Index
Adelfalk C. (1-P1.)
(28-P2.)
Anisimov A.P. (2-PL2.)
Rapoport A. (29-PL3.)
Anisimova A.A. (3-P2.)
Rybaczek D. (30-P2.)
Erenpreisa Je.
Sabisz M. (31-P2.)
(4-P2.)
Scherthan H. (32-PL1.)
(5-P2.)
Selga T. (33-PL3.)
(6-PL2.)
Shestakova I. (34-P3.)
Erenpreiss J. (7-PL3.)
Shkutele S. (35-P3.)
Friedl A. A. (8-LP1.)
Sibirny A. A. (36-PL3.)
Giorgobiani N. (9-P2.)
Sinha S. (37-P2.)
Greulich-Bode K. M. (10-PL1.)
Sjakste T. (38-PL1.)
Guzhova I. (11-PL3.)
Skladanowski A. (39-PL2.)
Hausmann M. (12-PL1.)
Skute N. (40-P3.)
Ianzini F. (13-P2.)
Sperga M. (41-P3.)
Isajevs S. (14-P3.)
Tchelidze P.
Ivanov A. (15-PL2.)
( 42-P1.)
Kalejs M. (16-PL2.)
( 43-PL1.)
Kalnina M. (17-P3.)
Tsarev I.
Knoch T. A. (18-PL1.)
( 44-PL3.)
Kvitko O.V. (19-P2.)
( 45-P3.)
de Laat W. (20-PL1.)
Wachsmuth M. (46-PL1.)
Leonchiks A. (21-PL3.)
Walen K. (47-P2.)
Liepinsh E. (22-PL3.)
Wheatley D. N. (48-PL2.)
Markovs J. (23-PL3.)
Zacharias H. (49-PL2.)
Maszewski J. (24-PL2.)
Zatsepina O. V.
Matsumoto S. (25-PL3.)
Plakhins G. (26-P2.)
( 50-PL1.)
Zybina T. G.
Rajaraman R.
( 51-PL2.)
(27-PL2.)
( 52-P2.)
1-P1. Meiotic telomere clustering requires actin for its formation and
cohesin for its resolution
Adelfalk Caroline 1, Edgar Trelles-Sticken,1 Josef Loidl,2 and Harry Scherthan1,3
1
Max-Planck-Inst. for Molecular Genetics, Ihnestr. 73, D-14195 Berlin, Germany
Institute of Botany, Rennweg 14, University of Vienna, A-1030 Vienna, Austria
3 Institute for Radiation Biology Bundeswehr, Neuherbergstr. 11, D-80937 Munich, Germany
2
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
2-PL2. Endopolyploidy as a morphogenetic factor of development
Anisimov A.P.
Far-Eastern National University, 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 CRDF and Russian Federation Ministry of
Education and Science (project REC-003).
3
3-P2. Morphofunctional parameters of nucleoli in polyploid cells of the snail
succinea lauta
Anisimova A.A, A.P. Anisimov
Far-Eastern National University, 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 Agprotein 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 CRDF and Russian Federation Ministry of
Education and Science (project REC-003).
4
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 mechanisms involved 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 Aulocantha as 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 depolyploidisation, 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
5
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 genomes establish
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.
6
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;
Paterson Institute for Cancer Research, Manchester, UK;
3
Faculty of Genetics Belarussian State University, Minsk, Belarus;
4
Tenovus Research Laboratory, Southampton, UK;
2
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.
7
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 MOSprotein interacting with cyclin B1, while rare endotelophases – show a para-haploid number of multistranded 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.
8
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.
9
8-PL1. The role of chromatin structure and nuclear architecture in the
cellular response to ionizing radiation
Friedl A.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-ofevents 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.
10
9-P2. Possible role of endogenous growth inhibitors in regeneration of
organs: searching of new approaches
Giorgobiani N, Tumanishvili G.
Laboratory of Develpmental Biology, Tbilisi State University, 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.
Endogenous regulators of proliferation are protein molecules, encoded by regulating
genes. Protein regulators induce specific signals to target cells. We study endogenous factors –
inhibitors isolated from: 1. Different organs of adult animals (heart, kidney, liver, brain); 2.
Homologous organs of phylogenetically different animals (heart of the helix, pigeon, rat, pig).
Factors obtained, represent thermo-stable protein complex (TSPC) that consists of low – 1217kD and high – 60-70kD molecular fractions. TSPC inhibits mitotic index, DNA and RNA
synthesis, suppressing transcription of nucleoli in particular.
Chromatography results of hydrophobic interactions have shown that low-molecular part
of TSPC in different investigated organs of the same animal species as well as in the same
organ (heart) of different investigated animal species have identical characteristics.
Thus, it can be supposed that the so-called phylogenetically conservative group of
thermo-stable proteins precipitate in proliferation and gene regulation. Therefore proposed
method of antibody production to TSPC is not unfounded. Antibodies to TSPC can be used for
cell proliferation in organs with the limited ability to regeneration that is myocardium.
11
10-PL1. Identification and characterization of telomere length and telomere
organization in metaphase and interphase cells
Greulich-Bode Karin M, Damir Krunic, Sharareh Moshir, and Petra Boukamp
DKFZ, Dept. Genetics of Skin Carcinogenesis, Heidelberg, Germany
Infinite growth and genomic instability are two main characteristics of cancer cells.
Both characteristics share a relation with one common chromatin structure: the telomere.
Telomeres, the outermost parts of the chromosomes consist of (TTAGGG)N repeats and are
– due to replication-dependent loss of these repeats – considered to be the „mitotic clock“ of
the cells. When they reach a critical length, cells stop to proliferate and senesce. Cancer
cells circumvent this process by expressing the ribonucleoprotein complex telomerase that
is able by de novo synthesis of (TTAGGG)N repeats to counteract further telomere erosion.
This endows the tumor cells with infinite proliferation, i.e. immortality.
Despite telomere length stabilization many tumor cells suffer from genomic instability
and newest findings suggest that not only telomere length, but also telomere organization or
distribution of the telomeres may be involved in this.
In order to study telomere length regulation on a single chromosome level, we
followed the faith of human chromosomes 3 or 15 after introduction into murine A9 cells
(telomere length –20kb) and after retransfer back into human cells (telomere length – 3kb).
Telomere length was investigated by quantitative fluorescence in situ hybridization (Q-FISH)
on metaphase spreads of the respective cell types. Under both conditions the telomeres
quickly adapted to the telomere length of the host cells demonstrating that telomere length
regulation – under physiologic conditions - is a complex process involving not only
proliferation-dependent but also genetic mechanisms. Interestingly, under forced conditions,
i.e. upon constitutive high expression of telomerase, telomeres elongated for several
passages and telomere length stabilized in different clones at different length (all larger than
the parental cells). This suggests that the length stability provided by endogeneous
telomerase can be overcome, however, telomerase-dependent telomere lengthening is a
finite process.
Although constitutive high expression of telomerase finally led to stable telomere
length, it was accompanied by chromosomal aberrations.
In order to investigate whether telomeres would play a role in these genetic changes
(genomic instability), we studied the 3D distribution and organization of telomeres in
interphase nuclei of HaCaT and HaCaT-TERT cells and observed telomere clustering
(called telomeric aggregates = TA’s) in the HaCaT-TERT cells. TA formation was even
increased in HaCaT cells carrying a constitutively expressed c-myc gene. c-Myc is known to
induce genomic instability and in agreement with that we now have evidence that transient
dysregulation of Myc is correlated with an immediate induction of TAs and a delayed
induction of chromosomal changes. Therefore, we conclude that telomeres play an
essential and versatile role in carcinogenesis. To further strengthen our findings, telomere
length and organization is presently investigated in situ on tissue sections and preliminary
data will be discussed.
12
11-PL3. Hsp70 chaperone as a survival factor in cell pathology
Guzhova Irina, Margulis Boris
Institute of Cytology Russ. Acad. Sci., Tikhoretsky pr. 4, St.Petersburg, Russia.
Heat shock or stress proteins (Hsps) represent the well-conserved mechanism of a cell
reaction to a variety of cytotoxic factors, both of xenobiotic and natural origin. Most of Hsps possess
chaperonic activity that means their ability to ensure the correct assembly of newly synthesized
polypeptides and removal of denatured proteins. In this process chaperones recognize hydrophobic
regions on a protein molecule; such regions are usually hidden inside the polypeptide in natural
conditions and can appear at the latter surface, if (i) protein structure is damaged, (ii) polypeptide
must be finally assembled, or (iii) the mature conformation is subject to change by a modifying factor.
The latter changes are often observed in the process of signal transduction. One of Hsps, Hsp70, is a
good example of inducible protein; its expression may be triggered by a great number of different
factors including those known to induce “normal” cellular processes, proliferation, differentiation and
apoptosis. Although Hsp70 is thought to be the inducible protein, its expression was found to be
rather high in certain human tissues and organs, for instance in muscle and heart. It is also important
that numerous human tumours contain large amounts of Hsp70. Most of anti-tumor therapeutic
means are aimed to induce the process of apoptosis in proliferating cancer cells without affecting
normal cells. Hsp70 by employing its chaperonic activity was found to bind molecules involved in the
regulation of apoptosis and to inhibit the process at different stages. The fact that Hsp70 expression
is high in cancer cells may become an obstacle for therapeutic approaches employed in oncology.
On the other hand, in case of over-expression Hsp70 was shown to appear at the surface of cancer
cells and participate in the presentation of tumour antigens to immune cells. Therefore, chaperonic
activity of Hsp70 can be a two-face factor in the fate of a cancer cell subjected to anti-tumour therapy
and this should be taken in account when planning the course of therapy.
Another field for the employment of protective capacity of Hsp70 is neuropathology. Many if
not all neurodegenerative processes are associated with the accumulation in neural cells of insoluble
aggregates consisting of misfolded or mutant proteins. These aggregates hamper intracellular
transport, inhibit metabolism and activate apoptosis through different pathways. It was shown that in
cells over-expressing Hsp70 gene or in transgenic animals the chaperone hindered aggregate
formation and enhanced cell viability. The discovery of that Hsp70 is able to pass cell membrane
prompted researchers to explore whether the protein introduced in cell culture can enter cells and
protect them from neuropathogenic factors. The results of these experiments show that the
introduction of Hsp70 was able to reduce the number and size of intracellular aggregates of mutated
proteins and to rescue cells from apoptosis. In the review chaperonic and protective capacities of
Hsp70 will be discussed in a connection to neurodegenerative process and carcinogenesis.
13
12-PL1. Nanosizing of specific gene domains by means of SMI microscopy
and COMBO-FISH labelling
Hausmann M1,2, Hildenbrand G.1, Finsterle J.1, Stein S.1, Spoeri U.1, Timme S.2,
Wiech T.2, Walch A.2, Werner M.2, Cremer C.1
1Kirchhoff-Institute
of Physics, University of Heidelberg, Im Neuenheimer Feld 227, D-69120
Heidelberg; 2Institute of Pathology, University Hospital Freiburg, Albertstr. 19, D-79104 Freiburg,
Germany;
The spatial organisation of the human genome is not random and the architecture of
chromosome territories and sub-chromosomal domains shows a functional correlation. The
radial arrangement of chromosome territories appears to be determined by gene density
and chromosome size and is evolutionarily conserved. Recent findings indicate that on the
micro-scale, i.e., in the dimensions of whole chromosome territories and entire subchromosomal domains, differences between individuals or between homologous regions of
different genetic activity are often not significant. Such differences, however, may occur on
the nano-scale, i.e. in functional units of the genome in the order of 100 nm. It may be
assumed that in these dimensions functionally active genome domains are more relaxed
and therefore larger than functionally inactivated domains.
In order to verify this hypothesis, high resolution microscopy of specifically labelled
genome domains is required. High resolution microscopy means fluorescence light
microscopy in the order of some ten nanometers which is considerably beyond the
diffraction limit of an objective lens of a fluorescence microscope. A practical approach to
measure the compaction of genome domains with high precision is SPDM (Spectral
Precision Distance Microscopy) using two well defined labelling regions of different spectral
signatures.
A more advanced technique measuring the diameter of a labelled genome domain
(gene or breakpoint domain) in the order of 100 nm with an accuracy of a few nanometers
only is SMI (Spatially Modulated Illumination) microscopy. The object is illuminated by a
standing wave-field of two coherent laser beams and a stack of 200 - 400 images is taken
from a cell nucleus moving step by step along the optical axis through the illumination field.
The modulation contrast determined by the object intensities in the images is a direct
measure for the diameter of a labelled region and can be rescaled into volume and
compaction values. The results are shown for several gene and breakpoint regions.
This highly improved volume resolution, however, opens questions on the influence
of the FISH labelling procedure on the detected genome domain. In order to better maintain
the genome nano-structure COMBO-FISH (COMBinatorial Oligo Fluorescence In Situ
Hybridization) has been developed. By means of an analysis of the human genome data
base an appropriate set of DNA or PNA oligo probes (homopurine or homopyrimidine
probes) can be configurated that specifically co-localize in the given region only and that
bind as a third strand to the native DNA double strand of the target. Due to smallness of the
probes and triple strand binding omitting any step of thermal denaturation of the DNA in the
nucleus, this procedure seems to gently maintain the nano-architecture of a genome
domain and can also be applied to vital cells (“FISH vital”). In combination with SMI
microscopy this opens novel aspects in the elucidation of the nano-architecture of the
genome. The feasibility is demonstrated for the example of the abl breakpoint region.
