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Інститут молекулярної біології і генетики НАН України
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irk-123456789-1537032019-06-15T01:26:14Z Abstracts GDRI meeting "From Molecular to Cellular Events in Human Pathologies" 2013 Article Abstracts / Вiopolymers and Cell. — 2013. — Т. 29, №. 5, доп. — С. 7-40. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/153703 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Abstracts / Вiopolymers and Cell. — 2013. — Т. 29, №. 5, доп. — С. 7-40. — англ. |
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Вiopolymers and Cell |
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7
CHI3L1 and CHI3L2 – one protein family, two opposite functions
Areshkov P. O., Avdieiev S. S.
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnogo Str., Kyiv, Ukraine 03680
chitotrio@gmail.com
Mammalian genomes code a set of homologous chitinase-like proteins. In human
there are six proteins of this family, with CHI3L1 and CHI3L2, the most
investigated among them. Overexpression of both genes was found in gliomas
[Shostak et al., 2003, Kavsan et. al., 2008]. It was reported that CHI3L1, similarly
to IGF1, promoted the growth of human fibroblasts and synoviocytes through
MAPK/ERK1/2 and PI3K/AKT pathways activation [Recklies et al., 2002]. In our
previous experiments we found that CHI3L2 activated those signaling pathways
but had opposite to CHI3L1 effect on 293 and U373 cell growth. Suppression of
cell proliferation by CHI3L2 treatment was caused by difference in ERK1/2
activation kinetics and their localization [Areshkov et al., 2012]. Moreover, using
specific inhibitors of different steps of clathrin-mediated endocytosis we showed
differences in CHI3Ls-associated MAPK and PI3K signaling initiation. CHI3L1
induces ERK1/2 and AKT phosphorylation through clathrin-coated vesicle
formation, whereas stimulation of MAPK and PI3K signaling by CHI3L2 could
be initiated by activated receptor clusterization and cell membrane invagination
only.
Recently, Miyatake et al. (2013) reported that lentiviral transduction of CHI3L2
did not decrease proliferation of mouse ATDC5 cells but significantly enhanced
cell dividing. Authors explained their results by difference in cell model (mouse
vs human cells) and manner of treatment (addition to culture medium vs
constitutive protein production). To test this hypothesis we obtained mammalian
cells that ectopically produced CHI3L1 or CHI3L2 proteins. Applying system of
lentiviral gene transduction, we confirmed our results and showed opposite
influence of CHI3L1 or CHI3L1 expression on cell growth. 293 cells stably
producing CHI3L2 possess decreased proliferation rate in comparison with cells
of wild type or 293 cells infected by “empty” vectors and, as expected, CHI3L1
production in 293 cells resulted in enhanced cell growth. Such function duality of
cancer-associated genes has been already described [Stepanenko et al., GENE,
2013, in press] and our results confirmed importance of cell type and activation
conditions for biological outcome.
8
A search of new treatment modalities for mantle cell lymphoma and
glioblastoma
Avdieiev S.1, Gera L.2, Havrylyuk D.3, Hodges R.2, Lesyk R.3, Ribrag V.4,
Vassetzky Y.4
1Dpt Biosynthesis of Nucleic Acids, Institute Molecular Biology and Genetics, National
Academy of Science of Ukraine, 150 Zabolotnogostr, Kyiv 03680, Ukraine
2Dpt Biochemistry and Molecular Genetics, University of Colorado, Denver, Anschutz Medical
Campus, Aurora CO 80045, USA
3Dpt Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National
Medical University, 69 Pekarska str, Lviv 79010, Ukraine
4CNRS UMR8126, University Paris-Sud 11, Institute Gustave-Roussy, Villejuif 94805,
France.
stasavdieiev@gmail.com
Aim. This study was initiated to analyze the effect of compounds with distinct
chemical nature, namely bradykinin (BK) antagonists and azolidinones, on
proliferation of different types of malignantly transformed cells: 293 cells, stably
transfected by CHI3L1 oncogene (293_CHI3L1), human glioblastoma cells U373,
rat glioma cells C6, mantle cell lymphoma (MCL) cells Mino. Methods. MTT-
based cell proliferation assay, western blot analysis. Results. Based on
cytotoxicity test data, it was found that BK antagonists BKM-570 and BKM-1800
revealed significant growth suppressor activity on 293_CHI3L1 and U373 cells.
LC50 of BKM-570 was 3,8 μM and 3,3 μM for 293_CHI3L1 and U373 cells,
while LC50 of BKM1800 was 25,8 μM and 20 μM, correspondingly. Prominent
growth inhibition was demonstrated also by BKM-570 on C6 cells with IC50 4
μM. Several azolidinones were involved in our investigation. 5-arylidene-2-
amino-4-azolone Les-28 lead to the substantial cell growth inhibition: IC50 of
Les-28 was 0,16 µM for 293_CHI3L1 cells and 15 µM for U373 cells. Pyrazoline
substituted thiazolone Les-4523 revealed high activity on C6 cells with IC50 0,13
µM. 50 % inhibition of Mino cells growth were observed after treatment by Les-
4523 at 1,58 µM. Conclusions. Thus, antiproliferative properties of two different
classes of molecules were shown in several distinct in vitro models of glioma and
MCL.
This work was supported in part by State Agency for Science, Innovations and
Informatization of Ukraine in frames of the project F33.4/001 "Identification of
promising molecular biomarkers for monitoring of human neurodegenerative and
oncological diseases".
9
Yeast and human SAGA acetyltransferase and deubiquitinase
activities shape all the transcribed regions of the genome
1Devys Didier, 1Bonnet Jacques, 1,2 WangChen-Yi, 2Kao Chen-Fu and
1Tora Laszlo
1IGBMC, CNRS UMR 7104, 1 rue Laurent Fries, 67404 Illkirch, France
2Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
devys@igbmc.fr
According to the text book model, co-activators with chromatin modifying
activities such as the SAGA complex are recruited at a number of well-defined
loci through interactions with DNA bound activators. At these sites, the induced
chromatin modifications are suggested to favor the recruitment of general
transcription factors and RNA polymerase II. Therefore, it is expected that
changes in gene expression upon depletion of these complexes should largely
overlap their genome wide distribution. This model has been challenged by recent
studies in Saccharomyces cerevisiae that revealed little overlap between SAGA
locations, as assessed by chromatin immunoprecipitation (ChIP) and by gene
expression defects measured by mRNA changes following downregulation of
individual SAGA subunits. Thus, we asked whether ChIP of SAGA specific
subunits would provide an accurate map of SAGA action. To answer this
question, we assessed the genome wide distribution of the SAGA
acetyltransferase and deubiquitinase activities in either yeast or human cells.
Surprisingly, upon inactivation of the SAGA acetyltransferase activity, the levels
of H3K9 acetylation at the promoters of almost all expressed genes were
significantly reduced. Similarly, upon inhibition of the SAGA deubiquitinase
activity, revealed increased histone H2Bub1 levels in the transcribed region of all
expressed genes in both organisms. These results together demonstrate that
contrary to the available genome wide binding data, both yeast and human SAGA
is recruited to all expressed genes on which they acts both at the promoter and the
transcribed regions. Therefore, besides SAGA coactivator function characterized
by a stable ChIP-able binding to its genomic sites, the two enzymatic activities of
SAGA have a global and genome wide action suggesting an important role in
shaping the transcribed regions of the genome.
10
The interaction of lipophilic derivatives of siRNA with hematopoietic
and tumor cells
Chernolovskaya E. L., Chernikov I. V., Meschaninova M.I., Dovydenko I. S.,
Venyaminova A. G., Zenkova M. A., Vlassov V.V.
Institute of Chemical Biology and Fundamental Medicine SB RAS,
Novosibirsk, Russian Federation,
elena_ch@niboch.nsc.ru
siRNAs are considered to be promising therapeutic agents for sequence-specific
silencing of disease-related genes. However, the problem of siRNAs delivery into
cells limits their biomedical application. Conjugation of siRNA to the molecules,
which can be internalized into the cell by natural transport mechanisms, can result in
the enhancement of siRNA cellular uptake. In this work we investigated the carrier-
free accumulation of nuclease-resistant siRNA equipped with lipophilic residues
tethered on the 5'-end of the sense strand in KB-8-5 cancer cells and normal
hematopoietic cells. We showed that conjugates of siRNA (0.2–5 µM) with
cholesterol effectively penetrated (up to 100 %) into the cells, whereas the uptake of
the unmodified siRNA was insignificant. The efficiency of the carrier-free cellular
accumulation of lipophilic siRNAs is dependent upon the type of lipophilic residues,
the type of the target cells and the length of the linker connecting the lipophilic
residue and siRNA strand. We found that efficacy of cellular uptake enhanced when
the length of linker between siRNA and lipophilic residue increased from 3 to 12
carbon atoms. As expected, the accumulation of lipophilic siRNA in preferential
blood mononuclear cells in serum free medium was higher than the accumulation in
the cells in whole blood. The biological activity of cholesterol-conjugated siRNAs
targeted to MDR1 gene was tested in KB-8-5 drug resistance cells. Incubation of the
cells in the presence of the conjugates resulted in the silencing of the target gene and
the restoration of the sensitivity of the cells to cytostatics. The pronounced reduction
of the number of living cells was observed at day 6 of cells incubation with
cholesterol-containing siMDR and 300 nM vinblastine. Thus, our conjugates are able
to penetrate into cells of different types without transfection reagents, to silence the
expression of the target gene and to reverse the multiple drug resistance of cancer
cells making their susceptible to chemotherapy.
This work was supported by RAS programs “Molecular and cellular biology” and
“Fundamental sciences to medicine”, by RFBR grant 11-04-01012, Ministry of
Science and Education of the Russian Federation ( No.14.740.11.1058) and by
Interdisciplinary grant of SB RAS (No.85).
