Index
1
Adelfalk C.(1-P1.)
Anisimov A.P.(2-PL2.)
Anisimova A.A. (3-P2.)
Erenpreisa Je.
(4-P2.)
(5-P2.)
(6-PL2.)
Erenpreiss J. (7-PL3.)
Friedl A. A.(8-LP1.)
Giorgobiani N. (9-P2.)
Greulich-Bode K. M.(10-PL1.)
Guzhova I. (11-PL3.)
Hausmann M.(12-PL1.)
Ianzini F. (13-P2.)
Isajevs S.(14-P3.)
Ivanov A.(15-PL2.)
Kalejs M.(16-PL2.)
Kalnina M.(17-P3.)
Knoch T. A.(18-PL1.)
Kvitko O.V. (19-P2.)
de Laat W. (20-PL1.)
Leonchiks A.(21-PL3.)
Liepinsh E. (22-PL3.)
Markovs J.(23-PL3.)
Maszewski J. (24-PL2.)
Matsumoto S. (25-PL3.)
Plakhins G. (26-P2.)
Rajaraman R.
(27-PL2.)
(28-P2.)
Rapoport A. (29-PL3.)
Rybaczek D. (30-P2.)
Sabisz M. (31-P2.)
Scherthan H.(32-PL1.)
Selga T. (33-PL3.)
Shestakova I. (34-P3.)
Shkutele S. (35-P3.)
Sibirny A. A.(36-PL3.)
Sinha S.(37-P2.)
Sjakste T. (38-PL1.)
Skladanowski A. (39-PL2.)
Skute N. (40-P3.)
Sperga M.(41-P3.)
Tchelidze P.
( 42-P1.)
( 43-PL1.)
Tsarev I.
( 44-PL3.)
( 45-P3.)
Wachsmuth M. (46-PL1.)
Walen K.(47-P2.)
Wheatley D. N.(48-PL2.)
Zacharias H. (49-PL2.)
Zatsepina O. V.
( 50-PL1.)
Zybina T. G.
( 51-PL2.)
( 52-P2.)
1
1-P1.Meiotic telomere clustering requires actin for its formation and cohesin for its resolution
AdelfalkCaroline1, EdgarTrelles-Sticken,1 Josef Loidl,2 and Harry Scherthan1,3
1 Max-Planck-Inst. for Molecular Genetics, Ihnestr. 73, D-14195 Berlin, Germany
2 Institute of Botany, Rennweg 14, University of Vienna, A-1030 Vienna, Austria
3 Institute for Radiation Biology Bundeswehr, Neuherbergstr. 11, D-80937 Munich, Germany
Meiosis reduces the chromosome number to the haploid, which compensates for the genome doubling at fertilization and instigates genetic diversity. During meiotic prophase, telomeres attach and transiently cluster to a limited sector of the nuclear envelope. Here, we show in living Saccharomyces cerevisiae meiocytes that telomeres move around the nuclear periphery during leptotene. During the bouquet stage telomere mobility becomes constrained to a limited sector of the nuclear periphery that usually is found near the spindle pole body (SPB). We observed that inhibition of actin polymerization, but not microtubule disruption, induced rapid dissolution of the meiotic telomere cluster indicating that meiotic telomere cluster formation is actin-dependent. In rec8Δ prophase I that lacks meiotic sister chromatid cohesion, we found that telomere clustering is arrested in a cohesin-dependent but DSB-independent manner. Surprisingly, cohesin turned out to be required for the colocalization of the meiotic telomere cluster with the spindle pole body: rec8Δ meiocytes have the base of the telomere cluster displaced from the SPB – this and bouquet exit was re-established by ectopic expression of the mitotic cohesin Scc1 in rec8Δ meiosis. Thus, our data indicate that functional cohesin is required to exit telomere clustering and to link the actin-dependent meiotic telomere cluster to the meiotic SPB.
2-PL2.Endopolyploidy as a morphogenetic factor of development
Anisimov A.P.