14
13-P2. Cells that Undergo Radiation-Induced Mitotic Catastrophe Have the
Potential to Survive for Many Generations Post-Treatment: A LSDCAS Study.
Ianzini F.1, 2,3, B.A. Keller2, P.J. Davis2 and M. A. Mackey 1, 2
Departments of 1Pathology, 2Biomedical Engineering, and 3Radiation Oncology, University of Iowa,
Iowa City, IA, USA.
Radiation-induced mitotic catastrophe (MC) occurs when cells enter into a premature
mitosis following radiation and it is thought to occur due to abnormally high levels of cyclin
B1 which accumulate during radiation-induced delays in late S and G2 phases (Ianzini and
Mackey, Int.J.Radiat.Biol., 1997, 72, 409). MC is associated with a delayed DNA damage
which occurs days following the radiation treatment (Ianzini and Mackey, Mutagenesis,
1998, 13, 337). Because this damage may represent a novel mechanism for the generation
of gene mutations, it is of interest to determine if a cell might survive MC, even at a low
frequency. In this report, experimental data obtained using the Large Scale Digital Cell
Analysis System (LSDCAS), an automated microscope system, shows that in irradiated
GM10115 cells (8 Gy - surviving fraction 0.13%), 0.07% of the cell population undergo MC
and continue to divide for up to 12 days following irradiation. Our data show that 94.8% of
the cells underwent abnormal cell division consistent with radiation-induced MC at the first
post-irradiation mitosis (mostly multipolar mitoses followed by cell fusion), while 5% of the
cells never entered into mitosis during the 12-day post-irradiation interval of observation,
thus indicating that MC is a major mode of radiation-induced death in this cell line. Cells that
survived MC underwent cell fusion after an attempted division, and then presented a
fragmented nuclear morphology. These results confirm other studies that demonstrated that
cells that have undergone radiation-induced MC can divide for several cell generations after
the abnormal mitosis (Prieur-Carrillo et al., Radiat.Res., 2003, 159, 705). Experiments are
underway using longer periods of observation to determine if the dividing cells observed in
these studies represent clonogenic cell populations. The importance of these findings will be
discussed in relation to the acquisition of radioresistance by a subpopulation of the
irradiated cells. (support: NIH RO1CA74899; NIH R33CA94801-01; Whitaker Foundation
Special Opportunity Award).
15
14-P3. Cigarette smoking increase NF-kBp65, but decrease HDAC2
expression in airway epithelium and alveolar macrophages in asymptomatic
smokers and COPD
Isajevs Sergejs, Immanuels Taivans, Uldis Kopeika, Gunta Strazda
Faculty of Medicine, University of Latvia, Sarlotes street 1a, LV-1001, Riga, Latvia
Airways inflammation and oxidant/antioxidant imbalance is a major cause of lung
damage in chronic obstructive pulmonary disease (COPD). An oxidative stress leads to
redox-sensitive nuclear factor-κB (NF-kB) activation, which regulates the transcription of
inflammatory mediators. The molecular mechanism by which inflammatory genes are
switched off is mediated by chromatin deacetylation by histone deacetylase (HDAC2).
The aim of our research was to compare inflammatory changes in central airways
and alveolar walls in nonsmokers, asymptomatic smokers and in smokers with COPD.
The study population was composed of 22 subjects undergoing lung resection for a
solitary peripheral carcinoma. They have been subdivided into three groups: 6 subjects
were nonsmokers with normal lung function, 8 subjects were smokers with normal lung
function and 8 subjects were COPD patients. Immunohistochemical methods were used to
identify NF-kBp65 and HDAC2 positive cells.
Subjects with COPD had more NF-kBp65 positive cells in central airways and
alveolar walls compared with asymptomatic smokers and nonsmokers. The NF-kBp65 was
activated both in macrophages and epithelial cells. In nonsmokers NF-kBp65 expression
was found mostly in cytoplasm, but in smokers in cell nucleus. There was a positive
correlation between the number of NF-kBp65 positive cells in central airways (p<0,001;
r0,68) and smoked pack-years. There was a negative correlation between the HDAC2
positive cells in alveolar walls and smoked pack-years (r=-0.69; p=0.028). HDAC2
expression was suppressed in smokers.
Smoking induced NF-kB activation with concomitent suppression of HDAC2
evidently seems as a reason for inflammation in central airways and lung parenchyma.
16
15-PL2. Delayed DNA double strand break repair in lymphoblastoid cells
that undergo mitotic catastrophe after radiation-induced MAPK activation
Ivanov Andrey1, Mark Cragg2, Jekaterina Erenpreisa3 and Tim Illidge1.
1 Paterson
Institute for Cancer Research, Manchester, UK
Tenovus Research Laboratory, Southampton, UK
3 Biomedical Research and Study Centre, Riga, Latvia.
2
P53 mutant tumour cells respond to genotoxic stress by bypassing G1 arrest and
halting in G2. Following release from G2 they undergo complex events grouped under the
term “mitotic catastrophe” whereby mitotic cycle is suppressed, delayed cell death begins
and polyploid cells are produced. Here we provide evidence that the abrogation of radiation
induced G2/M arrest, occurrence of mitotic catastrophe, production of polyploid cells and
delayed apoptosisis are triggered by activation of ERK MAPK cascade. These events are
fully abolished when ERK pathway is inhibited. The appearance and resolution of DNA
damage has been studied by observing the kinetics of formation and resolution of gammaH2AX foci and by comet assay. Subsequently the kinetics and distribution of Rad51 foci
were studied as a measure of attempted homologous recombination. The resolution of DNA
damage and the formation of Rad51 foci reached maximum in endopolyploid cells on days
5-6 after irradiation when delayed apoptosis was maximal indicating that cells were being
selected for survival at this time. Furthermore, the proportion of Annexin V positive polyploid
cells decreased as they continued ongoing rounds of DNA replication, suggesting
endoreduplication is involved in selecting cells resistant to cell death. Taken together, our
data indicate that the activation of ERK MAPK pathway may promote radiation induced
mitotic catastrophe. Also, endopolyploid cells produced after severe genotoxic stress have a
transient survival advantage that may potentially contribute to radioresistance of tumour that
undergo mitotic catastrophe.
17
16-PL2. Cancer/testis associated genes - a link to gametogenesis?
Kalejs Martins
Biomedical Research and Study Centre of the Latvian University, Ratsupites str. 1, LV-1067, Riga,
Latvia;
Cancer/testis associated genes (CTAs) is an extensively studied group of genes which is
characterized by an expression profile limited to germ cells in normal testis (less to ovaries and
trophoblast) on the one hand and various malignancies, on the other. 44 gene families have
been recognized as CTAs in various malignancies hitherto and will be shortly reviewed in the
lecture. But with a few notable exceptions, their function in gametogenesis is unknown and even
less is known on their function in tumours. However, it is interesting to note that a meiosisspecific endonuclease SPO11 which is required for recombination together with SCP1- a
component of the synaptonemal complex have been described as CTA genes and are crucial
during meiotic prophase 1.
There are several pathways in germ cell differentiation and early embryonic development
having specific features which potentially could be beneficial for cancerogenesis and cancer
progression (f.ex., recombinative DNA repair, high proliferative potential, telomerase activity,
evasion) and some share similar phenotype with cancer cells (f.ex., formation of giant cells in
cancer and trophoblast).
We suggest, after Old (2001), that an aberrant programme sharing some mutual players
and regulators with gametogenesis becomes activated or reactivated in p53 mutant tumour after
genotoxic stress. As each stage of the germ-line has its own protein profile, this could explain
the heterogeneity of CTA expression in various tumours at various times. In our material –
irradiated p53 mutant lymphoid tumours, we observe expression of a major meiotic cohesin
REC8 and a meiotic recombinase DMC1 which is thought to mediate association between REC8
and RAD51 which are also expressed. All three provide the ground for homologous
recombination and thus DNA repair. This process is characteristic for the meiotic prophase 1
both in spermatogenesis and oogenesis. In our material these three proteins appear to be
expressed in endopolyploid cells arising after radiation-induced mitotic catastrophe. In these
cells active DNA repair by homologous recombination takes place (Ivanov et al., 2003).
Employment of processes specific for meiosis 1 would give endopolyploid cells a survival
advantage alternative to catastrophic mitosis. Likewise meiotic cells, endopolyploid cells have
some potential to reduce DNA content and return to mitotic divisions. CTA genes of different
tumours, or at least part of them, could be involved in this programme.
18
17-P3. Historical view on development of special cell biology in Latvia
Kalnina M, S. Kukaine, A. Dalmane
Faculty of Medicine, University of Latvia, 1a Sarlotes street, LV-1001, Riga, Latvia
Though the frontiers of cell biology are extensive, already in 1884 Jean Baptiste Carnoy
(1836 – 1899) in his book “La biologie cellulaire” proposed three lines of cell research:
- general cell biology
- comparative cell biology
- special cell biology.
Compared to cell biology research in the rest of the world, in Latvia the development of
cell biology was slow. The research was begun by LU MF J.A. Eglitis (1902 – 1986) on tissue
cultivation („Audu kultūru pagatavošana”), published in the Latvian medical journal (Latvijas Ārstu
žurnāls) in 1935 (Issue Nr. 12), ended with his leaving for exile in 1944.
Cell biology research started again at the LU MF in 1947. It was undertaken by the
experienced cytologist K. Bogojavlensky
Latvia. K. Bogojavlensky’s
( 1899 – 1967). He set up the cytology school in
interests were in special cell biology and morphological criteria for
cell differentiation. He combined classical cytological methods with cytochemical reactions and
always treated a cell as a unified, integrated system. Influenced by A.Maksimov (1874 – 1926),
M. Zile (1863 – 1945) and K. Rudzitis (1899 – 1978) studied the connectiveness of conjunctive
tissue–mast cells, eosinophilic granulocyte and plasma cells.
Research into the interaction of
cells of the mucous membrane of the small intestine at the ultrastructural level was begun in the
second half of the last century (M. Kalnina 1974). In general cell biology and particularly, in
special cell biology both the contact of the inner cells with the outer cells and their interaction are
of critical importance. The interaction of mast cells, plasma cells and eosinophilic granulocyte is
characteristic of the mucous membrane of the intestine, for it is an essential part of the immune
system. Our work extends to electron microscopic
research on the mucous membrane of the
small intestine of seven ulcer sufferers. Ultra thin cuts have been analysed and photographed
with an electron-microscope JEM – 100C .
The surface of functionally active plasma cells is covered with outgrowths of various
lengths. They develop mutual contacts with eosinophilic granulocyte and mast cells.
Regular
contacts are found more often, but nexus junctions are also possible. Mast cells, which junctions
connect to plasma cells, are partially or completely degranulated. In this way special cell biology
research provides information on the immune–competent cells of the mucous membrane of the
small intestine.
19
18-PL1. From Sequence to Morphology
Towards a Holistic Understanding of Genomes
History and Perspectives
Knoch Tobias A
Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The
Netherlands; Biomedical Structure Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer
Feld 280, and Molecular Biophysics, Kirchhoff-Institute for Physics, University of Heidelberg, Im
Neuenheimer Feld 227, D-69120 Heidelberg, Germany
Genomes are one of the major foundations of life due to their role in information
storage, process regulation and evolution. However, the sequential and three-dimensional
structure of the human genome in the cell nucleus as well as its interplay with and
embedding into the cell and organism only arise scarcely. Various models were put forward
through history. To achieve a deeper understanding of the human genome the threedimensional organization of the human cell nucleus, here, the structural-, scaling- and
dynamic properties of interphase chromosomes are evaluated on all scales from a single
base pair to the nuclear morphology level in respect to achieving a consistent holistic
framework of genome organization.
Thus, by using Monte Carlo and Brownian Dynamics methods, the 30 nm chromatin
fiber was simulated according to the Multi-Loop-Subcompartment (MLS) model, in which
~100 kbp loops form rosettes, connected by a linker, and the Random-Walk/Giant-Loop
(RW/GL) topology, in which 1-5 Mbp loops are attached to a flexible backbone one finds
that both the MLS and the RW/GL model form chromosome territories but only the MLS
rosettes result in distinct subcompartments visible with light microscopy and low overlap of
chromosomes, -arms and subcompartments. The MLS morphology, the size of
subcompartments and chromatin density distribution of simulated confocal (CLSM) images
agree with the expression of fusionproteins from the histones H1, H2A, H2B, H3, H4 and
mH2A1.2 with the autofluorescent proteins CFP, GFP, YFP, DsRed-1 and DsRed-2 which
also revealed different interphase morphologies for different cell lines. Even small changes
of the model parameters induced significant rearrangements of the chromatin morphology.