11
DNA import competence and mitochondrial genetics
F. Weber-Lotfi1, D. V. Mileshina1,2, N. Ibrahim1,3, M. V. Koulintchenko1,2,3, G. G.
M. D'Souza4, V. Saxena4, Yu. M. Konstantinov2, R. N. Lightowlers3, A. Dietrich1
1 CNRS Institute of Plant Molecular Biology, Univ. Strasbourg, Strasbourg, France, 67084
2 Siberian Institute Plant Physiology and Biochemistry, SB-RAS, Irkutsk, Russia
3 Institute Cell and Molecular Biosciences, Medical School, Newcastle University, UK4 School
of Pharmacy, MCPHS University, 179 Longwood Ave, Boston, USA, MA 02115
andre.dietrich@ibmp-cnrs.unistra.fr
Mitochondrial genome expression is essential for organelle functional efficiency and
inter-compartment cross-talk. Manipulation of mitochondrial genetics is thus of
interest for a range of fundamental investigations and is appealing to treat
neurodegenerative diseases caused by organelle DNA mutations. We have previously
established that isolated plant and mammalian mitochondria can functionally import
double-stranded DNA through an active mechanism. We aim to understand the
mechanism(s) underlying mitochondrial competence for DNA uptake and to develop
cell uptake followed by mitochondrial targeting of functional gene constructs.
Methods: We developed DNA uptake experiments with mitochondria isolated from
potato (Solanum tuberosum) or from Saccharomyces cerevisiae mutants defective for
various nucleus-encoded mitochondrial proteins and carriers. qPCR and RT-qPCR
analyses assessed the level of cell-internalized construct and putative transcription.
Results: We used both biochemical approaches and S. cerevisiae genetic tools to
identify the still elusive inner membrane proteins participating in mitochondrial DNA
import. Strikingly, among the candidates from the inner membrane carrier family
selected on the basis of biochemical data with plant organelles, only the two minor
forms of the adenine nucleotide translocator turned out to be required for optimal
DNA translocation into isolated yeast mitochondria. Conversely, we highlighted a
putative contribution of proteins that control mitochondrial morphology in S.
cerevisiae. Building on the hypothesis that the competence for DNA uptake is also a
property of the organelles in vivo, we attempted to use nanocarriers to target DNA to
mitochondria in intact cells. We explored the use of a mitochondriotropic liposomal
formulation to deliver a DNA construct encoding a recoded green fluorescent protein
(gfp) gene controled by a rat mitochondrial promoter into the mitochondria in live rat
cells. In comparison to free DNA and vehicle controls, incubation of the cells with
liposome/DNA complexes led to significant incorporation of the construct and
generation of gfp mRNA. Conclusions: Taken together, our data imply that there are
significant variations in the mitochondrial DNA import mechanism between different
organisms and that even in a given organism multiple pathways might operate. Our
first in vivo results suggest that mitochondriotropic liposomes can deliver DNA into
mitochondria of live mammalian cells, potentially opening novel prospects for
mitochondrial transfection.
The work was financially supported by the CNRS, the University of Strasbourg and a
grant from the French Ministry for Research (Investissements d'Avenir / Laboratoire
d'Excellence MitoCross).
12
Defective regulation of miRNAs target genes in myoblasts from
facioscapulohumeral dystrophy patients
Petr Dmitriev1,2, Luiza Stankevicins1, Eugenie Ansseau6, Andrei Petrov5,
Ana Barat1, Philippe Dessen3, Thomas Robert3, Ahmed Turki2, Vladimir Lazar3,
Emmanuel Labourer4, Alexandra Belayew6, Gilles Carnac2,
Dalila Laoudj-Chenivesse2, Marc Lipinski1 and Yegor S. Vassetzky1
1 UMR 8126, Univ. Paris-Sud, CNRS, Institut de Cancérologie Gustave-Roussy,
F-94805 Villejuif, France
2 INSERM EA 4202 ERI25,
371 Avenue du Doyen Gaston Giraud F-34295 Montpellier, France,
3 Functional Genomics Unit, Institut de Cancérologie Gustave-Roussy,
F-94805 Villejuif, France
4Ambion Inc., USA
5IBC Generium
6Laboratory of Molecular Biology, University of Mons;
20 place du Parc, B700 Mons, Belgium
vassetzky@igr.fr
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant
hereditary neuromuscular disorder linked to the deletion of an integral number of
3.3 kb-long macrosatellite repeats (D4Z4) within the subtelomeric region of
chromosome 4q. Most genes identified in this region are overexpressed in FSHD
myoblasts including the double homeobox genes DUX4 and DUX4c. We have
carried out a simultaneous miRNome/transcriptome analysis of FSHD and control
primary myoblasts. Of 365 miRNAs analyzed in this study, 29 were found to be
differentially expressed between FSHD and normal myoblasts. Twenty-one
microRNA (miR-1, miR-7, miR-15a, miR-22, miR-30e, miR-32, miR-107, miR-
133a, miR-133b, miR-139, miR-152, miR-206, miR-223, miR-302b, miR-331,
miR-362, miR-365, miR-382, miR-496, miR-532, miR-654, miR-660) were
upregulated, eight downregulated (miR-15b, miR-20b, miR-21, miR-25, miR-
100, miR-155, miR-345, miR-594). Twelve of the miRNAs upregulated in FHSD
were also upregulated in the cells ectopically expressing DUX4c, suggesting that
this gene could regulate miRNA gene transcription. The myogenic miRNAs miR-
1, miR-133a -133b and miR-206 were highly expressed in FSHD myoblasts
which nonetheless did not prematurely enter myogenic differentiation. This could
be accounted for by the fact that in FSHD myoblasts, functionally important
target genes including cell cycle, DNA damage and ubiquitination-related genes,
escape myogenic microRNA-induced repression.
13
Strategies for the selection and identification of active sites inhibitors
with potential therapeutic value
Claire Loussouarn, Emilie Stephan-Queffeulou, Melody Shahsavarian,
Séverine Padiolleau- Lefèvre, Bérangère Avalle & Alain Friboulet
"Enzyme and Cell Engineering" Université de Technologie de Compiègne / CNRS
Rue Roger Couttolenc - CS 60319 - 60203 COMPIEGNE CEDEX – France
Alain.Friboulet@utc.fr
The research developed in our laboratory is devoted to the study of biocatalysis,
mainly by creating new functions not existing in nature to explain actual life
properties and to produce new tools for biotechnology and medicine. Catalytic
antibodies (abzymes) were first developed as tools in chemistry, or as laboratory
curiosities. Our group has shown that the structural and functional mimicry of the
idiotypic network of immune system can be exploited to elicit catalytic anti-
idiotypic antibodies. Monoclonal antibodies with esterase, amidase and protease
activity were thus obtained. The data obtained indicate that the idiotypic network
is capable, to a significant extent, to replicate sophisticated catalytic apparatus of
serine proteases, and further validates the use of mimicry of enzyme active
centers by the immune system for induction of catalytic antibodies.
On the other hand, different groups have clearly demonstrated the presence of
antigen-specific hydrolytic antibodies in a number of inflammatory, autoimmune
and neoplastic disorders, but also in the serum of healthy donors; their pathogenic
effects have been demonstrated occasionally. The origin of disease-associated
catalytic antibodies may have been "induced" by the antigen implicated in the
disease, may result from the loss of repressive control, or may be an expression of
the idiotypic network with exacerbated self-recognition in autoimmune diseases.
The field of catalytic antibodies has also brought lessons to understand the
molecular architecture necessary for regulating a catalytic activity, and search for
innovative approaches to inhibit "detrimental" activities or to elicit "beneficial"
catalytic antibodies are developed. In the present talk, results obtained in the
selection of peptidic and nucleic acid (aptamers) inhibitors will be presented,
using both a model monoclonal anti-idiotypic catalytic antibody, and enzymes
involved in leukemia’s.
14
RNA architectural modules and their detection in sequences
Eric Westhof
Architecture et Réactivité de l’ARN, Université de Strasbourg,
Institut de Biologie Moléculaire et Cellulaire, CNRS,
15 rue René Descartes, 67084 Strasbourg, France
e.westhof@ibmc-cnrs.unistra.fr
http://www-ibmc.u-strasbg.fr/arn/Westhof/
RNA architecture can be viewed as the hierarchical assembly of preformed
double-stranded helices defined by Watson-Crick base pairs and RNA modules
maintained by non-Watson-Crick base pairs. RNA modules are recurrent
ensemble of ordered non-Watson-Crick base pairs. Such RNA modules constitute
a signal for detecting non-coding RNAs with specific biological functions. It is,
therefore, important to be able to recognize such genomic elements within
genomes. Through systematic comparisons between homologous sequences and
x-ray structures, followed by automatic clustering, the whole range of sequence
diversity in recurrent RNA modules has been characterized. These data permitted
the construction of a computational pipeline for identifying known 3D structural
modules in single and multiple RNA sequences in the absence of any other
information. Any module can in principle be searched, but four can be searched
automatically: the G-bulged loop, the Kink-turn, the C-loop and the tandem GA
loop. The present pipeline can be used for RNA 2D structure refinement, 3D
model assembly, and for searching and annotating structured RNAs in genomic
data.
Cruz, J.A., and Westhof, E. (2011). Sequence-based identification of 3D structural
modules in RNA with RMDetect. Nature methods 8, 513-521.
15
Inhibitory signalling to the Arp2/3 complex steers cell migration
Roman Gorelik1,13, Irene Dang1,13, Carla Sousa-Blin1,13, Emmanuel Derivery1,
Christophe Guérin2, Joern Linkner3, Maria Nemethova4, Tamara Tchipysheva5,
Valeria Ermilova5, Sophie Vacher6, Julien G. Dumortier7, Florence A. Giger7,
Valérie Campanacci8, Isaline Herrada9, Anne-Gaelle Planson8, Susan Fetics8,
Véronique Henriot1, Violaine David1, Ksenia Oguievetskaia1, Goran Lakisic1,
Fabienne Pierre1, Adeline Boyreau11, Nadine Peyriéras11, Anika Steffen10,
Klemens Rottner10,12, Sophie Zinn-Justin9, Jacqueline Cherfils8, Nicolas B.