Far-EasternNationalUniversity, Vladivostok, Russia
Endoreproduction of certain cell groups leading to endopolyploidy (somatic polyploidy) is involved in the program of numerous animal and plant histogeneses and occasionally becomes an important additional factor affecting postnatal growth. Different authors emphasize different properties and functional aspects of endopolyploid cells in different tissues and organisms. To give an universal interpretation of endopolyploidy, we proposed that a single polyploid cell be better considered as an endoclone. In this case, evolutionary transformation of diploid cell clones into polyploid endoclones may be viewed as V. Dogel’s oligomerization on cell–tissue level. Oligomery, the basic property of endoclone, arises from its united organization, which requires structural and functional centralization and integration. According to this view, the most important derivative properties of this oligomerized system are the major peculiarities of polyploid growth strategy. They comprise intensification of function, economy, simplification of the intra– and supersystem regulations, increased gene reliability, shortening of development. The above peculiarities allow one to consider endopolyploidy as an additional means of integrative histogenetic regulations and as an important evolutionary factor acting through natural selection (“ontogenetic correlations” and “phylogenetic coordinations” according to I. Shmalgauzen). Thus, in general, endopolyploidy is an adaptive morphogenetic factor, but its role may differ depending on cell specialization and histogenetic particularities. This view on endopolyploidy is consistent with the data available on the properties and distribution of polyploid cells in histogeneses of various animal and plant species.
This study was supported by US CRDFand Russian Federation Ministry of Education and Science (project REC-003).
3-P2.Morphofunctional parameters of nucleoli in polyploid cells of the snail succinea lauta
AnisimovaA.A, A.P. Anisimov
Far-EasternNationalUniversity, Vladivostok, Russia
Examining various nucleolar parameters as markers of cell transcriptional and proliferative activity are widespread in various areas of applied investigations including tumor pathology studies. Nucleolar parameters may also reflect natural, non-pathological changes in cell status. The present study dealt with the number and integral area of nucleoli and their Ag-protein content in polyploid cells of the reproductive, albumen, and salivary glands in the snail Succinea lauta (Mollusca: Gastropoda). The size and activity of nucleoli generally increased with gene dosage (twice per each endomitotic cycle). The parameters studied were also found to depend on various other factors (endomitotic cycle stage, tissue development stage, cell differentiation rate and direction, age, rhythm of cell functioning, and state of the organism as a whole). Any of these accompanying polyploidization factors modified gene dosage effect and changed the ratio of ploidy value to nucleolar size and Ag-protein content. One should take these findings into account when examining various nucleolar parameters in endopolyploidy studies. Compared to the other parameters, the number of nucleoli displayed a lower correlation to ploidy degree. In the actively growing and functioning albumen glands, it did not change following an increase in cell ploidy level. In the interphase polyploid nuclei, one or two large nucleoli were usually observed. Cell growth and functional activity seemingly led to the association of nucleolar organizer regions. At the same time, in the endomitotic stage, partial, and not whole, dissociation of nucleoli occurred suggesting the retention of functional activity of nucleoli during endomitosis.
This study was supported by US CRDFand Russian Federation Ministry of Education and Science (project REC-003).
4-P2.Cytological mechanisms of genomes maintenance and segregation
in giant tumor cells
Erenpreisa Je1, Kalejs M1, Ianzini F2,3,4, Kosmacek EA3, Mackey MA2,3,Emzinsh D5, Illidge TM6
1LU BMC, Riga, Latvia; 2Dept. Pathology, Univ. Iowa, Iowa-city, USA; 3Dept. Biomedical Engineering, Univ. Iowa; 4Dept. Rad. Oncology, Univ. Iowa; 5 Oncology Centre of Latvia, Riga; 6 Paterson Cancer Res. Inst., Manchester, UK.