Thus, pathological diagnoses, are closely related to structural changes on the chromatin
level. The position of interphase chromosomes depends on their metaphase location, and
suggests a possible origin of current experimental findings. The scaling behaviour of the
chromatin fiber topology and morphology of CLSM stacks revealed fine-structured multi-
20
scaling behaviour in agreement with the model prediction and correlations in the DNA
sequence. Review and comparison of experimental to simulated spatial distance
measurements between genomic markers as function of their genomic separation also
favour an MLS model with loop and linker sizes of 63 to 126 kbp. Simulated and
experimental DNA fragment distribution after ion-irradiation revealed also best agreement
with such an MLS. Correlation analyses of completely sequenced Archaea, Bacteria and
Eukarya chromosomes revealed fine-structured positive long-range correlation due to
codon, nucleosomal or block organization of the genomes, allowing classification as well as
tree construction. This shows a complex sequential organization of genomes closely
connected to their three-dimensional organization. Visual inspection of the morphology
reveals also big spaces between the chromatin fiber allowing high accessibility to nearly
every spatial location, due to the chromatin occupancy <30% and a mean mesh spacing of
29 - 82 nm for nuclei of 6 - 12 µm diameter. This agrees with a simulated displacement of
10 nm sized particles of ~1 - 2 µm takes place within 10 ms, i.e. a moderately obstructed
diffusion of biological molecules in experimental agreement.
These results show that the local, global and dynamic characteristics of cell nuclei
are not only tightly inter-connected, but also are integrated holisticly to fulfill the overall
function of the genome. Future experiments and simulations benefit from taking this ever
further completed consistent framework into account in respect to project planning as well
as evaluation and hypothesis creation. Consequently, such a detailed understanding and
knowledge of genomes will ultimately lead to full functional virtual genomes in silicio
allowing predictions for diagnostics, disease treatment and the modification of genomes.
21
19-P2. Hunting the mechanisms of self-renewal of immortal cell populations
by means of real-time imaging of living cells
Kvitko O.V, Koneva I.I., Sheiko Y.I., Anisovich M.V.
Institute of Genetics and Cytology of the National Academy of Sciences of Belarus,
Belarus, 220072, Minsk, Akademicheskaya Str. 27.
Immortality of cancer cell populations may be dependent on a subpopulation of cells
with unlimited self-renewal potential. According to the concept of cancer stem cells
immortalizing events occur in normal stem cells. Alternatively, tumors can self-perpertuate
due to some periodical “rejuvenational” processes in which cells restore their proliferative
potential. Since many cancers are aneuploid because of chromosomal instability, candidate
processes of generating cells with the enhanced proliferative capacity may be asymmetric
bipolar and multipolar mitoses. Recently on the basis of computerized videomicroscopy of
cellular events during carcinogenesis a new process (termed neosis) of nonmitotic
asymmetric cytokinesis which ensues after nuclear budding has been proposed as a
producer of cells with extended mitotic life span (Sundaram et al., 2004). In our
computerized time-lapse study of the immortalized cell line from mouse embryos we found a
process of detachment of a part of cytoplasm (without a previous cell rounding which takes
place before mitosis). These detached parts often behaved like normal cells during short
periods (usually few hours), but then stopped to move and died, whereas the “main” cell
could live further during days and weeks. This cytoplasm separation occured in mitotic and
postmitotis cells, but the generation of new cells by a nonmitotic division never was
registered. Thus, on our immortalized cell system we have not found the evidence of
processes which could resemble neosis. At the same time, threepolar and asymmetric
dipolar mitoses happened frequently (1anomalous division per 20 symmetric mitoses). Cells
resulting from such anomalous mitoses as a rule divided by normal symmetric mitosis.
Moreover, in one thoroughly analysed individual cell genealogy a quickly proliferating cell
subpopulation descended from every of three sister cells which were generated by
threepolar mitosis.
It is possible that various processes of anomalous cell division
participate in the indefinite maintenance of immortal cell populations.
22
20-PL1. Spatial organization of gene expression
de Laat Wouter
Dept. of Cell Biology, ErasmusMC, PO Box 1738, 3000 DR Rotterdam, The Nettherlands;
Chromosome conformation capture (3C) technology provides a tool to study the
structural organization of a single gene locus. The technique involves quantitative PCRanalysis of cross-linking frequencies between two given DNA restriction fragments, which
gives a measure of their proximity in the nuclear space. Originally developed to analyse the
conformation of chromosomes in yeast, we adapted this procedure to investigate the
relationship between gene expression and chromatin folding at intricate mammalian gene
clusters. We found that the locus control region (LCR) and other cis-regulatory DNA
elements of the -globin locus, spread over 200 kb of DNA, cluster in space with the active
-globin genes to form a nuclear compartment, called the Active Chromatin Hub (ACH). No
ACH is formed in non-expressing brain tissue. Genes switch interaction with the ACH during
development, correlating with their transcriptional activity. A chromatin hub (CH) not
containing the genes is present in erythroid precursor cells and a functional ACH is only
formed upon differentiation. Transcription factors, like EKLF, play essential roles in
organizing the conformation of active gene loci. Here we will present new data on the role of
transcription factors in chromosome folding. We will also address how an ectopically
positioned beta globin LCR affects the transcriptional activity and structural organization of
the region surrounding its integration site.
23
21-PL3. Ubiquitin-Dependent Proteolysis of Cytochrome P450 CYP2E1
Enzyme
Leonchiks Ainars
Latvian University Biomedicine Centre, Riga, Latvia;
The vast majority of human population in the industrial countries is exposed to xenobiotics via
environment, diet, drugs and smoking. Lipophilicity of most xenobiotics enables absorption through
skin, lungs or gastrointestinal tract. Among other organs, the liver contains an impressive defence
system that modifies those xenobiotics that pass through it in the circulation to products more
extractable from the body. The hepatic cytochromes P450 superfamily represents monotopic
endoplasmic reticulum anchored hemoproteins participating in enzymatic oxidation of a wide variety
of xenobiotics. P450 superfamily with over 2000 variants is one of the major enzyme groups that are
involved in biotransformation of these xenobiotics, converting them to water-soluble chemicals
favouring excretion.
For one of them, the cytochrome P450 (CYP) 2E1 the known substrates represent such
diverse groups of xenobiotics as ethanol, benzene, aniline, dimethylformamide, flurane and their
derivatives. From other side, there are a number of toxic compounds that are activated by CYP2E1 to
reactive metabolic products like reactive O2 species that can attack the P450 and structurally or
functionally damage the enzyme.
Such a structurally damaged P450 is rapidly removed by the cellular system responsible for
degradation and removal of misfolded and damaged proteins. A pathway involved in this process is
the ubiquitin-proteasome system that recognizes a target protein by the covalent attachment of
multiple ubiquitin molecules, which provide a recognition signal for the 26S proteasome that plays a
central role in the controlled degradation of intracellular proteins. The proteasome processes target
protein substrate in an ATP-dependent fashion to yield small oligopeptides and free ubiquitin
molecules. The mechanisms of the regulation of P450s are still not fully understood and attract much
attention.
The aim of this study was to investigate these mechanisms of the posttranslational regulation
of the cytochrome P450 2E1 degradation, in particular, to investigate which sites are responsible for
protein stabilization. Six CYP2E1 mutants with Lys-Arg substitution and one mutant with the point
mutation at D394 site were constructed. H2.35 cells were transfected by the constructs and all
mutant proteins were expressed. Five cell lines were established, and the stability of mutated
proteins was examined in the presence of their substrates, as well as the role of mutations in the
degradation of protein by ubiquitin-proteasomal pathway was evaluated. Two mutants, S129AK140R-D394V and D394V, exhibited significant increase of the protein level and longer half-life. The
experiments suggested that the reason of this increase could be associated with the impaired
interaction of ubiquitinated 2E1 mutants with the proteasome.
24
22-PL3. Regulation of nuclear factor-kappaB in rat cardiomyocytes
Liepinsh Edgars, Reinis Vilskersts, Ivars Kalvinsh, Maija Dambrova
Latvian Institute of Organic Synthesis, 21 Aizkraukles Str., Riga LV-1006, Latvia;
NF-κB is an inducible nuclear transcription factor regulating the expression of many
genes. NF-κB activation may function as a master switch in a variety of immune and
inflammatory processes, including sepsis and transplant tolerance, and it is activated during
cellular responses to stress, hypoxia and ischemia-reperfusion (I/R).
NF-κB exists in the cytoplasm of most cells as a homo- or heterodimer of five
different proteins; the predominant form of NF-κB is a heterodimer of p50 and p65 proteins.
It is normally bound in the cytosol with a class of inhibitory proteins called IkBs. Activation of
NF-κB involves the phosphorylation, ubiquitization, and subsequent degradation of inhibitory
proteins leading to the activated NF-κB form which translocates to the nucleus to induce the
gene expression.
There are several studies exploring the role of NF-κB in heart diseases. However,
the regulation of NF-κB in cardiac cells for therapeutic purposes is still unclear. There is an
experimental evidence of the protective role of NF-κB activation in ischemic preconditioning.
On the other hand, inhibition of NF-κB translocation into cell nucleus is shown to bee
important for anti-inflammatory and anti-ischemic treatment. To test whether the role of NFκB as a promoter or antagonist of apoptosis depends on the stress stimulus, we determined
the influence of NF-κB activity on cell damage elicited by a variety of inducers within adult
rat cardiomyocyte primary culture.
Cardiomyocyte damage was induced by I/R subjecting cells to of anoxia and
reoxygenation, by exogenously added 100 μM H2O2 or the inflammation stimulator bacterial
lipopolysacharide (100 ng/ml). We used a guanidine compound ME10092 to inhibit the
activation of NF-κB translocation. We found, that stress factor induced over-activation of NFκB in isolated cardiomyocytes caused cell apoptosis and death. In contrary, the inhibition of
NF-κB translocation by ME10092 significantly increased number of viable and non-apoptotic
cells. On the other hand, the higher concentrations of ME10092, which completely reduced
the NF-κB activation till non-stimulated cell control level, decreased the number of viable
cells and increased the number of apoptotic cells. This finding suggests the balanced
activation of NF-κB translocation is essential for cardiac cell survival in stress conditions.
25
23-PL3. Autocrine regulation of palatine tonsil inflammation by substance
P: role of lymphocytes and macrophages
Markovs Jurijs, Sergejs Isajevs, and Gundega Knipshe
Faculty of Medicine, University of Latvia, 1a Sarlotes street, LV-1001, Riga, Latvia
It is increasingly recognized that immune and nervous systems are closely integrated by
secretion of neuropeptides to optimise defence systems within the lymphoid organs.
Substance P (SP) is a neuropeptide believed to be a major mediator of neurogenic
inflammation. Direct interactions between neuropeptides and immune cells (lymphocytes
express SP receptors) are facilitated by the innervation of lymphoid organs by afferent nerve
endings containing SP, VIP, somatostatin and neuropeptide Y. Although immunohistochemical
staining of lymphoid organs has demonstrated that SP is confined mainly to unmyelinated nerve
endings non-neuronal cells of the immune system can be a source of tachykinins.
The purpose of this study was to investigate the localization of SP in immunocompetent
cells of human tonsils in individuals suffering from recurrent tonsillitis and from hypertrophic
tonsillitis.
We collected tonsils from 12 patients who underwent tonsillectomy. They have been
subdivided into two groups: 6 subjects had recurrent tonsillitis and 6 subjects had hypertrophic
tonsillitis. Histopathologic and immunohistochemical examinations of tonsils were performed.
In reactive follicular hyperplasia large secondary follicules were found throughout the
tonsils. The lesions were characterized also by partial distortion of tonsil. At the same time the
mantle zone of the follicules undergo hypertrophy. SP was detected in tingible body
macrophages of the germinal centers and, in less extent, in lymphocytes of the mantle zone. In
recurrent tonsillitis patients the numbers/size of secondary follicules was decreased and mantle
zone become smaller in size. Main cellular expression of SP is found in resting mantle zone Bcells and macrophages, whereas activated lymphoblasts in the germinal center were negative.
Our findings led to hypothesis that hypertrophy of the follicules in patients with
hypertrophic tonsillitis could be mediated in part by SP released from activated B-cells of the
mantle zone. Therefore these cells display autocrine promoting activity and SP should be
included in the conceptual framework of the immune regulation of B-cell function.
26
24-PL2.
DNA
replication
of
prematurely
condensed
chromosomes
in root meristem cells of Vicia faba
Rybaczek Dorota and Janusz Maszewski
Department of Cytophysiology, University of Łódź, 90-231 Łódź, Poland
Nuclear events that make up the core of the cell-cycle comprise two opposite functional
states of chromatin, one adjusted to precisely replicate genomic DNA, and the other adapted to
segregate sister chromosomes to daughter cells at mitosis. Despite general similarity of the key
biochemical factors triggering S- and M-phase, each of these processes designates its own level
of structural organization, which brings about oscillation between the dispersed and condensed
form of chromosomal DNA. Although evident differences in chromatin architectures seem ample
to explain mutual exclusion of DNA replication and nuclear division, temporal separation of Sand M-phase (and the regulatory mechanisms that block cell cycle progression in response to
DNA damage or replication blocks) depend, primarily, on a series of multi-step cell-cycle
checkpoints, through which DNA-derived information is ‘transmitted’ to the large and diverse
group of chromatin components, including functionally distinct protein complexes responsible for
inhibiting cyclin-dependent kinases (CDKs).