David7, Ivan Bièche6, Antonina Alexandrova5, J. Victor Small4, Jan Faix3,
Laurent Blanchoin2, Alexis Gautreau1
CNRS Gif-sur-Yvette, France
group Cytoskeleton in Cell Morphogenesis, LEBS, CNRS UPR3082 Gif-sur-Yvette 91190,
France
alexis.gautreau@lebs.cnrs-gif.fr
Cell migration requires the generation of branched actin networks that power the
protrusion of the plasma membrane in lamellipodia. The Arp2/3 complex is the
molecular machine that nucleates these branched actin networks. This machine is
activated at the leading edge of migrating cells by the WAVE complex. The
WAVE complex is itself directly activated by the small GTPase Rac, which
induces lamellipodia. However, how cells regulate the directionality of migration
is poorly understood. Here we identify a novel protein that inhibits the Arp2/3
complex in vitro, Arpin, and show that Rac signalling recruits and activates Arpin
at the lamellipodial tip, like WAVE. Consistently, upon depletion of the
inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. The major
role of this inhibitory circuit, however, is to control directional persistence of
migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium
discoideum amoeba resulted in straighter trajectories, whereas Arpin
microinjection in fish keratocytes, one of the most persistent systems of cell
migration, induced these cells to turn. The existence of a Rac-Arpin-Arp2/3
inhibitory circuit embedded within the positive feedback loop that maintains
directional migration can account for this conserved role of Arpin in steering cell
migration.
16
Molecular and cellular effects of siRNA on RET/PTC3 junction
oncogene in papillary thyroid carcinoma
Hafiz Muhammad Ali1, Giorgia Urbinati 1, Hubert Chapuis 2, Didier Desmaële 2,
Patrick Couvreur 2, Liliane Massaad-Massade 1
1Institut Gustave Roussy UMR 8203 CNRS, Villejuif
2Faculté de Pharmacie UMR 8612 CNRS, Châtenay-Malabry, France
Liliane.massade@igr.fr
Papillary thyroid carcinoma (PTC) accounts of above 80-90% cases of thyroid cancer
and resulted by RET fusion with 12 other genes leading to 13 different onco-fusion
genes. Amongst; RET/PTC3 (RET fusion with ELE1) is the most metastatic type
occurring in 20–30 % PTC cases and represents an interesting target for small
interfering RNA (siRNA) strategies since it is present only in the tumour cells and not
in the normal cells. Our aim is: i) to target the RET/PTC3 oncogene by siRNAs, ii) to
assess the knockdown effects on cell growth and cell cycle regulation. Methods:
First, we established a stable cell line named RP3 cells derived from NIH-3T3 mouse
fibroblasts by transfecting a plasmid containing RET/PTC3 junction oncogene. An
efficient siRNA RET/PTC3 and an effective dose were selected on finding significant
RET/PTC3 gene (RT-qPCR) and protein (Western blot) inhibitions. In vitro siRNA
RET/PTC3 effects were tested on RP3 cell viability (MTT), toxicity (LDH),
invasion/migration in matrigel (IncuCyte), apoptosis (FACS, WB), RET/PTC3 gene
(RT-qPCR) and protein (WB) expressions. siRNA RET/PTC3-SQ bio-conjugate was
synthesized, corresponding nanoparticles were prepared and tested for their effects on
in vitro gene (RT-qPCR) and protein (Western blot) silencing. In vivo tumour growth
inhibition and gene and protein silencing efficiency of these nanoparticles was later
assessed as well, on administration in nude mice via intravenous route (cumulative
dose = 2.5 mg/kg). Results: In vitro siRNA RET/PTC3 was found to significantly
inhibit cell viability, LDH activity, invasion/migration, RET/PTC3 gene and protein
expressions compared to siRNA control and siRNA RET/PTC1 at 24h, 48h and 72h
post-transfection incubations. It was found to increase cell death in RP3 cells by
apoptosis and necrosis (Annexin/PI, WB). By Western blot, an increased caspase-3
expression was found from 24h to 48h while PARP1 expression was increased from
24h to 72h post-transfection. In vivo, the RP3 cells were found to be tumorigenic in
nude mice compared to mother NIH/3T3 cells. In vivo NPs siRNA RET/PTC3-SQ
were found efficient in significant (p<0.001) tumour growth, gene and protein
inhibitions. Conclusions: We established a siRNA efficient and specific to
RET/PTC3 oncogene that we are able to deliver by “squalenoylation”. These results
open new prospects in the treatment by siRNAs of PTC with RET/PTC3 junction and
offers a new non cationic plate-form for the siRNA delivery.
This work was supported by ANR “P2N Nanosqualonc”, ANR-11-NANO-0003,
http://www.anr.nanosqualonc.com
17
Interplay of the base excision repair and mismatch repair pathways in
active DNA demethylation
Inga Grin and Alexander A. Ishchenko
Université Paris Sud, Laboratoire « Stabilité Génétique et Oncogenèse » CNRS,
UMR 8200, Institut de Cancérologie Gustave Roussy, Villejuif, France.
Alexander.ISHCHENKO@gustaveroussy.fr
Active DNA demethylation in mammals occurs via hydroxylation of 5-
methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by the Ten-eleven
translocation family of proteins (TETs). 5hmC residues in DNA can be further
oxidized by TETs to 5-carboxylcytosines and/or deaminated by the
AID/APOBEC family proteins to 5-hydromethyluracil (5hmU). Excision and
replacement by regular C of these 5mC-derivatives is initiated by several DNA
glycosylases including: thymine-DNA glycosylase (TDG), methyl-binding
domain protein 4 (MBD4) and single-strand specific monofunctional uracil-DNA
glycosylase (SMUG1) in the base excision repair (BER) pathway. Recently, it has
been shown that non-canonical DNA mismatch repair system (MMR) can repair
both alkylation and oxidative damage to DNA. Here, we used a phagemid based
DNA substrate, containing defined oxidative base lesions and methylation marks
to identify and characterize different DNA repair pathways: BER, nucleotide
incision repair and MMR, which may be involved in active DNA demethylation.
Our data obtained from cell-free extracts repair assays support the role of MMR
in the active DNA demethylation in mammalian cells.
18
Deciphering the molecular mechanisms underlying the three-
dimensional folding of the Drosophila genome into physical domains
Caroline Jacquier,1 Tom Sexton,1 Eitan Yaffe,2 Ephraim Kenigsberg,2
Frédéric Bantignies,1 Benjamin Leblanc,1 Michael Hoichman,2
Hugues Parrinello,3 Amos Tanay,2 and Giacomo Cavalli.1
1Institut Génétique Humaine, UPR 1142, CNRS, 141 rue de la Cardonille, 34396 Montpellier,
France
2Department of Computer Science and Applied Mathematics and Department of Biological
Regulation, Weizmann Institute of Science, Rehovot 76100, Israel
3Montpellier GenomiX IBiSA, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France
caroline.jacquier@igh.cnrs.fr
The spatial organisation of the genome has been correlated with transcriptional
regulation for years: co-expressed genes share specialised sites within the nucleus
for their expression or repression, and regulatory chromatin loops bring distal
elements such as enhancers in contact with their target genes. Very recently,
technical advances (the “Hi-C” method) have allowed such chromatin interactions
to be assessed systematically. A major finding to come from the genome-wide
maps of such interactions is the conserved organisation of metazoan genomes into
well-demarcated physical domains, whereby interactions are strong within
domains and are sharply reduced when crossing domain boundaries. Such
domains contain co-expressed genes and overlap extensively with active and
repressive epigenetic marks, suggesting that the spatial and functional
organisations of the genome are closely linked. However, the mechanisms
establishing and maintaining these potentially important functional modules are
not known.
In drosophila, a systematic screen of chromatin profiles revealed a striking
enrichment for insulator proteins, such as CP190, Beaf-32 and CTCF, and for the
mitotic spindle protein Chromator at domain boundaries. These observations
strongly suggest that insulator binding sites actively define domain demarcations.
To test this hypothesis, we are establishing genome-wide maps of chromatin
interactions using the Hi-C method we developed in the lab, as well as chromatin
profiles of epigenetic marks in various genetic backgrounds lacking these
boundary-binding factors. These data will enable us to assess if chromatin folding
into physical domains is impaired in absence of these factors and if epigenetic
domains are consequently remodelled.
19
Progressive parkinsonism by acute dysfunction of excitatory amino
acid transporters in the rat substantia nigra
Maxime Assous, Laurence Had-Aissouni, Paolo Gubellini, Christophe Melon,
Imane Nafia, Pascal Salin, Philippe Kachidian, Lydia Kerkerian-Le Goff
CNRS/Aix-Marseille Université, IBDML UMR7288, 13009, Marseille, France
lydia.kerkerian@ibdml.univmed.fr
Parkinson's disease (PD) is characterized by the progressive degeneration of
substantia nigra (SN) dopamine neurons, involving a multifactorial cascade of
pathogenic events. Central players in PD pathogenesis, such as mitochondrial
dysfunction and oxidative stress, can affect the function of excitatory amino acid
transporters (EAATs), which play a major role in preventing excitotoxicity and
maintaining physiological levels of GSH. Here we explore the hypothesis that
dysfunction of excitatory amino acid transporters (EAATs) might contribute to
the vicious cycle sustaining degeneration of SN DA neurons. Acutely-induced
dysfunction of EAATs in the rat SN, by single unilateral injection of their
substrate inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), triggers a
neurodegenerative process mimicking several PD features. Dopamine neurons are
selectively affected, consistent with their sustained excitation by PDC measured
by slice electrophysiology. The anti-oxidant N-acetylcysteine and the NMDA
receptor antagonists ifenprodil and memantine provide neuroprotection. Besides
oxidative stress and NMDA receptor-mediated excitotoxicity, glutathione
depletion, autophagy and neuroinflammation characterize the primary insult. Most
interestingly, the degeneration progresses overtime with unilateral to bilateral and
caudo-rostral evolution. Transient compensatory changes in dopamine function
markers in SN and the target striatum accompany cell loss and axonal dystrophy,
respectively. Motor deficits appear when neuron loss exceeds 50 % in the most
affected SN and striatal dopamine tone is dramatically reduced. As a possible
cellular insight for neuron death progression, subthalamic nucleus metabolic
activity shows an asynchronous bilateral increase. These findings outline a
functional link between EAAT dysfunction and several PD pathogenic
mechanisms/pathological hallmarks, and provide the first acutely-triggered model
of progressive parkinsonism.