Endopolyploid tumor cells can, in small proportion, segregate into viable descendants. Therefore, it is important to determine the cytological mechanismsinvolved in the preservation the individuality and integrity of the genomes in giant cells. The term ‘segregation of genomes’, de-polyploidisation, was introduced by Grell in 1953 (KG Grell, Archiv für Protistenkunde 99:1-54, 1953). Grell described segregation of the genomes of polyploid nuclei of Aulocanthaas homologous units due to linkage of chromosomes of each haploid genome. Our studies were performed on 10Gy-irradiated p53-deficient cell lines - Ramos, Namalwa, WI-L2-NS, Jurkat, HeLa. These cell lines enter ‘catastrophic mitosis’ and form mono-nuclear and multi-nuclear giant cells. Mononuclear cells are partially polytenic, contain one centrally located nucleolus with fused fibrillar centres, centromeres are clustered around it. This nucleolus is in contact with a single composed (multi-centriolar) centrosome. In multinuclear cells, the radial connection of sub-nuclei to a single cellular centre by the DNA containing threads and microtubules is evident. In preparation for de-polyploidisation, endocycling giant cells re-arrange their genomes: get rid of micronuclei, and often segregate subnuclei (para-1n, 2n, 4n). Often subnuclei of one ploidy class are formed in giant cells prior to de-polyploidisation. 1C subnuclei seem further degenerate. The radial DNA connections of individual genomes to the cellular centre are especially well seen in HeLa cells which undergo repeated attempts of radial cell division, with condensation of subnuclei and intensive radial karyokinesis. Radial cytotomy follows and sub-cells may arise, often vitally looking, rarer apoptotic. The flower-like degenerative nuclear ‘ghosts’ may also form. Note that these divisions often comprise the odd number of segregating genomes and that individual chromosomes are not seen. Rather, the partially condensed short chromatin loops are seen. Corresponding to this peculiar ‘anaphase’ segregating genomes, we have also found peculiar ‘metaphase’, where a huge heterochromatic ring appears uniting all sub-nuclei, which settle perpendicularly to the ring (like individual chromosomes in an ordinary metaphase). These subnuclei can condense chromatin, but individual chromosomes are also not visible. In some of the cell lines studied, the participation of spindle apparatus in the radial segregation of genomes was found by beta and gamma-tubulin staining. In mononuclear (polytenised) giant cells, segregation of nucleoli and fibrillar centers precedes radial segregation of subnuclei. In Namalwa and Ramos cells, subnuclei segregate in endo-interphase. Segregated sub-cells immediately start mitotic divisions or enter endomitosis again. These preliminary results suggest that in endopolyploid cells, both mono-nuclear and multinuclear, individual genomesestablish permanent contact with a composed (multi-centriolar) centrosome and can be radially segregated as individual units. FISH studies are under way to further characterize the described phenomena.
5-P2.Micronucleation and chromatin diminution - two methods
of the DNA sorting in tumour cells undergoing mitotic catastrophe
Erenpreisa Je1, Ivanov A1,2, Ivanova M3, Gloushen S3, Cragg M4, Illidge T2
1 Lab. Tum. Cell Biol., Latv. Univ. Biomed. Centre, Riga, Latvia;
2Paterson Institute for Cancer Research, Manchester, UK;
3 Faculty of GeneticsBelarussianStateUniversity, Minsk, Belarus;
4 Tenovus Research Laboratory, Southampton, UK;
Tumour cells undergo mitotic catastrophe when exposed to genotoxic insult: they become arrested by the spindle checkpoint and through adaptation can produce endopolyploid cells. The latter are capable to undergo DNA repair by homologous recombination (HR) and/or non homologous end-joining (the two repair pathways are interconnected for the repair of radiation induced DSBs). Cytologically, HR was visualised by repair foci in combined immunostaining of g-H2AX and Rad51/52 proteins. The two types of DNA sorting from endopolyploid tumour cells with landmarks of HR were observed: (1) Micronucleation occurred during the period of aberrant mitoses and their failure (2-4 days post-irradiation), where 1-3 m micronuclei containing high concentration of DNA, -H2AX, and Rad 51 were detected. Micronuclei often showed non-synchronous with nucleus DNA synthesis as seen by pulse-label with 3 H-thymidine and native DNA conformation (in DAPI and green AO-fluorescence). Micronucleation