Most of what we learn about mechanisms that regulate the order and timing of cell cycle
transitions in yeasts, animals, or humans can be carried over to plants. A great number of
conserved proteins involved in the ‘critical’ G1/S and G2/M transitions include, but are not limited
to, retinoblastoma (RB) tumor suppressor protein, the heterodimeric adeno-virus E2 promoterbinding–protein-dimerization partner (E2F-DP) transcription factor, A- and D-type cyclindependent kinases and homologs of mammalian G1- and M-phase cyclins, Wee1 and CAK
kinases, cyclin-dependent kinase inhibitors (CKIs), proteins engaged in chromatin condensation
and the components of the ubiquitin-dependent proteolytic pathway. Our cytological and
immunobiochemical investigations were aimed to find out what kind of checkpoint controls and
phenotypic effects are produced during hydroxyurea-mediated inhibition of DNA synthesis and
caffeine-induced PCC in root meristem cells of Vicia faba. The obtained results point to the role
of Chk1 kinase as a transmitter of signals generated in chromatin at stalled replication forks and
DNA double strand breaks. Furthermore, it is documented for the first time that DNA replication
may start out in vivo in prematurely condensed chromosomes, provided that a cell has passed
the metaphase/anaphase transition.
27
25-PL3. Basic idea for spectral changes of dyes - a molecule and molecular
aggregates
Matsumoto Shinya
Faculty of Education and Human Sciences, Yokohama National University, 79-2 Tokiwadai,
Hodogaya-ku, Yokohama 240-8501 JAPAN
Many organic dyes have been applied in biochemical and biological fields as a probe
for visual and/or spectroscopic identification of morphological features. On the other hand,
organic dyes are also widely used as a functional dye for opto-electronical applications such
as optical recording, organic photoconductors and organic electroluminescent devices [1].
One of the most important subjects in both applications of organic dyes is to characterize
the spectral changes of dyes in a certain condition and to correlate them with the structures
and properties of systems of interest. Our group have conducted the study of the electronic
states of functional dyes in the solid state (crystals and crystalline thin films) from the
viewpoint of intermolecular interactions [2]. In this presentation, a basic idea for
characterizing the spectral changes of dyes will be addressed in terms of two important
factors. One is a change in the electronic states of a molecule itself. The electronic states of
a molecule are influenced by the chemical and physical conditions of the surroundings. The
other one is cause by molecular aggregation and this is well-known as an exciton interaction
[3]. Furthermore a brief consideration on the spectral changes of toluidine blue with their
correlation with chromatin structures will also be presented.
References:
[1] H. Zöllinger, Color Chemistry, 2003, Wiley-VCH, [2] (a) T. Kobayashi et al., Phys. Chem.
Chem. Phys., in press, (b) E. Horiguchi et al., Bull. Chem. Soc. Jpn., in press, (c) E.
Horiguchi et al., Chem. Lett., 2004, 33, 170-171, (d) S. Matsumoto et al., Chem. Commun.,
2003, 1910-1911, [3] M. Kasha, in Spectroscopy of the Excited State (B. D. Bartolo ed.),
1976, Plenum Press, 337-363.
28
26-P2. Expression of meiotic cohesins in irradiated lymphoblastoid celllines
Plakhins G1, Kalejs M1, Ivanov A2, Illidge T2, Erenpreisa Je1.
1
Biomedical Research and Study Centre of the Latvian University, Ratsupites str. 1, LV-1067, Riga,
Latvia;
2 Paterson Institute for Cancer Research, Manchester, UK
The ability of tumour cells to repair double-strand breaks (DSBs) after ionizing
irradiation is critical for their survival. After large genotoxic insult, the p53-functionally
deficient tumour cell undergo crisis of mitotic pathway (‘mitotic catastrophe’) producing
alternatively transient endopolyploid cells. A proportion of these cells avoid apoptosis and
can repair DNA DSBs by homologous recombination (Illidge et al., 2000; Ivanov et al.,
2003). An even smaller proportion of these cells further segregate into viable descendents.
However, the origin and mechanisms of clonogenic survival of these cell-lines remain
unclear.
One of the possible keys for molecular players resides in transcriptional and
translational activation by mt TP53 cell-lines of the meiotic kinase pathway (MOS/MAPK) in
irradiated mt p53 tumours and some of the features in the endopolyploid cells are similar to
meiotic prophase, after genotoxic insult (Erenpreisa et al., 2000; Ivanov et al., 16-Pl2).
Therefore, the expression of several meiosis-specific genes – meiotic cohesins (Rec8 and
STAG3), ‘Shugoshin’ (SGO1 and SGO2), which serves as a Rec8 protector from
segregation by Separase of sister chromatid centromeres in meiosis I, and recombinase
DMC1, providing linkage of Rec8 to recombinase Rad51, the indispensable complex for the
meiotic type DNA recombination and DSBs repair, were studied here.
In our experiments, on wt TP 53 TK6 and mt TP53 WI-L2-NS and Namalwa, we
have found transcriptional expression of Rec8, STAG3, DMC1 and SGO 1 and 2. After
10Gy irradiation, Rec8, SGO2 and DMC1 were significantly up-regulated in WI-L2-NS and
Namalwa, from d.3. In addition, translational up-regulation of Rec8 cohesing centromeres
was found by Western blot and immunofluorescence in endopolyploid cells in these celllines. TK6 did not survive over dd.3-4 to show these activities. Our working hypothesis is
that translational activation of meiotic genes related to DNA repair by homologous
recombination may be involved in the survival of mt TP53 lymphoid tumours after mitotic
catastrophe, which also occurs basically and is enhanced after genotoxic insult.
29
27-PL2. Neosis, stem cells and self-renewal in cancer
Rajaraman R1, D.L. Guernsey2, M.M. RAJARAMAN3
Department of Medicine1, Pathology** and Nova Scotia Cancer Center***, Dalhousie University,
Halifax NS, Canada
Cellular senescence, characterized by multinucleate/polyploid giant cells (MN/PGs)
is caused by telomere attrition, and is considered a tumor suppressor mechanism. However,
this is a leaky process. MN/PGs with inactivating p53, pRB or p16INK4A/p14ARF gene
mutations escape mitotic crisis and via an unknown mechanism, give rise to proliferating
mononuclear, aneuploid cells that display reactivation of telomerase, altered genotype and
phenotype, genomic instability, and are considered “immortal”. Exposure of tumor cells to
genotoxins induces similar events. Using transformed focus formation assay and video timelapse microscopy, we have shown that mononuclear transformed cells emerge from
MN/PGs via nuclear budding followed by asymmetric cyokinesis and thus, escape death by
mitotic catastrophe. We have termed this novel type of cell division neosis and the resultant
small cellls the Raju cells. Neotic colonies (Raju cells derived from one MN/PG, now called
neosis mother cell or NMCs) and not the non-transformed colonies from the same treated
plate, display neoplastic properties described above. Raju cells are the progenitors of
neoplastic cells. Mitotic derivatives of Raju cells accumulate further genetic alterations and
undergo aberrant differentiation and telomerase attrition leading to the next cycle of MN/PG
formation and neosis. Neosis yielding Raju cells with extended mitotic life span is repeated
several times in a progressively non-synchronous fashion during tumor growth. NMCs
formed after exposure of tumor cells to genotoxin produce Raju cells that are resistant to the
given genotoxin. Neosis is responsible for the escape of MN/PGs from spontaneous or
induced mitotic crisis, yielding resistant tumor cells with reactivated telomerase. This
contributes to the continuity of tumor cell lineage, and forces tumor progression via natural
selection*. Since tissue stem cells and their derivatives can undergo neoplastic
transformation, the relevance of neosis to self-renewal and cancer stem cells will be
discussed. A comprehensive hypothesis encompassing various theories of carcinogenesis
will be presented. *Cancer Biol Therap. 2004, 3:207-218.
30
28-P2. Plasticity in neosis
Rajaraman R.1*, M.M. Rajaraman2, D.L. Guernsey3,
Department of Medicine1, Division of Hematology1, Nova Scotia Cancer Centre2,
Dept. Radiation Oncology, QEII Health Sciences Center, Department of Pathology3, Dalhousie
University, Halifax NS. B3H 1X5.
Cellular senescence, characterized by multinucleate / polyploid giant cell (MN/PG) formation,
is caused by telomere attrition, leading to mitotic crisis, and is considered a tumor suppressor
mechanism. Exposure of tumor cells to genotoxins also induces a similar senescent state. However,
MN/PGs with inactivating p53, pRB or p16ink4A/P14ARF gene mutations escape mitotic crisis and, by
an unknown mechanism, giving rise to mononuclear cells with genomic instability, reactivated
telomerase, and extended mitotic life span, and are considered ‘immortal’ [10-12]. We have recently
reported that the emergence of mononuclear ‘immortal’ cells from MN/PGs occurs, before they die,
via nuclear budding, followed by asymmetric cytokinesis [13]. We have termed this novel type of cell
division neosis, which is different from mitosis and meiosis (Table 1) and the resultant small cells the
Raju cells [13]. Neosis is defined as a parasexual, somatic reduction division where the non-viable
polyploidy genome of MN/PG cells produce diploid or near diploid viable genomes of Raju cells. The
neosis mother cells (NMCs) and Raju cells share stem cell-like properties (stemness) in that NMCs
distribute asymmetrically the newly synthesized viable DNA is to the daughter Raju cells, while the
latter display transient stem cell-like properties including short mitotic cycles, maturation and
differentiation into tumor cells with extended, but, limited mitotic life span (ELMLS). The process of
karyokinesis via nuclear budding remaining constant, we have observed some degree of plasticity in
the cytokinesis in different systems. These include:
A: 1-5: Simultaneous cytokinesis: The non-viable polyploidy genome of NMCs undergoes
continuous nuclear budding without being immediately followed by cytokinesis. This leads to
syncytium-like multinucleate cell, which then undergoes cellularization by cytoplasmic cleavage
resulting in the formation of multiple Raju cells simultaneously, leaving a central island of the
multinucleate/polyploid nucleus of NMC with a little bit of cytoplasm. In p53-/- MEF/MGb, A5, this
residual NMC produced another but smaller batch of Raju cells after a couple of days.
B, 1-4. Sequential Pericellular cytokinesis - The newly formed Raju cell genome
migrates to the periphery of the large NMCs and then it is pinched of along with a small
amount of cytoplasm.
C, 1-4: Sequential Perinuclear.Cytokinesis - The newly formed Raju cell genome
is immediately surrounded by cytoplasm by cytoplasmic cleavage and the cell externalizes itself by
active movement, a phenomenon termed “birth dance [21]. It is likely that other variations might exist
in different systems.
We shall display some posters and time lapser videos of these variations.
31
29-PL3. Anhydrobiosis and dehydration stress of yeasts
Rapoport Alexander
Laboratory of Cell Biology, Institute of Microbiology and Biotechnology, University of Latvia, Kronvald
Blvd., 4, Riga LV-1586, Latvia
One of the most intriguing phenomena of live organisms is anhydrobiosis. This unique state when
cell metabolism is reversibly suspended for thousands and thousands of years can be reached by strong
dehydration of different procaryotic and eucaryotic organisms. Its investigation using as the model yeasts
gives us the possibility to understand main mechanisms of any eucaryotic cell transition into this state. At
the same time such studies reveal also a lot of very interesting general intracellular reactions directed to
the maintenance of live organisms viability at different unfavourable conditions of the environment as
well as at various natural and artificial stress treatments upon live systems.
Investigations of this state realized during last decades showed a lot of changes which take place
at molecular, structural and functional levels at the stage of cell transition into anhydrobiosis and during
its subsequent reactivation. They are linked with structural-and-functional reconstructions of practically
all organelles – cell wall, plasma membrane, vacuoles, endoplasmic reticulum, ribosomes, mitochondria,
nucleus. Different types of reversible and irreversible intracellular damages are revealed in the cells. Some
unusual protective reactions linked with activities of plasma membrane and nuclear membranes were
discovered. It was shown that one of the most important protective reactions is linked with condensation
of the chromatin in nucleus. It was concluded that besides the trehalose which usually was supposed as the
main intracellular protective compound there are also another important group of very active protective
substances – polyols. The hypothesis is proposed that in the case of the absence of trehalose polyols
(inositol, sorbitol, xylitol, glycerol and probably some others too) can substitute it for the protection of
molecular organization of intracellular membranes. In the case of the presence of adequate amounts of
trehalose polyols most probably protect intracellular macromolecules – proteins and nucleic acids. It is
very important that main intracellular protective reactions can be induced in live organisms also
“artificially” by some special treatments upon them. Such possibility is extremely important, for example,
in those cases when it is necessary to transfer into anhydrobiosis organisms which are non-resistant at
conditions of strong dehydration.
It is shown that conclusions which were made during investigations of different aspects of yeast
anhydrobiosis can give also the possibilities for working out principally new approaches which can be
very interesting already at the moment for further efficient development of different directions of modern
biotechnology.
32
30-P2. DSB-dependent phosphorylation of H2AX histones and induction of
PCC in hydroxyurea-treated root meristem cells of Raphanus sativus, Vicia
faba and Allium porrum
Rybaczek Dorota and Janusz Maszewski
Department of Cytophysiology, University of Łódź, 90-231 Łódź, Poland
Histone H2A variant, H2AX, is rapidly phosphorylated on the induction of DNA
double strand breaks (DSBs) by ionizing radiation (IR) and hydroxyurea (HU)-mediated
replication arrest, resulting in the formation of γ-H2AX foci along megabase chromatin
domains nearby the sites of incurred DNA damage. In an attept to establish a relationship
between species-specific nuclear architecture and H2AX phosphorylation in S/G2 phasearrested root meristem cells, immunocytochemical comparisons using antibody raised
against human γ-H2AX were made among three plants differing with respect to DNA
contents, Vicia faba and Allium porrum, both representing a ’reticulate’ type of DNA
package, and Raphanus sativus, characterized by a ‘chromocentric’ type of chromatin.