20
Telomere length regulation in Hansenula polymorpha
Alexander N. Malyavko1, Elena M. Smekalova1, Maria I. Zvereva1,
Andrey V. Mardanov2, Nikolai V. Ravin2, Konstantin G. Skryabin2,
Eric Westhof3, Olga A. Dontsova1
1Faculty of Chemistry, Lomonosov Moscow State University,
119999 Moscow, Russia and
Belozersky Institute, Moscow State University,
Leninskie Gory 1, Bldg. 40, 119991 Moscow, Russia.
2Centre “Bioengineering” of RAS,
Prosp. 60-let Oktyabrya, bld. 7-1, Moscow 117312, Russia
3Architecture et Réactivité de l’ARN, Université de Strasbourg,
Institut de Biologie Moléculaire et Cellulaire du CNRS,
15 rue René Descartes F– 67084 Strasbourg, France
malyavkoan@gmail.com
Aim. To characterize telomerase from Hansenula polymorpha. Methods.
Telomerase assay, telomeric PCR, pyrosequencing of PCR products. Results.
Telomerase sustains telomere homeostasis and provides unlimited proliferative
potential for unicellular eukaryotes, as well as stem, germ and cancer cells. The
two core components of telomerase complex are telomerase RNA (TER) and
telomerase reverse transcriptase (TERT). After each round of replication
chromosomes lose telomeric repeats. TERT maintains telomere length by reverse
transcription of the short template region of TER. We found that telomerase from
yeast Hansenula polymorpha elongated its substrate by one additional nucleotide
in vitro (compared to the expected sequence from the predicted template region).
We showed that this nucleotide (dT) was reverse transcribed from adenine, which
was positioned beyond the predicted template boundary. The product of such
elongation could not be recognized by telomerase as a substrate that should
compromise effective telomerase action at telomeres. Sequencing of PCR
products of telomeres provided evidence for the incorporation of this noncognate
dT nucleotide into telomeres in vivo. The involvement of this event in telomere
length regulation was confirmed by mutational analysis of the template region of
H. polymorpha TER. Conclusions. H. polymorpha telomerase utilizes reverse
transcription of the noncognate nucleotide to control telomere elongation.
Materials of this work are part of the article, which is currently in press
(Smekalova et al. “Specific features of telomerase RNA from Hansenula
polymorpha”. RNA).
21
The tRNA3
Lys packaging complex involves association of human
mitochondrial LysRS with the polyprotein GagPol from HIV-1: a new
pharmacologic target?
José Dias, Lydia Kobbi, Martine Comisso, Aurélien Nail, Anne Szklarz, and
Marc Mirande
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France
Marc.Mirande@lebs.cnrs-gif.fr
Cytosolic and mitochondrial LysRS are encoded by alternative splicing of a single
gene and can only be distinguished according to their very N-terminal sequences.
Beyond its role in translation, mitochondrial LysRS (mLysRS) is also hijacked
from the host cell following HIV-1 infection to carry the primer tRNA3Lys into
the virions1. Using monospecific antibodies, we previously showed that only
mLysRS is taken up in viral particles along with tRNA3Lys, the primer for
reverse transcription of the HIV-1 genome. We screened all the viral proteins to
identify the partners of LysRS responsible for the formation of the tRNALys
packaging complex. We showed that mLysRS associates with the Pol domain of
GagPol. This interaction is highly specific, as assessed by the KD value of about
5-10 nM between mLysRS and Pol. More specifically, the transframe (TF) and
integrase (IN) domain proteins of Pol interact with the catalytic domain of
LysRS2. A model of the assembly of the mLysRS:tRNA3
Lys:GagPol packaging
complex is proposed, which is also consistent with the release of its different
components after maturation of GagPol into the virions. Maturation of the
precursor of mLysRS upon its mitochondrial targeting is a prerequisite to form a
complex with tRNA3. These data open new perspectives for the search of a new
class of inhibitors of the HIV-1 development cycle that would block the
packaging of tRNA3Lys into viral particles.
1Kaminska et al. J. Virol. (2007) 81: 68-73.
2Kobbi et al. J. Mol. Biol. (2011) 410, 875-886.
3Dias et al. Biochemistry (2012) 51, 909-916.
22
Structure of mammalian proto-oncogene eEF1A2 and its functional
implications
B. Negrutskii, V. Shalak, A. Yaremchuk, T. Crepin, O. Novosyl’na, D. Vlasenko,
M. Tukalo, A. El’skaya
State Key Laboratory of Molecular and Cellular Biology,
Institute of Molecular Biology and Genetics NAS of Ukraine.
150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680
negrutskii@imbg.org.ua
eEF1A2 is the isoform of mammalian translation elongation factor which is also
involved in the carcinogenesis. Comparison of spatial structures of the eEF1A
isoforms reveals different organization and oligomeric properties of eEF1A2 and
eEF1A1. Crystal structure of preferentially monomeric, naturally folded and post-
translationally modified eEF1A2 in the complex with GDP uncovers major
structural and functional difference in nucleotide binding and exchange
procedures in the mammalian and prokaryotic analogues. Novel mechanism of the
guanine exchange factor eEF1B action in mammalian cells is described.
23
Stress granules and the proliferative rate of epithelial cells
Loic Hamon, Ouissame Bounedjah, Bénédicte Desforges, Patrick A. Curmi,
David Pastre
Institut National de la Santé et de la Recherche Médicale (INSERM),
UMR829; Université Evry-Val d'Essonne;
Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques,
Evry 91025, France.
david.pastre@univ-evry.fr
Following severe oxidative, heat or osmotic stresses, and also after some viral
infections, micrometric cytoplasmic bodies composed of mRNAs and many
RNA-binding proteins appear in the cytoplasm of eukaryotic cells. For more than
one decade, these non-membranous granules called “stress granules” were the
subject of intense research. The most popular model is that stress granules are
formed after the massive release of mRNAs from polysomes with the help of self-
attracting RNA-binding proteins like TIA-1 and G3BP, this process being
microtubule-dependent, at least for oxidative stress after arsenite exposure. These
granules then could allow the reprogrammation of the translational response via
the sequestration of mRNAs encoding housekeeping proteins in stress granules
and the exclusion of mRNAs encoding important proteins like heat shock proteins
in order to allow their translation. Here we will show that elevated polyamine
contents and highly dynamical microtubules are important factors that allow the
formation of stress granules in proliferating cells, in contrast with their quiescent
counterpart. Many interesting aspects remain to be tackled especially regarding
the potential advantages/disadvantages resulting from the formation of stress
granules in proliferating cells.
24
The prion protein: an unexpected link between base excision repair
and neurodegeneration
Anne Bravard1, Frédéric Auvré1, Jacqueline Bernardino-Sgherri1,
Damiano Fantini1, Zhou Xu2, Ludmilla Sissoëff2, Olivier Etienne1,
Mathieu Daynac1, GianLuca Tell3, François Boussin1, Jean-Philippe Deslys2, and
J. Pablo Radicella1
1Institute of Cellular and Molecular Radiobiology, CEA, Fontenay-aux-roses, France ;
2Institut des Maladies Emergentes et des Thérapies Innovantes, CEA,
Fontenay-aux-roses, France ;
3Department of Biomedical Sciences and Technologies, University of Udine, Italy.
pablo.radicella@cea.fr
A conformational change in the highly conserved and ubiquitously expressed
prion protein (PrPc) to yield an abnormal form (PrPSc) is associated with the
generation of prions, the infectious agent of transmissible spongiform
encephalopathies. Whether the resulting pathology is due to the accumulation of
PrPSc aggregates or to a loss of function of the normal PrPc is still an open
question since, despite decades of investigation, the physiological role of the
normal PrPc remains elusive. A protective effect against oxidative stress has been
shown, but the underlying mechanisms have not been determined. Here, by using
animal and cellular models, we unveil a key role of PrP in the DNA damage
response. We found that exposure of neurons to a genotoxic stress activates PRNP
transcription leading to an increased amount of PrP in the nucleus where it
interacts with APE1, the major mammalian endonuclease essential for base
excision repair (BER), and stimulates its activity. Preventing the induction of
PRNP results in accumulation of abasic (AP) sites in DNA and impaired cell
survival after genotoxic treatment. The reduced activity of APE1 in brains from
Prnp-/- mice is associated with a defect in the repair of induced DNA damage in
vivo. Brains from mice at early stages of prion infection also display a reduced
APE1 activity, suggesting that loss of the PrP protective function on DNA plays a
role in neuronal death and could be implicated in many neurodegenerative
processes.
25
Increased mobility of broken MLL gene ends in human lymphoid cells
treated with DNA topoisomerase II poison etoposide
Sergey I. Glukhov1, Mikhail A. Rubtsov1, Daniil A. Alexeyevsky4,
Andrei V. Alexeevski3,4,5, Olga V. Iarovaia2, Sergey V. Razin1,2,6
1Dpt Molecular Biology, Faculty of Biology, Lomonosov Moscow State University,
Moscow, Russia
2Institute of Gene Biology RAS, Moscow, Russia
3A.N. Belozersky Institute for Physical and Chemical Biology, Lomonosov Moscow State
University, Moscow, Russia
4Dpt of Bioengineering and Bioinformatics, Lomonosov Moscow State University,
Moscow, Russia
5Scientific Research Institute for System Studies (NIISI RAN), Moscow, Russia
6LIA 1066 French-Russian Joint Cancer Research Laboratory,
Villejuif, France-Moscow, Russia
sergey.v.razin@usa.net
The mixed lineage leukaemia (MLL) gene is frequently rearranged in secondary
leukaemias, in which it could fuse to a variety of different partners. Breakage in
the MLL gene preferentially occurs within a ~8 kb region that possesses a strong
DNA topoisomerase II cleavage site. It has been proposed that DNA
topoisomerase II-mediated DNA cleavage within this and other regions triggers
translocations that occur due to incorrect joining of broken DNA ends. To further
clarify a possible mechanism for MLL rearrangements, we analysed the frequency
of MLL cleavage in cells exposed to etoposide, a DNA topoisomerase II poison
commonly used as an anticancer drug, and positioning of the broken 3’-end of the
MLL gene in respect to inherent chromosomal territories. It was demonstrated
that exposure of human Jurkat cells to etoposide resulted in frequent cleavage of
MLL genes. Using MLL-specific break-apart probes we visualised cleaved MLL
genes in ~17 % of nuclei. Using confocal microscopy and 3D modelling, we
demonstrated that in cells treated with etoposide and cultivated for 1 h under
normal conditions, ~9 % of the broken MLL alleles were present outside the
chromosome 11 territory, whereas in both control cells and cells inspected
immediately after etoposide treatment, virtually all MLL alleles were present
within the chromosomal territory. The data are discussed in the framework of the
“breakage first” model of juxtaposing translocation partners. We propose that in
the course of repairing DNA topoisomerase II-mediated DNA lesions (removal of
stalled DNA topoisomerase II complexes and non-homologous end joining),
DNA ends acquire additional mobility, which allows the meeting and incorrect
joining of translocation partners.