of -H2AX/Rad51/ DAPI bodies was several-fold more extensive in wt p53 tumour (TK6) than its mutated parent (WI-L2-NS) and was proportional with amounts of apoptosis at this time period. Evidently, formation MN displays the failure of HR to cope with DNA damage through mitotic repair capacities (as was earlier suggested by Haaf et al. 1999), rendering the cells prone to initiation of apoptosis. Another method of DNA elimination has been reported previously (Erenpreisa et al., J Cell Biol Int 2000, 24:621-633) and is characteristic only for the proportion of large endopolyploid cells in TP53-mutated tumours, which have survived onto the second week after irradiation. These cells undergo a period of extensive DNA repair and nuclear reconstruction culminated in de-polyploidisation and formation of viable descendents (7-12 days post 10 Gy irradiation). Large portions of degraded chromatin are actively and repeatedly extruded from live cells through actin and microtubule funnels, prior/parallel to final disintegration of giant cells. The extruded DNA is fragmented (AO-red; -H2AX-positive). EM shows sequestration of this chromatin within autophagosomes. Now we demonstrate that the diminuted DNA is associated with deranged Rad 51 suprastructures and Rad52 aggregates. It can be suggested that endopolyploid tumour cells are capable to use homologous recombination repair for another method of sorting DNA, where, possibly, the whole genomes are degraded and eliminated from live cells enabling their further segregation into viable sub-cells.
6-PL2.Recapitulation of protozoan life-cycles by tumour cells
and the origin and evolution of meiosis: a hypothesis
Erenpreisa Je.
Lab. Tum. Cell Biol., Latv. Univ. Biomed. Centre, Riga;
Mitotic catastrophe as a result of DNA damage leads to the production of transient endopolyploid cells in many p53 functionally deficient tumours. Curiously, in human lymphoblastoid cell-lines that we have studied, expression of several meiotic genes was found during this process. The response typically includes polyploidising abortive mitoses and endomitosis, which culminate in a peculiar division of giant cells from which several daughters may return to near diploidy and mitoses. This sequence is reminiscent of events of the asexual life-cycle in some protozoans (Raikov, 1982; Kondrashov 1994, 1997). In most protozoans, sexual process (meiosis and syngamy) exist; however, in some, the sexual life-cycle is not permanent and occasionally they revert to a more primitive process. Interactive combination of these processes shows how the origin and evolution of meiosis from mitosis might have occurred as initially suggested by Cleveland (1947). While higher organisms have developed no workable method for relieving polyploidy and giant cells in most cases become terminal and degenerate, in the basically haploid flagellate Barbulanympha, as reported by Cleveland, polyploidy (tetraploidy) is invariably reduced by meiosis, and no degeneration results. The important part of such a life-cycle is a two-step somatic meiosis, which is neither preceded nor followed by any kind of syngamy. However, meiotic prophase is preceded by a monopolar endomitosis. Endomitosis also precedes segregation of primary polyploid nucleus of radiolarian Aulocantha into isospores (not gametic cells), and axial structures were revealed between chromatids in endomitotic chromosomes (Grell and Ruthman, 1964). Cachon et al. (1973) suggested therefore that in endomitotic telophase pairing of somatic bivalents occurs and coined endomitosis ‘meiosis without karyogamy’. Up to hitherto, molecular regulation of endomitosis and its role in development and tumour cells is obscure (Edgar and Orr-Wiever, 2001). Curiously then was our finding that endomitotic nuclei in irradiated lymphoid TP 53-mutant tumours contain the meiotic kinase MOS-protein interacting with cyclin B1, while rare endotelophases – show a para-haploid number of multi-stranded chromosomes. Moreover, up-regulation of meiotic cohesions, Dmc1, and recombinational DNA repair was found in these giant cells, preceding reduction divisions (Ivanov et al., 15-PL2; Plakhins et al., 26-P2). It is hypothesised that p53-mutant cells are able to recapitulate aberrantly the evolutionary program of asexual meiosis (somatic reduction), efficiently repair DNA double-strand breaks, and reduce polyploidy. The process extends the life-span of tumour cells, although the clonogenicity of these offspring now requires much further study.