Another approach was aimed at determining possible correlations between the extent of
HU-induced phosphorylation of H2AX histones and the quantities of root meristem cells
induced by caffeine to enter aberrant mitotic division (premature chromosome condensation;
PCC). It was concluded that the higher order structure of chromatin may contribute to the
accessibility of molecular factors engaged in the recognition and repair of genetic lesions.
Consequently, as opposed to V. faba and A. porrum, a diffuse chromatin in chromocentric
cell nuclei of R. sativus may become more vulnerable both to generate DNA DSBs and to
recruit molecular elements needed to arrange the cell cycle checkpoint functions, and thus,
more resistant to factors which allow the cells to enter PCC spontaneously.
33
31-P2. Drug-induced premature senescence (DIPS) is activated by DNA
topoisomerase II inhibitors in human non-small cell lung carcinoma A549 cells
Sabisz Michal, Lukasz Skora and Andrzej Skladanowski
Laboratory of Molecular and Cellular Pharmacology, Department of Pharmaceutical Technology and
Biochemistry, Gdansk University of Technology, Gdansk, Poland
In this study, we evaluated the role of DIPS in the antiproliferative effect of different DNA
topoisomerase II inhibitors toward human lung carcinoma cells. Lung cancer cells frequently
overexpress topoisomerase II and show increased sensitivity to topoisomerase II inhibitors in human
cancer patients. We compared biological effects induced by different classical DNA topoisomerase II
inhibitors (doxorubicin, m-AMSA, C-1305) which stabilize covalent DNA-topoisomerase II complexes
and catalytical inhibitor, compound ICRF-187, which does not induce direct DNA damage.
We showed that A549 cells exposed to IC80-IC90 doses of studied drugs ceased cell
proliferation after 1-2 cell divisions and accumulated mostly in G2/M after 48-72 hr of drug treatment.
Curiously, after prolonged post-incubation of drug treated cells (2-3 weeks), a small fraction of
growth-arrested cells re-started normal proliferation.
Growth-arrested cells become very big with flat morphology and biochemical markers of
cellular senescence, such as expanded lysosomal compartment and increased activity of
senescence-associated galactosidase (optimum pH 6). The TRAP assay showed decreased activity
of telomerase in cellular lysates from drug-treated cells, with no apparent changes in telomere
functioning in A549 cells exposed to studied drugs. We also observed increased number of aberrant
mitoses, with frequent multipolar metaphases and anaphases with lagging chromosomes. Western
blotting analysis showed accumulation of p53 and p21 proteins in senescent cells. Interestingly, after
initial increase of both cyclin B1 level and Cdk1 activity, both proteins were downregulated at later
time periods of drug treatment. We also observed induction of stress signalling associated with
activation of ERK 1/2 kinases.
Together, we here show that A549 cells treated with DNA topoisomerase II inhibitors
progress through 1-2 abnormal mitoses and arrest cell proliferation with biochemical and
morphological markers of senescent cells. However, after prolonged post-incubation in drug-free
medium, a small fraction of growth-arrested cells is able to re-enter cell cycle and continue cell
proliferation. We conclude, that induction of DIPS by DNA topoisomerase II inhibitors does not lead to
irreversible growth arrest of tumour cells. This might be particularly important since induction of
cellular senescence program or DIPS in cancer cells by antitumour drugs represents a promising
alternative approach to treat human cancers.
34
32-PL1. Comparative analysis of chromosomal events and DNA repair in
first meiotic prophase
Scherthan H1,3, G. Petukhova2, M. Bellani2, R.D. Camerini-Otero2 and B. Liebe1
1Max-Planck-Inst.
for Molecular Genetics, Ihnestr. 73, D-14195 Berlin, Germany;
Inst. of Diabetes and Digestive and Kidney Diseases, National Inst. of Health, Bethesda,
Maryland 20892, USA;
3Inst. of Radiation Biology Bw, Neuherbergstr. 11, D- 80937 Munich, Germany.
2 National
Chromosome pairing during meiosis I depends on DNA double strand breaks (DSBs)
which are formed during leptotene when telomeres begin to cluster in a limited region of the
nuclear periphery. Different duration of telomere clustering (bouquet stage) has been noted
to correlate with delayed disappearance of intermediate recombination markers in the sex
with longer bouquet stage duration, i.e. in human females. Here, we investigated a possible
link between the duration of meiotic telomere clustering and progress of recombinational
DSB repair in male mouse meiosis. Using knockout models for genes involved in
recombination we show that rapid transit through post-replicative mid-preleptotene stage
requires the recombination proteins Spo11, Dmc1, Hop2, Mlh1 and Atm. Initiation of
telomere clustering and recombination are independent events in the mouse, while DSBdependent ATM signaling influences duration of bouquet stage, as the elevated bouquet
frequences in Atm-/- meiosis are reverted to normal in absence of Spo11 and DSBs.
Absence of the homology sensor protein Hop2 and defective non-homologous end-joining
pathway of DNA repair did not influence meiotic telomere dynamics. The significant increase
of both mid-preleptotene and bouquet frequencies in most recombination mutants relative to
wild type as well as to Sycp3-/- and Hop2-/- spermatogenesis, respectively, suggests that
duration of the mid-preleptotene stage depends on the assembly of pre-recombination
chromatin, while duration of the bouquet stage is integrated with progress of
recombinational DSB repair in mammals.
35
33-PL3. Programmed cell death and stress response of plant leaf cells
Selga T*, M.Selga, V.Pāvila
Laboratory of Plant Cell Biology, Faculty of Biology, University of Latvia Kronvalda blvd. 4, LV 1848,
Riga, Latvia
Last decade provided lot of evidence about programmed cell death (PCD) in plants.
Main efforts were devoted to characterise basic types of PCD in plants. Aim of our work was
to compare two types of PCD – caused by leaf senescence (yellowing) and induced by the
ethylene.
Pea (Pisum sativum L.), cucumber (Cucumis sativus L.) and tobacco (Nicotiana
tabacum L.) plants were grown in greenhouse. Part of 40 day old plants were sprinkled with
Ethephon at a concentration of 4x10–2 M. Mesophyll cells were analysed with confocal laser
scanning microscope Zeiss CLSM 410, Fluorescence microscope LOMO, TEM Philips 301
and SEM Jeol.
Senescence was typical with high seretory activity of palisade parenchyma cells in basal
part of the cell. Nuclei actively functioned as endoplasmic reticulum – they interacted with
Golgi bodies.
Crista of mitochondria were tubular and matrix density remarkably decreased
pointing to the lack of proteins. Mitochondria were enclosed in large vesicles and
transported into the central vacuole. In chloroplasts the number of thylakoids decreased,
large plastoglobules appeared and decreased density of stroma. Chloroplasts formed
stromules which were attached to other chloroplasts, mitochondria and nucleus.
Spraying of plants with ethylene releaser Ethephon increased the number of ER tubules and
Golgi bodies. Golgi bodies were in apical part of palisade parenchyma cells. In addition,
Golgi bodies and vesicles were frequently co-localised with chloroplasts and mitochondria.
Crista of mitochondria became swollen. Mitochondria divided and frequently their division
was inhibited and appeared mitochondria with elongated cristae. Matrix density sligtly
increased due to swelling of crista. Plant leaf cells contained highly organised multiorganellar complexes. Each complex contained centrally located nucleus and was
surrounded by chloroplasts and mitochondria. During yellowing and Ethephon induced PCD
outer membranes of chloroplasts and mitochondria were attached to outer membrane of
nucleus. These nuclei showed pattern of chromatin condensation and fragmentation.
36
34-P3. Interaction of mosaic virus-like particles carrying HBV preS
sequences with cell surface in cultures: Do these epitopes work as specific
addresses?
Shestakova Irina, Ilona Domracheva, Sanita Shkutele, Velta Ose-Klinklava and
Regina Renhofa
Latvian Institute of Organic Synthesis, BMC University of Latvia, Riga, Latvia
To study the role of HBV surface proteins in the mechanism of HB virus infection several
investigations with fused proteins were performed. On the base of maltose binding protein
was formed a set of recombinant preS proteins and their accumulation in the permeabilized
cell nucleus was observed. As result was shown that the most effective domain for this
action localises around the preS 2 region. Multivalent and co-operative attachment of
permeabilized HepG2 with a variety of HBV surface proteins was found by using of preS
proteins conjugated to polystyrene beads. In this case one hexapeptide sequence within
preS1 was found as the receptor-binding viral domain epitope.
The aim of our study was to test interaction of mosaic virus-like capsids with several
preS proteins on the surface with alive human hepatocyte (HepG2) as well as with mouse
hepatoma (MG 22A) cells. We interested whether and how our VLP are disturbing cell
living-cycle, but main question for us was are they able to bind specifically to human
hepatocytes in comparison with neuroblastoma(SH-SY-5Y)cells. In other words
does act preS proteins as specific addresses or not? What sequence(s) is(are) responsible
for recognition and interaction with cell receptors?
To answer these questions we performed experiments with a set of phage Q- derived
VLP with different length of preS1 and full length preS2 exposed on the surface of particles.
Modified virus like capsids were tested in primary screening assay in solid-phase ELISA
with fixed alive cells. Interaction of VLP with cells was analysed also by fluorescent
microscopy when used selected fluorescently labelled particles. First results show that VLPs
with full-length preS1 on the surface interact strong and specific with hepatocytes opposite
to other type of cells.The next step will be the experiments with shortened variants of
preS1,preS2 or both.
Results are promising and first of all they characterise such VLP as successful tool for
molecular biology.
37
35-P3. Development of mosaic virus-like particles on base of RNA phage Q coat and HBV preS sequences
Shkutele Sanita, Indulis Cielens, Dace Skrastina and Regina Renhofa
BMC University of Latvia, Riga, Latvia
Virus like particles belong to modern non-viral vectors and have become of great
importance in scientific and in applied aspects.
Chimeric VLPs, with foreign (eucaryotic) proteins on the surface, may be used not
only as delivery tools with specific addresses, but also for the exposition of these epitopes.
Possibility to display foreign determinants on the surface of VLPs makes them applicable
for development of new diagnostics and vaccines, and, particularly, for the use as research
instruments in molecular biology, immunology, as models to study infection mechanisms in
“molecular medicine”.
RNA-bacteriophage Q- based mosaic VLPs were obtained in the end of 90s. This
was done harbouring foreign epitopes within the A1-extension. This method “copies” the
nature, because each bacteriophage contains few A1 molecules into capsid.
Now we have developed other methods to become chimeric VLP: N- and C- fusion
of the coat protein, amber and opal suppressions, two- gene systems and produced a set of
capsids with HBV subtype ayw preS sequences on the surface. Immunisation of Balb/C
mice with these chimeric particles showed high anti-Q- as well as anti-preS 1 specific
antibody response compared with peptide amino acids 21-47 alone.
Competitive ELISA proved that these preS sequences are on the surface of
particles.

Thus we have mosaic virus-like particles for further purposes.
38
36-PL3. Signaling mechanisms in yeast catabolite repression and
pexophagy
Stasyk A.A1, Olena G. Stasyk1, Taras Y. Nazarko1, Mykola M. Maidan2, Johan M.
Thevelein2, James M. Cregg3, Andrei A. Sibirny1,4
1Institute
of Cell Biology, NAS of Ukraine, Drahomanov Str. 14/16, Lviv 79005, Ukraine; 2Katholieke
Universiteit, Leuven, Belgium; 3Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA;
4Institute of Biotechnology, Rzeszow University, Poland.
In yeasts, glucose exerts transcription (catabolite) repression and induces autophagic
degradation of peroxisome (pexophagy). Mechanisms of these phenomena still remain unknown.
We identified a hexose transporter gene homologues in the methylotrophic yeast H.
polymorpha, HpGCR1 and HpGCR2, deficiency in which abrogates repression triggered by
hexose sugars. The closest HpGcr1p homologues, S. cerevisiae glucose transporter-like sensors
Snf3p and Rgt2p, are not involved in the glucose repression. HpGcr1p is not a functional glucose
transporter, is expressed at a low level, and itself is subject to glucose repression. Remarkably,
gcr1 mutants are also affected in induction/derepression of peroxisomal enzymes by methanol in
the absence of glucose. Pexophagy occurred much slower in gcr1 mutants but was not totally
alleviated. The effect of HpGCR2 deficiency on repression is significantly weaker than that of
HpGCR1. Similarly to HpGcr1p, HpGcr2p is also expressed at a low level, but its expression is
constitutive and independent of the carbon source.
We also analyzed the effect of Δmig1 and Δmig2 mutations on the repression mechanism
in H. polymorpha. The corresponding genes are homologues of the main S. cerevisiae
transcriptional factors involved in glucose repression, MIG1 and MIG2. We demonstrated that,
relative to Δgcr1, H. polymorpha Δmig1 and Δmig2 exert little, or no effect on glucose repression.
Our results suggest that the molecular mechanism of glucose repression, with regard to its
signaling and transcriptional factors, is distinct in different yeasts. In the methylotrophic yeast H.
polymorpha, two glucose transporter/sensor homologues participate in signal transduction for
repression, where two MIG homologues are not the main transcriptional repressors.