26
DNA-protein and protein-protein interactions in nucleotide excision
repair and their regulation
N. I. Rechkunova1, Y. S. Krasikova, E. A. Maltseva, M. V. Sukhanova, O.I. Lavrik1
Institute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk, Russia
1Department of Natural Sciences, Novosibirsk State University, 630090, Novosibirsk, Russia
nadyarec@niboch.nsc.ru
Aim. Nucleotide excision repair (NER) is one of the major mechanisms to prevent
genomic DNA instability. This process removes a wide range of lesions distorting the
double helix and bulky chemical adducts resulting from environmental factors or
chemotherapeutic agents. The coordination of the assembly of the NER complexes is
achieved through multiple DNA-protein and protein-protein interactions. The interactions
of key NER proteins, XPC-RAD23B, XPA, and RPA with DNA structures mimicking
NER intermediates and their cooperation within DNA-protein complexes have been
analyzed. Methods. The topography of the NER protein complexes with damaged DNA
was determined by photoaffinity labeling technique using DNA structures containing
photoreactive 5I-dUMP residues in the certain positions either in damaged or in
undamaged strands and fluorescein group linked to uridine residue as a lesion.
Electrophoretic mobility shift assay (EMSA) and fluorescent depolarization measurements
were used for the analysis of DNA-protein binding. Results. Positioning of human and
yeast DNA damage recognition complexes, XPC-RAD23B and Rad4-Rad23,
respectively, on damaged DNA was determined. Under conditions of equimolar binding
to DNA both proteins exhibited the highest level of crosslinks to 5I-dUMP located exactly
opposite the damaged nucleotide. Both proteins bind to the damaged 15 nt bubble-DNA
structure mimicking in size the “transcription bubble” DNA intermediate with the highest
affinity. RPA and XPA are very abundant proteins that are absolutely required for NER
functioning. Both RPA and XPA proteins stimulate XPC binding to the damaged DNA
and enhance the level of XPC-DNA crosslinks. An influence of the substitution of lysine
in DNA binding cleft of XPA by glutamate (XPA K141E, K179E, and K141/179E;
recombinant plasmids were kindly provided by Dr. Hanspeter Naegeli, University of
Zürich) on the interaction with various DNA structures was analyzed. Both K179E and
K141E mutations result in moderate decrease in DNA binding affinity and do not
influence on the protein positioning on partially open DNA duplex. Tandem mutation
K141/179E dramatically reduced XPA affinity to DNA. The results allow suggesting the
key role of XPA orientation for positioning the NER preincision complex. Poly(ADP-
ribose)polymerase-1 (PARP-1) is one of the candidate to participate in NER regulation.
Using EMSA, the influence of PARP1 on binding of XPC-RAD23B and XPA to model
DNA-structures modulated by PAR synthesis was revealed. Conclusions. Obtained data
fill the gap between biochemical results for XPC-RAD23B and X-ray structure for yeast
ortholog Rad4-Rad23 and exhibit a significance of protein-protein interactions for the
correct assembling of the NER machinery. PARP1 can be regarded as the universal
regulator in DNA repair processes.
This work was supported by RFBR (grants 12-04-31323, 12-04-33162, 13-04-00538, 13-
04-93107, 13-04-40197-comfi); the Russian Ministry of Education and Science (contract
14.B37.21.0188); the Program of the RAS Presidium ‘Molecular and Cellular Biology’
27
Search for molecules with combined antioxidant and DNA-binding activities
E. Rostoka1,2, S. Isajevs2, E.Buraka1,2, L. Baumane1, N. Sjakste1,2
1Latvian Institute of Organic Synthesis, Aizkraukles Street 21, Riga LV1006, Latvia;
2Faculty of Medicine, University of Latvia, Raina bulv. 19, Riga LV1050, Latvia
Search for substances protecting DNA against oxidative damage is a topical problem. In
our work we have tried to assess possible antioxidant and DNA-protecting activities in a
vast group of compouds: natural compounds - baicalein, quercetin, kaempferol, indole-3-
carbinole, luteolin, lycopene, myricetin, separately and combinations and artificial 1,4-
dihydropyridines (1,4-DHP) synthesized in Latvian Institute of Organic Synthesis. Several
natural compounds (baicalein, luteolin, quercetin, indole-3-carbinol) decreased NO
production in different tissues and organs of intact rats. Intraperitoneal injection of
lipopolysascharide (LPS) to the animals caused a drastic increase of NO production levels
in all tissues studied. The highest production of nitric oxide was detected in liver.
Baicalein and luteolin decreased NO outburst in several organ. In contrast, administration
of the indole-3-carbinol enhanced the LPS-induced increase of NO production, quercetin
produced a similar effect. No influence of indole-3-carbinol and quercetine on iNOS
expression in liver was observed in the control group. Luteolin up- regulated the gene
expression. Baicalein decreased level of the gene expression. Level of transcription was
still decreased when baicalein was given in combination with luteolin. The LPS induced
iNOS gene expression on high level. Quercetin and kaempferol significantly decreased the
LPS-triggered iNOS mRNA expression. Enhancement of the iNOS mRNA expression by
indole-3-carbinol was observed. The same trend was observed when indole-3-carbinol
was supplemented by quercetin. Baicalein decreased the gene expression triggered by
LPS, the effect was better pronounced if it was combined with luteolin also in sepses
model. Interestingly, in brain cortex indole-3-carbinol inhibited iNOS mRNA expression
in control group. LPS triggered a marked increase in iNOS gene mRNA copies compared
to LPS-untreated animals. Indole-3-carbinol and lycopene enhanced this effect. In the
1,4-DHP study Metkarbaton appeared to be the only effective hydroxyl radical scavenger
in Fenton reaction among twenty tested 1,4-DHP. AV-153-Na, AV-154-Na, AV-153-Li,
PP-150-Na, PP-54-NH4 manifested ability to protect plasmid DNA against damages
caused by peroxynitrite in vitro. Compound AV-153 evidently interacted with DNA, as
addition of the sonicated rat liver DNA to AV-153 solutions caused pronounced
hyperchromic and bathochromic effects on the spectra. Reciprocally addition of the
compound to DNA solutions caused hypochromic effect in DNA spectra. Similar effects
were observed when intact pTZ57R plasmid was used for titration. Oxydative
modifications of DNA bases by peroxynitrite did not change much binding affinity of the
compound. However the effect was much stronger when sonicated plasmid was taken for
titration. The binding mode did not change when ionic strength of the solution was
changed from 5mM to or 150mM of NaCl, it became stronger at 200 mM. Interaction of
the compound with AT-rich DNA of Staphilococcus aureus ot GC-rich of Micrococcus
luteus were much weaker compared to interaction with DNA of similar AT and GC
content. Data of in vivo studies will be also reported.
The work was supported in part from the European Regional Development Foundation
project No 2010/0315/2DP/2.1.1.1.0/10/APIA/VIAA/026
28
New repair pathways for free radicals induced complex DNA damage
Ibtissam Talhaoui1, Doria Alilia, Armelle Vigouroux2, Bulat Aikeshev1, Nicolas E.
Geacintov3, Solange Morera2, Alexander A. Ishchenko1, Murat Saparbaev1
1Groupe «Réparation de l’ADN», Université Paris Sud, Laboratoire « Stabilité Génétique et
Oncogenèse » CNRS, UMR 8200, Institut Gustave Roussy, Villejuif, France
2Laboratoire d’Enzymologie et de Biochimie Structurales (LEBS), CNRS, Gif-sur-Yvette
Cedex, F-91198, France
3Chemistry Department, New York University, New York, NY 10003-5180
Murat.SAPARBAEV@gustaveroussy.fr
Oxidative DNA lesions are believed to be a major type of endogenous damage
leading to human degenerative disorders including cancer, cardiovascular disease
and neurological syndromes. The clinical features of inherited human DNA repair
deficient disorders such as Cockayne syndrome and Fanconi anemia point to
complex nature of endogenous oxidative DNA damage which may include bulky
adducts (cyclopurines, exocyclic bases), interstrand and intrastrand DNA
crosslinks. Conversely, severe biological effects of ionizing radiation and drugs
such as bleomycin, mitomycin and neocarzinostatin are correlated with clustering
of lesions within a single helical turn of the DNA molecule. At present, the
precise repair mechanisms for the complex DNA damage (CDD) are poorly
understood. Oxidized DNA bases are substrates for two overlapping pathways:
base excision repair (BER) and nucleotide incision repair (NIR). In the BER
pathway a DNA glycosylase cleaves the N-glycosylic bond between the abnormal
base and deoxyribose, leaving either an abasic site or single-stranded break in
DNA. Alternatively, in the NIR pathway, an apurinic/apyrimidinic (AP)
endonuclease incises oxidatively damaged DNA in a DNA glycosylase-
independent manner, providing the correct ends for DNA synthesis coupled to the
repair of the remaining 5´-dangling damaged nucleotide. In the present work we
studied alternative repair pathways for bulky G-T intrastrand crosslinks and
spiroiminodihydantoin (Sp) adduct when present in DNA. Here we demonstrate
that bacterial, yeast and human AP endonucleases can directly repair G-T
crosslinks and Sp residues in the NIR pathway. Furthermore, we describe genetic
dissection of human AP endonuclease 1 functions in BER and NIR pathways and
construction of NIR-deficient mammalian cell lines. The potential biological roles
of the BER and NIR pathways in counteracting complex DNA damage are
discussed.