7-PL3.Role of sperm DNA damage assessment in infertility workup
Erenpreiss J1,2, J. Orbidans3
1Latvian University Biomedicine Centre, Riga, Latvia; 2Lund University, Fertility Centre, Malmo University Hospital, Malmo, Sweden; 3Clinics „Piramida”, Riga, Latvia
Infertility is one of the major both social and medical concerns in a modern western society, affecting approximately 15% of couples. A male factor is a sole or contributing factor causing infertility in approximately half of the cases. Detoriating male semen quality and inter-regional differences in Europe has been shown.
Sperm chromatin status is of a paramount importance for a normal fertilization process. It has been shown that if a proportion of sperm cells with damaged DNA in ejaculate exceeds 30% as detected by the Sperm Chromatin Structure Assay (SCSA), neither natural conception nor in vivo conception by means of the Intrauterine Insemination (IUI) can be achieved. Assisted reproductive techniques (ART) like in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) can overcome this restrictive threshold for in vivo conception, although the controversies and debate on this issue still remain.
The clinical usefulness of the techniques available for the sperm chromatin/DNA structure assessment will be reviewed. Controversies on the role of sperm DNA damage in ART will be discussed. The controversial issue on the possible negative consequencies of using semen samples with high loads of sperm DNA damage for ART will be highlighted.
8-PL1.The role of chromatin structure and nuclear architecture in the cellular response to ionizing radiation
FriedlA.A.
Radiobiological Institute, University of Munich, Germany
In recent years, our understanding of the biochemical mechanisms of double-strand break (DSB) repair has increased substantially. A thorough understanding of the chain-of-events taking place during repair in the context of the three-dimensional functional architecture of the nucleus requires, however, that topological factors also be considered. These factors include the position of the damage with respect to functional chromatin organisation and to other damaged sites, potential movement of damaged chromatin within the nucleus, and the dynamics of DSB signalling and repair proteins.
One approach to analyse these factors relies on targeted induction of DSB by using an ion micro-irradiation facility, where the site to be damaged can be pre-determined by the experimenter, and where signals observed after irradiation can be related to the initial damage site. In the present set-up of the Munich micro-beam, we achieve an accuracy of irradiation of about 500 nm, i.e. small enough to target specific substructures of the nucleus. By use of a scanning mode, geometric irradiation patterns can be applied. At present, by immunofluorescent detection of damaged chromatin sites we investigate the mobility of these sites. In addition, the recruitment of signalling and repair factors to damaged sites, and the dependence of these processes on factors such as damage density, cell type, cell cycle phase etc. is studied.
In additional work, we investigate how nuclear architecture affects the generation of chromosome aberration in S. cerevisiae after irradiation and after induction of DSB using site-specific endonucleases. Finally, we investigate how chromatin structure affects the spatial distribution of DSB induced by densely ionising radiation.
9-P2.Possible role of endogenous growth inhibitors in regeneration of organs: searching of new approaches
Giorgobiani N, TumanishviliG.
Laboratory of Develpmental Biology, TbilisiStateUniversity, Tbilisi, Georgia
To date, regeneration of organs with low proliferative potential, such as myocardium, features as most acute problem of modern biology and medicine. It was supposed that there is no cambial reserve in the process of cardiomyogenesis. Thus, it seems very probably that all postnatal cardiomyocytes cannot divide, due to irreversible block in G0 phase of the cellular cycle. Remarkably, that single mitotic cardiomyocytes were registereed in regenerating heart by Rumyantsev in 1970. Then after, various mitotic potential of auricular and ventricular cardiomyocytes and DNA synthesis have been reported in different works. Evidence of mitotic activity of cardiomyocytes in postnatal myocardium has been shown in some investigations at the beginning of 2000. Authors have indicated presence of both caryo- and cytokinesis figures in myocardium after infarction (4% in border and 1% in distant zones) in postmortal human material. Several approaches are developing for the treatment of ischemic and post-infarction heart. Three of them should be emphasized in particular: 1) cardiac transplantation; 2) transplantation of bone marrow cells (stem cells plasticity); 3) elaboration of antibodies to endogenous growth inhibitors. Here we suggest last-mentioned method for consideration.