In other yeast species, Pichia pastoris and Yarrowia lipolytica, we have identified four novel
genes involved in control of yeast pexophagy: YlTRS85 (early secretory pathway protein, the
component of TRAPP I and TRAPP II complexes), PpATG26 (coding for enzyme sterol
glucosyltransferase), PpATG28 (species-specific protein) and PpPDG1 (involved in control of
both peroxisome biogenesis and degradation).
39
37-P2. Endoapoptosis: a novel form of cell death that helps continuity of
tumor cell lineage.
Sinha S1, R. Rajaraman1, 2, G. Faulkner1, M.M. Rajaraman3
Department of Medicine, Microbiology & Immunology, Faculty of Medicine, Dalhousie University, and
Department of Radiation Oncology, Nova Scotia Cancer Center, Halifax, NS., Canada. B3H 1X5.
Apoptosis results in cell death and the lineage of that given cell is brought to an end.
Tumor cells are often resistant to anti-cancer agents, although the mechanism is not fully
understood. We report that multinucleate polyploid cells (MN/PGs) found in preneoplastic
and neoplastic cell lines eliminate one or more defective daughter cells by endoapoptosis or
death of a daughter cell within the cytoplasm of the mother cell, thus maintaining the
continuity of cell lineage. Endoapoptosis is similar to apoptosis and is characterized by
DNA fragmentation, chromatin marginization and condensation, cytoplasmic shrinkage, cell
fragmentation into endoapoptotic bodies, residual DNA synthesis in the dying nuclei, and
the dismantling of the dying cell, often leaving an empty vacuole, all of which occur inside
living cell. Endoapoptosis may occur after mitotic division or neotic division. Endoapoptosis
results in abortive cell division by asymmetric intracellular compartmentalization of the living
and dying domains of a cell and the survival of the major fraction of cytoplasm that retains
the reproductively viable genome. This process of selective suicide of a non-viable genome
or a daughter cell can occur repeatedly until two viable genomes are formed. Post-mitotic
endoapoptosis can be experimentally induced after exposure to genotoxins; post-mitotic
endoapoptosis may help continuity of tumor cell lineage, while the post-neotic
endoapoptosis may or may not. In bi-nucleate or multinucleate cells formed by fusion of
irradiated and non-irradiated control cells, the irradiated cell genome dies via
endoapoptosis. Although this is very similar to the demise of the phagocytosed apoptotic
cell, electron microscopic evidence indicates otherwise and the endoapoptotic cell nucleus
is often very close to the viable nucleus. In post-mitotic endoapoptosis, the dying nucleus is
about the same size of the living nucleus, but in post-neotic endoapoptosis, the dying cell is
very tiny compared to the live genome, indicating that this is a dying Raju cell. If all the Raju
cells dye along with the polyploid genome of the neosis mother cell, then tumor cell lineage
is terminated. Even if one of the Raju cells survive, then the contiguity of tumor cell lineage
is maintained.
40
38-PL1. Identification of DNA sequences involved in the tight complexes with
DNA-protein complexes on different stages of the barley shoot development
Sjakste T1, M. S. Röder2, A. Levina3, N. Sjakste3
1Institute
of Biology, Miera str. 3, LV 2169, Salaspils, Latvia, 2IPK, Correnstrasse 3, Gatersleben D06466, Germany, 3Latvian University, Sharlotes 1a, Riga, LV1001, Latvia
Rearrangements in DNA packaging as in DNA interactions with functional protein groups
reflect the switches in gene expression during plant development. Mapped molecular markers
were successfully used to label DNA fragments in chromatin profiling experiments using DNA
complexes with different protein groups: nuclear matrix, DNA complexes with tightly bound
proteins (TBPs), and, nucleoprotein-celite chromatography (NPC-chromatography) fractions.
The presence of specific DNA fragments in various fractions obtained by the different
isolation procedures was monitored by PCR-amplification of the 22 and 24 markers
previously mapped along barley chromosomes 1H and 7H. All methods revealed the tissue
specific developmental reorganization of tight DNA protein complexes along the
chromosomes. Apparently, different methods detected interactions of different protein groups
with DNA. It was shown that in the young leaf large stretches of the chromosome become
completely released from the tight DNA-protein complexes as compared to dry grains.
Senescence of the leaf is followed by involvement of numerous chromosome sites in the tight
DNA-protein complexes. On the contrary, during senescence of the coleoptile some
fragments are released from the tight DNA-protein complexes. Taking into account eventual
significance of the genome areas involved in the nucleus rearrangements during plant
development for evaluation of the utility of genetic markers situated therein we managed to
characterize the DNA sequences involved in or released from complexes with tightly bound
proteins. The DNA was extracted from barley shoot leaves, coleoptiles and roots. The tight
DNA-protein complexes were isolated from restrictase-digested DNA by fractionation on
nitrocellulose or by exhaustive digestion by DNaseI followed by purification of the DNA
fragments protected by the TBPs. The obtained DNA fragments were cloned in plasmids,
sequenced and characterized. Analysis of the DNA sequences involved in complexes with
TBPs will be presented in the talk.
41
39-PL2. The role of endopolyploidy in tumour cell sensitivity to genotoxic
stress
Skladanowski Andrzej
Laboratory of Molecular and Cellular Pharmacology, Department of Pharmaceutical Technology and
Biochemistry, Gdansk University of Technology, Gdansk, Poland
Changes in chromosomes number and DNA ploidy are very common in many human
tumours, which suggest that tendency to polyploidization is an integral feature of carcinogenesis.
These genetic defects may further evolve as a result of treatment with antitumour drugs, in
particular alkylating compounds and DNA topoisomerase inhibitors.
In some situations, tumour cells treated with DNA damaging agents enter
endoreduplication cycle which involves DNA replication without corresponding cell divisions.
This leads to increased cellular DNA ploidy and the production of chromosomes with doubling
series of chromatids. Molecular mechanisms which regulate the process of endoreduplication
are still far from clear. Based on the available data, one can propose at least two types of
abnormalities frequently associated with endoreduplication: i) aberrant functioning of cell cycle
checkpoints (DNA damage checkpoint, spindle checkpoint and tetraploidy checkpoint); ii)
abnormalities in centrosome number and activity which may result in incomplete segregation of
chromosomes during anaphase B. Another event which is absolutely required for
endoreduplication is resetting replication origins for a subsequent DNA replication. Recent data
show that cyclin B1/Cdk1 inactivation can drive replication origin resetting in mitosis, even in the
absence of anaphase promoting complex APC/C function and mitotic exit. Endopolyploidy is
observed in non-stressed situations in plant cells and during differentiation of megakaryocytes.
Interestingly, there are striking similarities between DNA damage-induced endoreduplication and
natural polyploidization observed in plant cells and megakaryocytes. These include requirement
of inactivation of cyclin B1/Cdk1 kinase, increased expression of p21 protein, incomplete karyoand cytokinesis as well as requirement of type II DNA topoisomerases for efficient decatenation
of chromosomes during successive endocycles.
Traditionally, endoreduplication induced by antitumour compounds is considered as a
'dead end' for cell proliferation and is believed to culminate in cell death of polyploid cells. More
recent data suggest that a small fraction of polyploid cells may regain the proliferative potential
and re-enter cell cycle. The presence of polyploid cells after exposure to DNA damaging agents
is often associated with drug resistance. Therefore, an important question remains whether
these surviving tumour cells acquire additional genetic changes, which would increase their
genomic instability and result in more resistant/aggressive phenotypes.
42
40-P3. Methylation and fragmentation of nuclear DNA in plant cells
Skute Natalija
Daugavpils University, Daugavpils, Latvia
In this cell, methylcytosin (5mC) contributes up to 30% of the total cytosine residues.
Enzymatic methylation of cytosine residues in nuclear DNA is a very important phenomenon
in plant cells. The nuclear DNA methylation and fragmentation in proliferating and ageing
cells of the whole organ in the etiolated barley was studied.
There are maximally and symmetrically methylated duplex of nuclear DNA in early
replication in the division cells of the first leaf of etiolated barley shoots. It was shown that
these cells contain regular modulation of asymmetry of the complemtary strands with
respect to the 5mC residue. The early and late replicated portions of DNA differ in the
degree of methylation asymmetry of complementary strands.
During blocking of replication by hygroxyurea the percentage of postreplicative
methylation of nuclear DNA increases in the first cell cycle, and this process contributes, to
a new replication cycle in the following cell cycles.
5 azacytidine (5azaC) acts as an inhibitor of replication methylation in animal cells. It
was shown that 5azaC inhibits the replication of nuclear DNA in cell cycles of barley shoots
cells. 5azaC induces some gene expression in the cells of barley shoots. The role of nuclear
DNA methylation in apoptotic - like cells of barley shoots was shown. During the incubation
of barley shoots with 5azaC, the level of DNA methylation decreases in the cells of ageing
coleoptile. At the same, the degree of fragmentation of nuclear DNA and decondensation of
hromatine increases. This process is connected with oxygen radicals formation in plant
cells. Some cell processes (for example, mitochondrial pore formation) are involved in this
development and antioxidants influence this process in ageing plant cells.
The mechanisms of this phenomenon in plant cells are discussed in the present
presentation.
43
41-P3. Analyses of Cell Proliferation Kinetics, Angiogenesis and Expression
of Transmembrane Glycoprotein in Papillary Renal Adenomas and
Carcinomas
Sperga Maris, Regina Kleina
Center of Pathology, Latvia; Riga Stradins University, Department of Pathological Anatomy
Renal cell tumors histogenetically arise from different parts of nepfron and in its
cytoskeleton there are components of epithelial and mezenchymal orign with different
immunohistochemical reactivity. The aim of the study is to compare the proliferation markers
Ki- 67, PCNA with angiogenesis (CD31) and expression of CD-44 (transmembrane
glycoprotein) molecule in papillary renal cell adenomas (PRCA), papillary renal cell
carcinomas (PRCC) without and with metastasis and in secondary metastases of lymph
nodes.
We have examined three PRCA, three PRCC without and with metastasis and three
cases of metastasis. Immunohistochemical analyses were done according LSAB method
(DAKO).We have evaluated immunohistochemical staining results both inside the benign
and malignant tumor and around them. Proliferation activity was assessed by detecting the
Ki- 67 (Clone MIB-1) and PCNA (Clone PC10). Endothelial cell marker CD-31 was used to
measure angiogenesis of neoplasm. CD-44 (clone DF1485) positive tumor cells were semi
quantitatively scored according to four grades.
Labeling index of PRC adenoma with Ki-67 was 1,8% but in surrounding areas only
0,2% of nuclei in tubular epithelium were marked. PCNA showed 6% but renal parenchyma
shoved 2,1% of marked nucleus. The density of vessels with CD31 was 13 capillaries per
1mm2 but in surrounding areas there were 10 capillaries per square unit. Transmembrane
glycoprotein (TG) immunoexpression was weak (+) but its neighboring areas varied from
negative till (+). Accordingly WHO classification papillary cell carcinoma were evaluated in
stage G1(n=1) and G2(n=2). Ki-67 expression in PRCC of G2 was 18% and around it 0,3%.
PCNA shows 30% of marked cells and 2% of renal tubules beside neoplasm. CD- 31
labeled 40 vessels in tumor per 1mm2 but outside- 16 capillaries. TG was weak (+) in
cytoplasm and mild (++) in membranes of cancer cells. In G1 stage Ki-67 shoved 19%,
PCNA 26%,CD31 21 capillaries per 11mm2, TG was with mild expression (++). Tumors with
spreading into regional lymph nodes were of G2 (n=1) and G3 (n=2) stage.
Immunohistochemical characteristics in carcinomas of both grades were similar. Ki-67 45%,
44
PCNA 85%, CD31: 34 capillaries per 1mm2. TG were with mild expression (++) in
cytoplasm but strongly positive (+++) in membranes. In metastases immunoexpression was
such: Ki-67 30%, PCNA 89%, CD31: 60 capillaries per 1mm2, TG varied from mild
expression in cytoplasm till strong expression (+++) in membranes. It is still problematic to
differentiate papillary RC adenoma from carcinoma although last WHO classification has
postulated its size. There is a lot of controversial dates about CD31,CD 44 expression in
them. Our study proved that proliferate activity and expression of CD 31,CD 44 differs in
papillary adenomas and PRC cancers with spreading.
Conclusions:
1. Amount of CD31 positive capillaries increase progressively from benign to malignant
papillary renal cell tumors and reach their maximum in the metastasis of PRCC in stage G3.
2. Marked expression of transmembrane glycoprotein (CD 44 positive) correlates with the
progression,
dedifferentiation level of papillary renal cell cancer and its spreading to lymph nodes.
3. High proliferate activity, enlarged microcirculation area of neoplasm and intensive
transmembrane glycoprotein expression are factors of an unfavorable prognosis of papillary
renal cell carcinoma.