29
Poly(ADP-ribose) glycohydrolase in DNA repair and replication:
towards potential applications in anticancer strategies ?
Giuditta Illuzzi1, Elise Fouquerel1,2, Aurélia Noll1, Jean-Christophe Amé1,
Marie-José Nzambi-Khonde1, Carole Beck1, Barbara Camuzeaux1,
Françoise Dantzer1, Valérie Schreiber1
1IREBS-Biotechnology and Cell Signalling, UMR7242-CNRS, Université de Strasbourg,
Labex Medalis, 300 Bd Sébastien Brant, BP 10413, 67412 Illkirch, France.
2Present address: University of Pittsburgh Cancer Institute, University of Pittsburgh,
Pittsburgh, Pennsylvania 15232, United States
valerie.schreiber@unistra.fr
Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in a
wide number of biological processes including DNA repair, transcription, cell
differentiation or cell death. The regulation of poly(ADP-ribose) produced in
response to DNA damage by the poly(ADP-ribose) polymerases (PARPs), and
degraded by poly(ADP-ribose) polymerase (PARG) is critical for the damaged cell
fate. The role of the founding member of the PARP family PARP-1 is highly
documented in the DNA damage response. Moreover, PARP inhibitors are involved
in clinical trials to potentiate the action of anticancer clastogenic drugs as well as for
their cytotoxic effect on tumours harbouring mutations in genes involved in double
strand breaks repair by homologous recombination, such as BRCA1/2. Far less is
known about PARG and the role of its different isoforms in the cell response to DNA
damage. A major goal of our laboratory is to determine whether PARG could also be
considered as a promising target for anticancer strategies. We have shown previously
that the absence of PARG increases radiosensitivity and affects the repair of
radioinduced single (SSB) and double (DSB) strand breaks. We have also
demonstrated a functional link between PARG and the repair/replication factor
PCNA: binding to PCNA contributes to PARG recruitment to laser induced DNA
damage sites and to replication foci. This latter observation prompted us to investigate
the contribution of PARG in DNA replication. Whereas PARG appeared dispensable
for normal replication, PARG-deficient cells showed increased sensitivity to the
replication inhibitor hydroxyurea (HU). These cells showed defect in S-phase restart
after prolonged but not short HU-treatment, conditions known to trigger replication
fork collapse and formation of double strand breaks that are repaired by homologous
recombination. Our current studies are aimed to determine the underlying
mechanisms involving PARG and poly(ADP-ribose) in the cell recovery from
replication stress.
Project funded by CNRS, Université de Strasbourg, Labex Medalis, Ligue contre le
Cancer (Equipe Labellisée), EDF.
30
Association of the PSMA3 gene polymorphisms with multiple sclerosis
Tatjana Sjakste1, Jolanta Kalnina1,2, Natalija Paramonova1, Olga Sugoka1
1Genomics and Bioinformatics, Institute of Biology of the University of Latvia;
2Faculty of Medicine, University of Latvia;
tanja@email.lubi.edu.lv
Multiple sclerosis (MS) is the most common autoimmune disorder of the central
nervous system. The cause of MS is still poorly understood and different
metabolic pathways seem to play a role in disease development. Failure of
ubiquitine proteasome system (UPS) efficiency had been recently implicated to
MS pathogenesis. The aim of the present study was to analyse the association
between MS in Latvian population and allelic variants of the PSMA3 gene
encoding one of T1A peptidase, which is a 20S core alpha subunit of a
constitutive and immune-proteasomes. Methods: The rs2348071 SNP of the
PSMA3 gene (c.543 + 138G > A) has been genotyped in 281 MS patients (201
women) being diagnosed on relapsing – remitting (188 subjects) or secondary
progressive (93 subjects) course of the disease, versus 191 control subjects (117
women) without inflammatory and any autoimmune disorders. Results: In
controls the rs2348071 locus showed allele and genotype presentation similar to
other Europeans with MAF about 30 % and genotype GG being most frequent
(53 %). In both female and male cohorts of MS patients the minor allele A was
observed slightly more frequent than in controls (P < 0.05), frequency of both GG
and AA homozygotes was decreased (about 30 % and 5 % respectively) in favour
of heterozygote GA genotypes (from 61 % to 71 % in patients of relapsing –
remitting and secondary progressive course of disease respectively) that was
significantly higher than in controls (P < 0.0001; OR = 3.539 [95 % CI 2.409 –
5.198] according to co-dominant model). The PSMA3 c.543 + 138 G > A
nucleotide substitution potentially could affect binding of several transcription
factors, sequence similarity to splicing signals and other modulators of gene
expression and finally influence 20S proteasome functionality. Conclusion: The
rs2348071 heterozygous genotype appears to be the MS risk factor in Latvian
population.
The work was supported in part from the European Regional Development
Foundation project No 2010/0315/2DP/2.1.1.1.0/10/APIA/VIAA/026
31
Both integration of plasmid DNA into genome and constitutive
overexpression of CHI3L1 promote chromosome instability and phenotype
changes in 293 cells
Stepanenko A.A.1*, Andreeva S.V.1, Mikitenko D.A.1, Huleyuk N.1, Vassetzky Y.S.2,
Kavsan V.M.1
1State Key Laboratory of Molecular and Cellular Biology,
Institute of Molecular Biology and Genetics,
150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680
2CNRS, Université Paris-Sud 11, Institut de Cancérologie Gustave Roussy,
94805 Villejuif, France
a.a.stepanenko@gmail.com
To study the patterns of CCAs/NCCAs after genotoxic stress (integration of
foreign DNA into genome and overexpression of oncogene CHI3L1) and to
establish the link between changes of karyotype and malignant phenotype, we
used two distinct 293 cell lines (variant 1 and variant 2) with different
morphology, proliferation level, aggressiveness, and degree of CIN. Empty vector
stably transfected 293_pcDNA3.1 cells variant 1, 293_pcDNA3.1 cells variant 2,
and 293_CHI3L1 cells (nonclonal) were derived from 293 cells variant 2.
293_CHI3L1 cells clone 1 and clone 2 were obtained from 293 cells variant 1.
293 cells variant 1 and variant 2 differed in many marker chromosomes and
degree of CIN (CCA/NCCAs) and resembled different phases of evolution (step-
wise phase and punctuated/discontinuous phase) according to Heng et al. [Heng et
al., 2010-2013]. Analysizing 293 cell derivatives we conclude that genotoxic
stress (as integration of foreign DNA into genome and overexpression of
oncogene CHI3L1) potentiates CIN during somatic cell evolution and
tumorigenesis. Karyotypes of cell lines after genotoxic stress evolved
stochastically and were individual with different patterns of CCAs/NCCAs. The
pattern of CCAs/NCCAs of cells depended on the nature of genotoxic stress,
phase of cells evolution, the nature of applied stress, and time lapse (number of
passages), which cells were in culture after genotoxic stress before karyotyping.
Artificial manipulations of specific molecular mechanisms (e.g., CHI3L1
oncogene overexpression) into a dominant form strongly promoted cancer
evolution, drastically reducing genome heterogeneity. The pattern of
CCAs/NCCAs reflected tumorigenic properties of cells. The interplay between
CCAs and NCCAs is a driving force for cellular genetic heterogeneity,
phenotypic plasticity, and clonal population diversity that is essential to cancer
evolution.
32
The study of Hsp60 role in thyrod cancer progression
1Sidorik L.L., 1Kapustian L., 1Chornyy S., 2Gurtovyy V., 3Pogrebnoy P.,
2Khozhaenko Yu., 4Gagarkin N., 2Usenko V. S.
1Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine
2Pathomorphological laboratory BIONTEK, Dnipropetrovs’k, Ukraine
3Kavetsky’ Institute of Experimental Pathology, Oncology and Radiobiology,
NAS of Ukraine, Kyiv, Ukraine
4Mechnikoff’ Clinical Hospital, Dnipropetrovs’k, Ukraine
sidorik@imbg.org.ua
Background. A wide range of tumor cells or tissues have been shown to express
atypical level or localization of Hsp60. Hsp60 expression in breast or gastric cancer is
associated with poor prognosis and resistance to chemotherapy or radiation therapy.
Hsp60 expression in thyroid cancer has not been studied extensively. The aim of our
study was to evaluate the possible changes in Hsp60 cellular content and localization
in thyroid gland’ cells in normal state and upon pathology by quantitative
immunohistochemical analysis and to exam the correlation between anti-Hsp60
autoantibodies level and the degree of thyroid gland lesions in this group of patients.
Methods. Sera from 49 patients (46 females and 3 males, 20–57 years old, 12 –
nodular hyperplasia of thyroid gland, 12 – Hashimoto’s thyroiditis, 18 – follicular
adenoma, 6 –papillary and 1 follicular carcinoma) and 12 healthy donors (as a
control) were used for determination of anti-Hsp60 antibodies level by ELISA. To
evaluate the content and localization of Hsp60 in thyroid tissue of such patients and
upon pathology we used immunohystochemical analysis. As a control, autopsy
material of 12 thyroid tissue without morphological signs of thyroid pathology was
used. Hsp60 expression was evaluated according to the percentage of positively
stained cells. Results. The increased anti-Hsp60 autoantibodies level has been
detected in sera more than 50 % of patients with non-malignant thyroid gland
pathology (Hashimoto’s thyroiditis, nodular hyperplasia, follicular adenoma) and in
86% of TC patients sera by ELISA method. This finding correlates with the degree of
thyroid gland lesions in this group of patients obtained by the immunohistochemical
study. Conclusion. Significant increase of anti-Hsp60 antibodies level was
determined in sera of patients with thyroid pathology. The highest titers of anti-Hsp60
antibodies in sera of patients with thyroid cancer has been revealed. Changes of
Hsp60 expression and cellular localization in thyroid cancer tissue have been detected
in comparison with normal ones. The increase of Hsp60 expression and elevated level
of anti-Hsp60 autoantibodies was associated with morphological signs of pathology -
lymphoid infiltration and sclerotic changes of tissue. The working model of Hsp60
involvement in thyroid cancer progression is proposed.
