staub Sternieri

Cell Biology International, Vol. 25, No. 2, 2001

59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

Sullivan Suter Szczepanowski Tanaka Tirone Towers Tsurumi Tvrdik Uhlmann Voigt Walzenegger Wanzel Weingart Wheatley Wickl Yamaguchi Yanow Zbytek

78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95