45
42-P1. Mapping of UBF isoforms by electron tomography: toward
supramolecular organization and behavior of GFP tagged UBF1 and UBF2 during
inhibition of rRNA synthesis
Tchelidze Paul
Laboratory of Developmental Biology, Faculty of Biology,
Tbilisi States University, Tbilisi, GEORGIA
Upstream binding factor (UBF) is a key co-regulator of the polymerase I activity
assumed to be responsible for bending and looping of rDNA at the gene promoter,
assembling of active initiation complex and triggering of rRNA synthesis. A dimmer of UBF
can bind rDNA not only in promoter sites but was found to be consistently associated across
entire intergenic spacers and gene segments structurally supporting the package of rDNPfibers at active NOR’s. The structural role of UBF isoforms (UBF1 and UBF2) in spatial
organization of rDNA within the nucleolar transcription sites (fibrillar centers) has not been
revealed yet. In this study we have designed a reliable detection technique based on
electron tomography and applied it to high-resolution 3D-mapping of GFP-tagged UBF1 and
UBF2 within fibrillar centers of A549 and KB human cancer cell lines. The technique
proposed is useful either for electron tomography or TEM, since nuclear ultrastructure retain
an adequate preservation, allowed precise detection of UBF isoforms on counterstained
ultrathin sections. Localization of UBF1 and UBF2 molecules was found to be strongly
restricted to fibrillar centers where they are organized in 10-25 nm fibrils folded in the looplike manner. We have further demonstrated that the UBF isoforms are distributed distinctly
within the fibrillar centers. The spatial folding of UBF1- and UBF2-positive fibers within the
fibrillar centers was not correlated with the distribution of Pol I molecules (…). Finally, we
have demonstrated that selective inhibition of polymerase I does not affect the loop-like
organization of UBF positive fibers. For the best of our knowledge, these findings present
the first direct experimental evidence of structural organization of UBF isoforms and their
rearangement at the conditions of blockage of rRNA synthesis.
46
43-PL1. Microconstruction of the Nucleolus: Emerging New 3D
Approaches
Tchelidze Paul
Laboratory of Developmental Biology, Faculty of Biology,
Tbilisi State University, Tbilisi, GEORGIA
The stepwise molecular events related to ribosome biogenesis are spatially highlyorganized within the structural framework of the nucleolus. The nucleolus is the most prominent
compartment of the nucleus, primarily known as the site
of ribosomal genes (r-genes)
transcription, rRNA processing and ribosome assembling. The cell nucleoli are formed around
few hundred copies of ribosomal genes (r-genes, rDNA), gathered in clusters of
tandem
repeats, that are distinguished in mitosis as discrete chromosomal segments named nucleolar
organizer regions (NORs). At the structural level, the nucleolus generates in interphase as rRNA
synthesis/processing products accumulated around decondensed rDNA following the vector of
ribosome formation processes. This giant but extremely compact microconstruction is
maintained by the scaffolding elements of the nucleolar matrix, sharing the whole nucleolar
volume among functional sub-territories (sub-compartments) designated to the transcriptional
and processing machineries. Indeed, nucleolar functions are morphologically embodied into
three basic substructures, distinguishable according to their appearance in electron microscopy.
They are classified as:1) pale staining fibrillar center/s (FC); 2) adjacent to FC dense fibrillar
component (DFC) and 3) located on the nucleolar periphery, relatively opaque granular
component (GC). To date it is well established that FCs represent interphase counterpart of
mitotic NORs, when transient area between FC and DFC is traced as transcriptional active rgenes territory. Arguably fibrillar network of FC is made up by rDNP-fibers of relaxed specificity
distinguishable from the nucleosomal chromatin. In fact, the nature of specific package of active
ribosomal chromatin (r-chromatn) fibrils is left largely unclear. Meanwhile DFC and GC
correspond to processing domains, occupied respectively by nascent pre-rRNP fibrils and
preribosomal particles. The overall nucleolar architecture is featuring as rather flexible as well.
Variations in rRNA production provoke major structural modifications of the nucleolus as
observed during the cell cycle, or in the presence of transcriptional inhibitors/activators. At the
ultrastructural level, drastic remodeling of the nucleolar subcomponents occur during activation
or inhibition of the transcription machinery. Nucleolar subcompartments are furnished (supplied)
by the specific sets of resident proteins (structural proteins, enzymes, transcription factors)
dedicated to coordination of ribosome production and maintenance of overall structural integrity
of the nucleolus. Recent proteonomic studies revealed that over 350 proteins appear to be at
47
work, acting for both the global organization and regulation of the nucleolar fuctions. Genes of
rRNA are transcribed by the transcriptional machinery of RNA polymerase I (Pol I), an enzymatic
complex solely dedicated to this task. The synthesis of pre-rRNA starts from the r-gene promoter
region, at the site of committed template, where several rDNA specific factors (SL1 and UBF in
human) cooperate Pol I to form an initiation complex. A important target for this tight regulation
is the transcription factor UBF (Upstream Binding Factor), whose ability to trans-activate rDNA
promoter and mediate protein-protein interactions is crucial for assembly of an active initiation
complex.In fact, visualization of the r-genes expression according to their activity offers a key to
the structure-molecular modeling of rRNA transcription and maturation machineries. Meanwhile
the complex and dynamic nature of the nucleolus makes 2D-electron microscopic studies mostly
insufficient to dismantle the functional orders in 3D-organization of r-genes. The only methods
enable to overcome the expecting “spatial” problems are certainly based on 3D/4D imaging
approaches. The possibility to analyze precise 3D information at a resolution comparable to that
of TEM arose with electron tomography. Recently a significant advance have been made toward
elucidation of three-dimensional structure of Pol I machinery. Using electron tomography of antiPol I labeled cells a volumic distribution of Pol I was mapped and spatial structure of transcribing
r-genes within FC was eventually modeled. While Pol I is displaying a strictly defined position
within transcription unit it appears as unopposed candidate to start 3D-mapping of proteins
involved in the initiation complex and rDNA compactization. On the other hand an important
relationship assumed between UBF and Pol I while another polymerase associated factor
(PAF53) may bind to UBF. This suggest that these two protein complexes could organize active
r-genes in cooperative fashion. Having sampled the 3D organization of Pol I molecules it’s much
easier now to overlap their map/pattern with localization of other transcription factors (UBF, SL1
etc) and spatial structure of r-genes. Here we have focused on spatial organization and
rearrangement of two spliced variants of nucleolar transcription factor UBF in the course of
selective inhibition of rRNA synthesis. Although many studies were dedicated to intranucleolar
localization of UBF this is the first report reviewed in details the precise 3D-organization of a
GFP fusion variants of UBF1 and UBF2, regarding to spatial package of ribosomal chromatin
fibers within fibrillar centres.
48
44-PL3. Image spectral cytometry for sperm chromatin testing:
new approaches
Tsarev I1, Ju Erenpreiss2, T. Freivalds3, Y. Dekhtyar4, Je. Erenpreisa1
1LU
3LU
Biomedicine Centre, Riga, Latvia; 2 Lund University, Fetility Centre, Malmo, Sweden;
Clinical and Experimental Medicine Institute, Riga, Latvia; 4RTU, Riga, Latvia.
Importance of sperm DNA integrity for predicting of fertilization outcome, both in vivo and in
vitro has been proved in many studies, and assays for its determination are recommended for the
infertility workup. However, sperm chromatin maturity and stability may also be an important
parameter of sperm quality, probably no less than DNA integrity, but no easily applicable test for
estimation of sperm chromatin packaging exists so far.
Recently, we have elaborated the cationic dye Toluidine Blue (TB) test (at pH 3.5) as an
alternative to existing methods for sperm DNA integrity evaluation. In this study, we developed two
new methodical approaches for evaluation of sperm chromatin stability and structure using
modification of this TB test.
The first approach, based on pH shift, has been tried on the group of 58 patients, comparing
staining results at pH 4.5 with that at pH 3.5. In this way, ionic binding between DNA and sperm
chromatin proteins can be evaluated. We have found that all sperm samples can be divided into two
groups - those with stable and unstable chromatin. In the first case, no increase in the number of TB
darkly stained sperm cells - is caused by elevation of TB pH, while this was the case for the second
group. Sperm chromatin stability was weakly associated with background DNA integrity status. We
also found no significant correlation between sperm chromatin stability and standard sperm
parameters with the only exception for motility in samples with low level of DNA damage. Therefore
sperm chromatin stability, detected by TB pH shift assay, seems to possess its own value and
should be further estimated for its prognostic power in fertilization outcome - both in vivo and in vitro.
Second approach is based on the data obtained earlier by Erenpreisa et al. (1992) on
somatic cells stained by TB, which showed that impairment of DNA integrity (in early apoptosis)
causes increase of TB absorption in the blue-green (metachromatic) part of spectrum, while
additional disorder of the chromatin packaging (in late apoptosis), induces additional increase of
absorption in the red (batochromic) part of spectrum. Therefore we used the ratio of red/green optical
density of TB-test (pH 3.5) stained sperm cell nuclei in order to discriminate of the cells with
disordered chromatin structure. 120 sperm samples from infertile and fertile patients have been
analysed in this way. The obtained data expressed as the 3 grade polynom and its mathematical
analysis allow to discriminate the proportion of these cells. This particular group should be further
studied as an object of clinical interest.
49
45-P3. Toluidine Blue pH-shift assay for sperm chromatin structure: first
results in clinical studies
Tsarev I.1, Erenpreiss Ju. 2,3, Hlevicka S1. and Erenpreisa Je. 1
1LU
Biomedicine Centre, Riga, Latvia, 2Scanian Fetility Centre. Malmo, Sweden, 3 Riga Stradins
University
Recently, we began to elaborate Toluidine Blue (TB) pH-shift assay for sperm
chromatin stability assessment. In this study, we have evaluated our method on the group of
82 patients, including 20 donors, 38 patients from barren couples undergoing clinical
examination, and 24 patients with diagnosed infertility. In addition, patients undergoing
clinical investigations were divided in two groups: with normal standard semen parameters
(according to WHO) and with at least one parameter being abnormal (23 and 15 samples,
respectively). The data were compared for pH 3.5 and 4.5. Dark, intermediate, and light
sperm cell nuclei were discriminated by optical density using special algorithm in the green
channel of CCD-camera.
The proportion of TB-darkly-stained cells at pH 3.5 wast the highest in groups of
infertile patients and patients with abnormal standard semen parameters (43,5±19.5% and
47±17.9%, respectively). Proportion of darkly-stained cells in the patient group with normal
sperm parameters was 26%, that is significantly lower than that in infertile patient group
(p<0.001), while significantly higher than in a donor group - 19% (p=0.02). Provoking
chromatin stability by TB pH shift to 4.5, the proportion of darkly-stained cells increased
most dramatically in the samples of infertile patient group, (from 43.5% to 73%), while it
remained the same or increased slightly in all other groups. Also, the proportion of light cells
decreased most significantly in infertile patient group (from 31% to 7,4%) and the patient
group with abnormal standard semen parameters (from 50,5% to 19,8%), while in other
groups remained over 25%. Thus, TB-pH shift assay clearly demonstrates significant
differences in chromatin structure and stability between different patient groups. In
particular, patients with confirmed infertility demonstrate high instability in their sperm
chromatin conformation after provocation by pH shift, which is otherwise "hidden" in the
usual TB (3.5) test.
In conclusion, Toluidine blue pH-shift assay for sperm chromatin stability is sensitive
and easy usable tool for sperm chromatin structure assessment. It has its own value and
seems to be potentially usable for fertility evaluation and prediction of fertilisation outcome .
50
46-PL1. Aspects of nuclear and chromatin organization as seen with
fluorescence correlation spectroscopy and related methods
Wachsmuth Malte1,2, Katalin Fejes Tóth2, Thibaud Jegou2, Tobias A. Knoch2, Sabine
M. Görisch3, Karsten Rippe2
1Institut
Pasteur Korea, Seoul 136-791, Korea
Institute of Physics, University of Heidelberg, D-69120 Heidelberg, Germany
3Max Delbrück Center for Molecular Medicine, D-13125 Berlin, Germany
2Kirchhoff
The interaction of chromatin with proteins and supramolecular complexes involved in
gene expression, replication, or repair processes is controlled by its dynamic organization,
as it defines the DNA accessibility. As an example, in order to establish a transcriptionally
competent and decondensed chromatin conformation, the acetylation of histones is a
central factor.
Tracking of PML and Cajal bodies as well as artificial Mx1 bodies revealed a
mechanism of nuclear body movement consisting of two components: diffusion of the body
within a chromatin corral and its translocation resulting from chromatin diffusion in the order
of micrometers. The Brownian movements of chromatin segments themselves on smaller
scales were investigated with high spatial and temporal resolution with FCS in H1-EGFPexpressing mammalian cells. The application of polymer-based models allows to
characterize structural and dynamic aspects of chromatin organization with spatial
differentiation. Trichostatin A (TSA) is a deacetylase inhibitor and the treatment of cells with
TSA led to a clearly visible decondensation of chromatin in the whole nucleus, either seen in
cells expressing histone-EGFP fusion proteins or after DAPI staining. Chromatin features
subchromosomal structures with typical sizes of 1.2 μm as determined by ICS which double
their size upon TSA treatment.
51
47-P2. Polyploidy with aberrant nuclear division (amitosis) produces
genetically changed cell growth
Walen Kirsten
California Department of Health Services, VRDL 850 Marina Bay Parkway, Richmond, CA 94804
A cellular process that produces genetically changed cells in normal, nearsenescent, human diploid cell cultures has been described (2,3,4,5). The following
sequence of cell phenomena occurred in virus infected cells and spontaneously: a)
endoreduplication of DNA to cells with large, polyploid nuclei; b) amitosis/fragmentations of
the polyploid nuclei to diploid-sized nuclei that gave rise to multinucleated cells (MNCs);c)
induced and spontaneous budding of the diploid-sized nuclei from the MNCs resulting in
karyoplasts that were surrounded by a rim of cytoplasm and cell membrane; d) new cell
growths with changed morphology and extended lifespans (EL cells) from karyoplasts
capable of normal mitotic divisions, and e) the cells in the new colonies were karyotypically
normal diploid (1,5).