33
Specificity of DNA import into isolated mitochondria from plants and
mammals
M. V. Koulintchenko1,2, N. Ibrahim2, E. S. Klimenko1, Yu. M. Konstantinov1,
F. Weber-Lotfi2, A. Dietrich2
1 Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch of the Russian
Academy of Sciences, 132 Lermontova Str., Irkutsk, Russian Federation, 664033
2 CNRS Institute of Plant Molecular Biology, University of Strasbourg, 12 General Zimmer
Str., Strasbourg, France, 67084
mk100171@yahoo.com
The aim of the project is to study different features of DNA import into plant
mitochondria and into human mitochondria. Methods. We developed DNA uptake
experiments with isolated plant mitochondria, using as substrates various sequences
associated or not with the specific TIRs present at each end of the 11.6 kb linear
plasmid from rapeseed (Brassica napus L.). Further substrates for mitochondrial
import were the S1 and S2 linear plasmids from maize (Zea mays). Results. The
efficiency of the import of large DNA molecules into plant mitochondria depends on
the sequence and (ii) the specificity of DNA import can be mediated by the presence
of certain elements in their sequence, especially TIRs at the ends of the molecules.
Conversely, the efficiency of DNA import into mammalian mitochondria seemed to
depend neither on the DNA sequence, nor on its size. We addressed the possible
universal role of the TIRs of mitochondrial linear plasmids from Z. mays and Brassica
in the mechanism of DNA import into plant mitochondria. Using a vector containing
the TIRs (327 bp) from the 11.6 kb B. napus plasmid, we obtained several DNA
constructs and tested the importance of the size and DNA structure on the import
efficiency into plant mitochondria. The DNA sequences of Z. mays linear plasmids,
S1 and S2 were also cloned and used as substrates for import assays. Using
radioactively labeled DNA substrates and the in organello potato (Solanum
tuberosum) mitochondrial import system, it was established that the import efficiency
has a non-linear dependence on DNA size: DNA fragments of 6–7 kb in size can be
imported into S. tuberosum mitochondria more effectively than molecules with a 4 kb
size. It was also shown that import into plant mitochondria of DNA molecules of
“medium" sizes, i.e. between 4 and 7 kb, barely has any sequence specificity: neither
TIRs from the 11.6 kb Brassica plasmid, nor TIRs from the Z. mays S-plasmids
influenced DNA import into S. tuberosum mitochondria. Conversely, the role of the
TIRs from the 11.6 kb linear plasmid in the import of large DNA molecules was
established earlier for Brassica rapa mitochondria. Conclusions. The data obtained
support a species-specific import mechanism of the mitochondrial linear plasmids,
and more generally of large DNA molecules, into plant mitochondria, which needs
further investigation.
The work was financially supported by a grant from the Russian Fund for Basic
Research (12-04-01400) and the French Ministry for Research (Investissements
d'Avenir / Laboratoire d'Excellence MitoCross).
34
Role of DDC1 as a link between check point and DNA repair in yeast
Saccharomyces cerevisia
M.V. Sukhanova, C. D’Herin1, P.A. van der Kemp2, S. Boiteux1, O.I. Lavrik
Institute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk, Russia
1CBM, UPR 4301, CNRS, 1A rue de la Férollerie, Orléans, France
2CEA, iRCM, UMR217, CNRS, 18 route du Panorama, 92265, Fontenay aux Roses, France
lavrik@niboch.nsc.ru
Aim. The aim of this study was to identify key nick sensor proteins operating in yeast
Saccharomyces cerevisiae by using original approach that was developed under study of
nick sensor proteins in mammalian cells. Methods. Photoreactive DNA structures
mimicking intermediates of base excision repair (BER) were used for affinity
modification of interacting proteins in the crude cell extracts. The protein identification
was performed by MALDI–TOF-MS peptide mapping. The cell extracts were produced
from wild–type yeast Saccharomyces cerevisiae and mutants deleted in specific genes.
Results. To characterize proteins that interact with BER/single strand break repair DNA
intermediates in cell extracts, we used photoreactive DNA duplex containing nick
carrying photoreactive group. This photoreactive DNA was incubated with the yeast cell
extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked
proteins were identified by MALDI–MS as catalytic subunit of DNA polymerase epsilon
and Ddc1 checkpoint protein. Labeling of DNA polymerase epsilon catalytic subunit with
the nick-containing DNA indicates that this DNA polymerase is involved in DNA repair
synthesis in yeast. Ddc1 crosslinks to DNA nick independently of the other components of
checkpoint clamp, Mec3 and Rad17, suggesting Ddc1 alone is able to recognize DNA
single-strand break. In addition, the absence of Ddc1 protein greatly influences the overall
pattern of proteins crosslinked to DNA nick and stimulates proteolysis of topoisomerase 1
crosslinked to DNA. We suggested that this effect is due to Ddc1 capacity to prevent
proteolytic degradation of the DNA–protein adducts. DNA duplexes containing 3′-
recessed (5′-ss/dsDNA) or 5′-recessed end (3′-ss/dsDNA) carrying photoreactive group
were used for photoaffinity labeling of proteins in cell extracts. It was found that Ddc1
was crosslinked by the 5′-ss/dsDNA independently of the other components of checkpoint
clamp. The detectable crosslinking of subunits of Ddc1-Mec3-Rad17 clamp with 3′-
ss/dsDNA was not observed. The p70 subunit of RPA (RPA p70) was the predominant
crosslinking product with 3′-ss/dsDNA. Cell extracts deleted for Ddc1 (ddc1) did not
display labeling of full-length RPA p70 with neither 5′-ss/dsDNA nor 3′-ss/dsDNA. It is
interesting that RPA p70 undergoes a proteolytic cleavage in ddc1 extract. Addition of
purified Ddc1 or C-terminal fragments of Ddc1 to ddc1 extract slightly inhibits of RPA
p70 cleavage. Conclusions. The interaction of Ddc1 with DNA nicks may provide a link
between the DNA damage checkpoint and BER pathways in yeast. Genetic and
biochemical studies in S. cerevisiae determined that Ddc1-Mec3-Rad17 checkpoint clamp
and RPA act at the early steps of the checkpoint response to recognize DNA
repair/replication intermediates. Our results reveal a novel and potentially important
property of Ddc1 in preventing of proteolytic degradation of RPA and other key DNA
binding proteins.
The work was supported by RFBR (grants 11-04-00559, 12-04-92601, 12-04-33162, 13-
04-93107, 13-04-40197-comfi); Program of RAS “Molecular and Cell Biology”.
35
Biological effects of therapeutic siRNA against TMPRSS2-ERG fusion
oncogene for the cancer prostate treatment
Giorgia Urbinati, Quentin Rousseau, Hafiz M. Ali, Liliane Massaad-Massade
Institut Gustave Roussy UMR 8203 CNRS France
Liliane.massade@igr.fr
Prostate cancer is one of the most common tumors in men worldwide and the second
leading cause of death among all cancer types in western countries. Combined with
surgery, hormone therapy is the first line treatment administrated for hormone-
dependent prostate cancers, however when the pathology turns into a castration
independent phase, the treatments become less specific. New specific molecular
targets would then be highly useful to develop innovative personalized medicines.
Among these targets Tomlins et al. decrypted in 2005 a chromosomal rearrangement
resulting in the fusion of TMPRSS2 gene with members of the E26 Transformation-
Specific (ETS) family and in particular E26 related gene (ERG). The TMPRSS2-ERG
genomic fusion is found in 50 % of localized and 30 % of metastatic prostate cancers
and this particular chromosomal rearrangement lead to more aggressive cancer
phenotypes and worst cancer-specific survival rate. Our aim is: i) to target the
TMPRSS2-ERG oncogene by siRNAs, ii) to assess the knockdown effects on cell
growth and cell cycle regulation. Material and Methods: the human VCaP cell line
that harbours the TMPRSS2-ERG rearrangement was used. siRNAs specifically
directed against TMPRSS2-ERG oncogene were designed to knockdown ERG in the
VCaP cells. They were transfected in VCaP then, total RNA was extracted and First-
strand cDNA was generated with M-MLV (Reverse Transcription), real time PCR Q-
RT-PCR was used to assess the knockdown efficiency. Western blot was performed
using ERG antibody and β-actin was used as loading control. Genes affected by the
ERG knockdown were identified by microarray (MA) analysis followed by Q-RT-
PCR validation. Then, siRNA TMPRSS2-ERG effects on cell cycle growth (MTT
tests), cell cycle and apoptosis (flow cytometry) were studied. Results: ERG mRNA
and protein levels were dramatically down-regulated after siRNA transfection in
VCaP cell line. Microarray analysis showed regulation of apoptosis related genes by
TMPRSS2-ERG siRNA which was confirmed by Western blot and fluorescent kit
assays and regulation of other genes involved in intracellular protein trafficking. In
addition, cell viability was compromised and angiogenesis appeared to be affected.
Conclusion: We success to design efficient and specific siRNAs TMPRSS2-ERG
against ERG oncogene. Now, we are bioconjugated them with lipids to obtain
nanoparticles in order to evaluate their potential antitumor effects in vivo.
This work was supported by ANR “P2N Nanosqualonc”, ANR-11-NANO-0003,
http://www.anr.nanosqualonc.com
36
miRNA boost in the tumor and miRNA drop in the blood serum
caused by treatment with RNase A promote an attenuation of tumor
malignancy
Zenkova M. A., Mironova N. L., Patutina O. A., Brenner E. V., Kurilshikov A. M.,
Vlassov V. V.
Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia
marzen@niboch.nsc.ru
In recent years, ribonucleases are regarded as perspective anticancer drugs. Novel
data, suggesting an important role of miRNAs in mediating tumor growth and
invasion, have provided researchers with a new area to search for possible molecular
ribonuclease targets. Previously, antitumor and antimetastatic properties of pancreatic
ribonuclease RNase A were demonstrated and therapeutic efficacy was shown to
correlate with increase in ribonuclease activity of the blood plasma of experimental
animals (Patutina O.A. et al., Biochimie 2011). We hypothesized that RNase A
affects some regulatory pathways of tumorgenesis through the cleavage of circulating
oncogenic miRNAs. Here by high-throughput SOLiD sequencing technology we
performed analysis of genome-wide profiles of miRNAs in tumor and serum of mice
bearing Lewis lung carcinoma after the treatment with RNase A. Sequencing data
revealed that treatment with RNase A resulted in an apparent alteration in the levels
of 123 tumor-derived miRNAs and 139 serum miRNAs. We observed drop of most
miRNAs in blood serum and very unusual miRNA boost in tumor tissue. Data of
qPCR of selected miRNAs mir-29b, mir-21, mir-10b, mir-451a, mir-17, mir-18a,
mir145, mir-31 and let-7g confirmed the observed effect of RNase A. The miRNA
boost in tumor tissue was accompanied by the overexpression of miRNA processing
genes drosha/RNASEN, xpo-5, dicer1 and eif2c2 (Ago-2). It was assumed, that while
penetrating into cells, RNase A or its proteolytic fragments, may act as a transcription
factor like Angiogenin promoting miRNA overexpression. In bloodstream RNase A
may cleave circulating non-coding RNAs such as tRNAs, snoRNAs, rRNAs and
snRNAs with generation of short RNAs that can compete with miRNAs for binging
with Ago-2 and displace them thus facilitating further miRNA degradation by RNase
A. Thus tumoricidal activity of RNase A is explained by the change of miRNA
profiles in tumor tissue and bloodstream and switch off the miRNA signature from
malignant to more normal promoting the attenuation of tumor malignancy.
This work was supported by RAS programs “Molecular and cellular biology” and
“Fundamental sciences to medicine 2012-24”, Scientific schools №2972.2012.4,
Fellowship of the President of Russian Federation for young scientists 2307.2012.4 and
by RFBR grant 11-04-01012.
37
Ku80 interaction with apurinic/apyrimidinic sites depends on the
structure of DNA ends
A.A. Kosova1,2, S.N. Khodyreva1,2, O.I. Lavrik1,2
1Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, 630090, Russia
2Novosibirsk State University, Novosibirsk, 630090, Russia
kosova.anastasiya@gmail.com
Apurinic/apyrimidinic (AP) sites are among the most frequent DNA damages. Of
particular interest is repair of AP sites in clustered DNA damages presented by
combinations of AP sites, oxidized bases and single strand breaks within 1-2 turns
of DNA helix. Such lesions arise in DNA under the action of ionizing radiation or
radiomimetic drugs. The aim of this study was to identify the human cell extract
protein specifically interacting with (AP) site in partial DNA duplex containing
5′- and 3′-dangling ends and mimicking clustered DNA damage. Methods. The
Schiff-base dependent cross-linking of proteins to AP DNA (borohydride
trapping) in combination with gel electrophoresis and MALDI-TOF-MS was used
to identify the protein. Chromatography was used to enrich the cell extract in
target protein and to purify the protein. Results. For identification of the protein
we used the fraction of HEK 293 cell extract proteins eluted from heparin-
sepharose. The human cell extract protein which forms a major covalent adduct
with an apparent molecular mass of 100 kDa with AP DNA duplex with dangling
ends was identified as Ku80 subunit of Ku antigen by peptide mass mapping
based on MALDI-TOF-MS data. However, if AP DNA duplex has blunt ends, the
predominant product of cross-linking has an apparent molecular mass of 90 kDa.
The protein forming this adduct was previously identified in our laboratory as
Ku80. Appearance of Ku80 adduct with lowered electrophoretic mobility
characteristic for AP DNA with dangling ends can reflect existence of two
different modes of Ku80 binding with DNA or highly efficient cross-linking of
DNA with another Ku80 isoform. To discriminate these alternatives, we purified
Ku antigen from HeLa cells near to homogeneity. Purified Ku antigen was shown
to form the covalent adducts with the same mobility as observed in cell extracts.
Conclusions. Ku80 subunit of Ku antigen can specifically interact with AP DNA
forming the Schiff base mediated adducts, which electrophoretic mobility depends
on the structure of DNA ends. The difference in electrophoretic mobility can be
caused by cross-linking of AP DNA to distinct target amino acids that appears to
reflect unequal positioning of AP DNAs in the complex with Ku antigen.
This work was supported by RFBR, project 13-04-01426, GDRI program, the
RAS program “Molecular and Cellular Biology”, and the Ministry of Education
and Science of Russia, project 14.B37.21.0188.
38
Structure and function of oncogene-transformed immortal cells
1Kavsan V., 2Кulagova Т., 3Кuznetsova Т., 2Semenkova G., 1Stepanenko A.,
4Vassetzky Y.
1State Key Laboratory of Molecular and Cellular Biology,
Institute of Molecular Biology and Genetics,
150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680
2Belorussian State University,
Minsk, Belorussia
3N.I. Pirogov Russian National Research Medical University,
Moscow, Russia
4CNRS UMR8126, Institut de Cancérologie Gustave Roussy,
Villejuif, France
Vadym06@yahoo.com
Previously we have characterized the new oncogene CHI3L1, overexpressed in
glioblastoma, and obtained malignant 293_CHI3L1 cells, stably producing
CHI3L1 angiogenic oncoprotein. 293_CHI3L1 cells proliferated faster and
acquired higher ability to anchorage independent growth. Here we report the
atomic force microscopy and functional characteristics of these cells. The
constitutive CHI3L1 expression leads to the increased resistance to the damages
by oxidative substances and promotes chromosome instability in 293 cells.
According to the data and last clinical investigations, anti-cancer therapy should
be aimed not to the individual genes, but to the physiological effect they caused.
We propose complex treatment of gliomas including multi-target inhibitors,
which can be delivered to the brain tumor by specific nanoparticle vector.
39
Ca/calmodulin-dependent phosphorylation of endocytic scaffold
ITSN1
D. Ye. Morderer, O. V. Nikolaienko, I. Ya. Skrypkina, O. V. Rymarenko, S. V.
Kropyvko, L. O. Tsyba, A. V. Rynditch.
State Key Laboratory of Molecular and Cellular Biology,
Institute of Molecular Biology and Genetics NAS of Ukraine.
150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680
dmytromorderer@gmail.com
ITSN1 is an endocytic scaffold protein with a prominent function in synaptic
transmission. It is known that Ca signaling is crucial for regulation of functioning
of synaptic proteins. The aim of this work was to test the possibility of
Ca/calmodulin-dependent phosphorylation of ITSN1. Methods. Affinity
chromatography, in vitro kinase reaction, Western blotting, gel staining with
fluorescent stains. Results. We show that the fraction of calmodulin-binding
proteins is able to phosphorylate recombinant fragments encoding the coiled-coil
region and SH3 domain-containing region of ITSN1 in the presence of Ca ions
and calmodulin. Conclusions. The coiled-coil region and SH3 domain-containing
region of ITSN1 undergo Ca/calmodulin-dependent phosphorylation in vitro,
suggesting possible regulation of ITSN1 by Ca signaling.
This work was partially supported by
Programme of Joint European Scientific Associations of NAS of Ukraine (Project
“Human pathology, from the molecular to the cellular level”, F1-2013) and
State Fund for Fundamental Researches (Project F33.4/001).
40
Coculture of spheroids of human fibroblasts and HeLa cells to study
tumor-stroma interaction
Shkarina K. A., Cherednyk O. V., Tykhonkova I. O., Khoruzhenko A. I.
State Key Laboratory of Molecular and Cellular Biology
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680
i.o.tykhonkova@imbg.org.ua
Background. In vivo the cancer develops in the complex microenvironment
major cellular component of which are stoma fibroblasts. The question about
relationship between cancer and stroma cells requires additional comprehensive
analysis. Earlier it was shown that fibroblasts are capable to inhibit the growth
and proliferation of tumor cells in the early stages of oncogenesis, whereas in the
later stages activated tumor -associated fibroblasts, demonstrate the ability to
stimulate proliferation, invasion and angiogenesis. Three-dimensional culture is
promising way of modeling the different stages of carcinogenesis in vitro. This
approach allows to reproduce some morphological and molecular characteristics
of initial tissue more precisely than traditional monolayer culture. Aim. To
develop a model to study the interaction between tumor and stromal cells in three-
dimensional culture.Methods. Cultivation of HeLa cell lines and human dermal
fibroblasts in monolayer and three-dimensional culture, immunofluorescent and
immunohistochemical analysis. Results. In this work we present an approach
based on a direct interaction between the cells of multicellular tumor spheroids
and spheroids of fibroblasts. Subsequent immunofluorescence analysis allows to
determine an origin of cells in the area of their contact.
Conclusions. This model will be useful to study the basic mechanisms of
carcinogenesis, and to find targets for anticancer therapy.
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/HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. Stvoreni PDF dokumenti mogu se otvoriti Acrobat i Adobe Reader 5.0 i kasnijim verzijama.)
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/NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.)
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/ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.)
>>
/Namespace [
(Adobe)
(Common)
(1.0)
]
/OtherNamespaces [
<<
/AsReaderSpreads false
/CropImagesToFrames true
/ErrorControl /WarnAndContinue
/FlattenerIgnoreSpreadOverrides false
/IncludeGuidesGrids false
/IncludeNonPrinting false
/IncludeSlug false
/Namespace [
(Adobe)
(InDesign)
(4.0)
]
/OmitPlacedBitmaps false
/OmitPlacedEPS false
/OmitPlacedPDF false
/SimulateOverprint /Legacy
>>
<<
/AddBleedMarks false
/AddColorBars false
/AddCropMarks false
/AddPageInfo false
/AddRegMarks false
/ConvertColors /ConvertToCMYK
/DestinationProfileName ()
/DestinationProfileSelector /DocumentCMYK
/Downsample16BitImages true
/FlattenerPreset <<
/PresetSelector /MediumResolution
>>
/FormElements false
/GenerateStructure false
/IncludeBookmarks false
/IncludeHyperlinks false
/IncludeInteractive false
/IncludeLayers false
/IncludeProfiles false
/MultimediaHandling /UseObjectSettings
/Namespace [
(Adobe)
(CreativeSuite)
(2.0)
]
/PDFXOutputIntentProfileSelector /DocumentCMYK
/PreserveEditing true
/UntaggedCMYKHandling /LeaveUntagged
/UntaggedRGBHandling /UseDocumentProfile
/UseDocumentBleed false
>>
]
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice
|