The extraordinary event in the cell transformation process outlined above is the
derivation of diploid cells (2N) from polyploid cells that ranged from 4N to 16N. This type of
break-down of polyploid cells (over 100N) to non-random whole genome segregations (2N,
4N and 8N) occurs as a constitutive process in mammalian placentas (6). From known
somatic genome reduction division (akin to meiosis) of the polyploid macronucleus in ciliate
protozoans, it was suggested that the process of whole genome segregations had been
preserved through evolution (6,7). For these organisms various forms of internal spindles
have been described. Similar somatic cell reductional divisions are known from primary cell
cultures of hamster, mice and cell hybrids (8, 9,10). However, these types of precise
genome segregations were achieved by multipolar spindles. For human cells such spindle
apparatus occurs in immortal cell lines which are products from EL cells having gone
through a second transformation process. Multi-polar mitosis does not occur in normal,
human diploid cell growth, because of more stringent control mechanisms in such cells as
compared to in hamster and mice cell growths (11).
Another event in the cell transformation process is also a constitutive phenomenon,
nuclear budding in form of karyoplasts. When human red cells mature the nucleus is
extruded. In this process as described above the nucleus becomes surrounded by
52
cytoplasm and cell membrane. The fact that two crucial steps (genome reductional division
by amitosis and nuclear budding) in the process of cell transformation occur naturally in
human cells suggests that in the corresponding environment (stress; X-ray, chemicals,
virus, nutritional deficiency, etc.) this process that lead to new cell growth can be induced.
Conclusion: Polyploidy is the initial step in a multi-step process that gives rise to
genetically changed human diploid cell growth. The genetic variation (mutations) necessary
for the first genetic change from normal to EL cells has at least the following likely sources;
DNA endoreduplication errors, chromosomal unequal exchanges, uni-parental chromosomal
segregations and methylation changes.
References;
1)
Walen, KH (1982), In Vitro 18:203-212.
2)
Walen, KH (2000), Cytogenet Cell Genet 90:167 (abstract).
3)
Walen, KH (2002), Cancer Genet Cytogenet 133:45-54.
4)
Walen, KH (2004), In Vitro Cell Dev Biol 40:150-158.
5)
Walen, KH (2005) Submitted; In Vitro Cell Dev Biol
6)
Zybina, EV, Zybina, TG (1996), Internat Review Cytol 165:53-119.
7)
Blackburn, EH, Karrer, KM (1986), Annu Rev Genet 20:501-521.
8)
Schmid, W (1966), Exp Cell Res 42:201-204.
9)
Teplitz, RL, Gustafson, PE, Pellet, OL (1968), Exp Cell Res 52:379-391.
10) Pera, F, Rainer, B (1973), Chromosoma 42:71-86.
11) Holliday, R (1996), Cancer Surveys 28:103-114.
53
48-PL2. Cancer cells, death and ploidy in arginine deficiency
Wheatley Denys N.
BioMedES, Leggat House, Keithhall, Inverurie, Aberdeen AB51 0LX, UK.
Cancer can be treated with enzymes that degrade arginine. However, the effects of
arginine deprivation on normal as well as tumour cells have not been very thoroughly
researched. The main difference is that many tumour cells cannot move out of cycle in the
way that normal cells can, although the reason(s) for this is (are) not clear. Removal of any
(semi)essential amino acid should have similar effects, but this is not necessarily the case.
Removing most of them also has a different effect from removing just one, such as arginine.
Some of the ways that damage can result in such cells has been through chromosome
breakages, but this is old work and we have no modern studies that indicate the
mechanism(s) involved.
There is also a way in which the death usually caused in tumour cells - such as the
mouse leukemic line, L1210 - by arginine deprivation can be avoided. This is probably due
to the presence of giant cells, present in cells growing in normal cultures as well as in
treated tumour cell populations made arginine deficient. These should be selected out
because they can survive deprivation and are not usually cycling in the normal way, as
small cells die off. We should then be able to watch them regenerate as cultures in normal
medium, that is, if these cells can go through “reduction division” to create new small cells,
with normal cycle properties.
54
49-PL2. Quantitative Genome Modification in Development
Zacharias Helmut
24631 Langwedel, Germany.
Theodor Boveri (1892) has shown differentiation to occur in embryonic cells. He explored a
horse parasitic nematode that Edouard van Beneden had introduced. Ascaris megalocephala
univalens has been convenient for early cytogeneticists, because it has only n=1 chromosome. The
second cleavage eliminates some 85 % of germ line specific DNA from the primordial soma cell.
Chromatin diminution happens also in Crustacea. Germ line specific sequences become expelled
from primordial somatic nuclei in early ontogeny of some freshwater copepods. Sigrid Beermann
recorded such DNA differences from Cyclops divulsus (42 %), C. furcifer (52 %) and C. strenuus (59
%).
DNA endoreplication is another instance of genome modification in somatic cells, most
conspicuously demonstrated by giant polytene chromosomes. The chironomid midge Acricotopus
lucidus (subfamily Orthocladiinae) shows 3 brilliant polytene elements that originate from a mitotic
2n=6 complement. Germ line nuclei possess additional chromosomes that are completely eliminated
from somatic nuclei by an asymmetric anaphase. However, one of the two sets of germ line restricted
elements is also eliminated from developing gonads in the 1st larval instar. This chromosome paucity
in the germ line becomes rectified by the differential mitosis that precedes meiosis.
Selective DNA endoreplication is an epi-phenomenon of polytenization, well investigated in
some Drosophila species. Amplification means the multiplication of specific sequences, e.g. the
chorion gene cluster in the follicle cells of D. melanogaster. In contrast, DNA underreplication of
larger genomic domains is a convergent mechanism to elimination, since it suspends repetitive
sequences from nuclear enlargement. Emil Heitz (1934) was first to describe such a situation with D.
virilis: While euchromatic sections of the chromosomes increase to gigantic size during the growth of
the nuclei, the heterochromatic sections are not able to do this. In the Samoan D. nasutoides (2n=8),
suppressible DNA is mainly comprised in the mitotic isochromosome 4, which represents > 60 % of
the genome. Measurements of single nuclei revealed that underreplication is induced during
endocycle 1 or 2.
A summary on genome modification can be seen in the hypotrichous ciliate Stylo-nychia
lemnae. Its macronucleus anlage performs polytenization between two elimination events.
Suggested mechanisms are excision and repair for diminution of intercalary chromatin, differential
kinetochore protection for elimination of whole chromosomes, and specific origin access for selective
endoreplication.
55
50-PL1. SPATIAL AND TEMPORAL ORDER OF THE NUCLEOLUS
ASSEMBLY IN EARLY MOUSE EMBRYOS
Zatsepina Olga V.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow,
Russia
In early (preimplantation) mammalian embryos, building-up of the functional nucleolus is a
step-wise process, which includes assembly of the nucleolar precursor bodies (NPBs), formation of
the RNA polymerase I transcription complex, and activation of transcription of the rRNA genes. A key
role in this process is apparently played by the NPBs - numerous discrete entities, which anchor the
active rRNA genes. However, the molecular mechanisms regulating resumption of rDNA transcription
and the nucleolus assembly still remain far to be understood. To shed more light on the problem, in
our work we address the following questions. (1) What is the general arrangement of the rRNA genes
in transcriptionally inert (one-cell and early two-cell) versus active (late two-cell) mouse embryos. To
answer this question, we examined the distribution of rDNA by fluorescence in situ hybridization with
probes complementary to rDNA repeat units. (2) What is the dynamics of reactivation of the genes
that encode the major nucleolar proteins, such as RNA polymerase I and UBF (both protein are
involved in regulation of the rRNA gene transcription), fibrillarin (is required for processing of the
primarily RNA transcripts), and B23/nucleophosmin (a pre-ribosome assembly factor). To reach the
goal, real-time PCR analysis of the protein gene expression and the protein expression by Western
blots with specific antibodies were performed.
The results obtained show that in one-cell and early-two cell mouse embryos, where no
rDNA transcription occurs, rDNA forms small in number and compact clusters, which are located in a
close proximity to the NPBs. However, almost in all pronuclei examined, a portion of the NPBs was
not associated with the rRNA genes. Resumption of rDNA transcription in late two-cell embryos was
concomitant with a profound relaxation of the rDNA clusters, which still remain associated with the
NPBs. Nevertheless, like in the transcriptionally silent embryos, from one to several NPBs in a
nucleolus do not structurally interact with rDNA. These observations, for the first time, show that the
population of the NPBs is heterogenous in its ability to associate with the rRNA genes and thus,
apparently, to support the assembly of the nucleolus. The real-time PCR analysis of mRNA
transcripts for the major nucleolar proteins demonstrates that the certain number of transcripts
remains stable and is transferred to zygote from MII oocytes. Transcription of the RPA116, UBF, B23
and fibrillarin genes as judged by their transcript number per cell is very likely initiated in one-cell
embryos, and then becomes gradually down regulated. The minimal number of rRNA transcripts per
cell is characteristic for the blastocyst stage. Taken together, these data argue in favor of the idea
that expression of the major nucleolar protein genes precedes the resumption of rRNA synthesis.
56
51-PL2. Cell reproduction and genome multiplication in the proliferative and
invasive trophoblast cell populations of mammalian placenta.
Zybina TG, Zybina EV
Instiutute of Cytology RAS, St.-Petersburg, Russia
Recently a great attention has been drawn to regulation of trophoblast invasion into
uterine wall. Mammalian placenta includes a range of trophoblast cell populations that differ
in their peculiarities of cell reproduction and genome multiplication. Spatio-temporal "timetable"
of
ways
of
cell
reproduction
(mitosis,
restitutional
mitosis,
endomitosis,
endoreduplication) of trophoblast cell populations is described here. As a rule, proliferative
populations of mitotically active trophoblast cells (diploid and low-polyploid) are located out
of contact with maternal tissues. In rodent placenta they mainly switch from mitotic cycle to
polyploidizing (restitutional) mitoses and reach 4c-8c. Thereafter they switch to
endoreduplication and reach 16c-64c. Following a series of endoreduplication cycles a part
of this cell population sets apart and penetrates deeply into decidualized endometrium and
myometrium, their capability of replication losing progressively. By contrast, the highly
polyploid (in rodent - 256c-2048c) invasive trophoblast cells stay at the border with
semiallogenic maternal tissues. Interestingly, the deepest invasion into the uterine wall
requires an optimum but not the highest ploidy level and complete termination of replicative
processes. It cannot be ruled out that polyploidy may compensate DNA damage that may
result from allogenic cell interaction. Besides, switch to endoreduplication leads to
irreversible differentiation that makes impossible renewal of mitotic division (Nagl, 1978,
1995, Brodsky, Uryvaeva, 1985). Meantime, relatively low level of ploidy does not prevent
their migration through the uterine wall tissues. In this case complete repression of mitoses
and DNA replication after a series of endoreduplication cycles makes hardly probable
renewal of mitotic activity in the deeply invading trophoblast cells thereby preventing the
possibility of their ectopic expanding in the maternal tissues during the normal pregnancy.
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52-P2. Polyploidizaion and depolyploidization of trophoblast cells of
Microtus rosiaemeridionalis studied with the use
of gonosomal chromatin arrangement.
Zybina TG, Zybina EV, Bogdanova MS, Stein GI
1Institute
of Cytology RAS, St.-Petersburg, Russia
X and Y chromosomes of the field vole Microtus rosiaemeridionalis contain prominent
blocks of heterochomatin that form large condensed chromatin bodies in interphase nuclei.
These gonosomal chromatin bodies (GCBs) may be used as a natural chromosome marker
in interphase nuclei for the study of the ways genome multiplication of the invasive
trophoblast cells. The aims of the present work was to clarify the regularities of GCB
multiplication and arrangement a) in the course of genome multiplication of giant trophoblast
cells of the field vole and b) in the course of fragmentation of giant nuclei that results in
formation of multinucleate trophoblast cells at the end of their differentiation.
Cytophotometrical measurement of DNA content in the nuclei, nuclear fragments and
simultaneously in the GCBs was made in the Feulgen-stained secondary giant trophoblast
cells (SGTCs) of field vole M. rosiaemeridionalis at the 10th and 14th day post coitum using
image analyze system Videotest (St.-Petersburg, Russia).
In the highly polyploid (16c-256c) SGTCs DNA content in GCBs rose proportionally to
the ploidy of nuclei. Meantime, the number of GCBs did not exceed 2 at the different ploidy
levels that reflects polytene nature of giant trophoblast cells. At the late stage of
differentiation the giant trophoblast cells undergo fragmentation that results in polykaryocyte
formation. In the nuclear fragments GCB DNA content decreased mostly proportionally to
DNA content in the whole fragments. The latter corresponded to 2 c, 4 c and 8 c. In most
cases 1-2 GCBs got in the nuclear fragments of different ploidy levels. A high correlation
was found between the total DNA content in GCBs as well as between DNA content in the
separate GCBs and the ploidy levels of fragments. The data obtained demonstrate a
regular, whole-genome chromosome distribution into nuclear fragments. A possible
mechanism of nuclear fragmentation that ensures most probably balanced genome of
nuclear fragments but do not lead to renewal of mitotic cycles in the giant trophoblast cell
population is discussed.
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