Abstracts
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| Дата: | 2014 |
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Інститут молекулярної біології і генетики НАН України
2014
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irk-123456789-1546082019-06-16T01:31:07Z Abstracts 2014 Article Abstracts / Вiopolymers and Cell. — 2014. — Т. 30, № 5, доп. — С. 5-44. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/154608 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Інститут молекулярної біології і генетики НАН України |
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Abstracts / Вiopolymers and Cell. — 2014. — Т. 30, № 5, доп. — С. 5-44. — англ. |
| series |
Вiopolymers and Cell |
| first_indexed |
2025-07-14T06:39:23Z |
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2025-07-14T06:39:23Z |
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1837603398134792192 |
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The role of TFF3 in the development of thyroid cancer
1,2Ābols A., 1Ducena K., 1,2 Andrejeva D., 1,2Endzeliņš E., 3Vilmanis J., 3Narbuts
Z., 4Tārs J., 4Eglītis J., 1,3Pīrāgs V. and 2Linē Aija
1 University of Latvia;
2 Latvian Biomedical Research and Study center;
3 Pauls Stradins University Hospital;
4 Latvian Oncology Center, Riga Eastern Clinical University Hospital
arturs@biomed.lu.lv
Aim: Trefoil factor 3 (TFF3) is overexpressed in a variety of solid epithelial cancers
including breast cancer where it has been shown to promote migration, invasion,
proliferation, survival and angiogenesis, whereas it is downregulated in the majority of
thyroid tumors derived from follicular cells. However, its role in the development of
thyroid cancer remains unknown. The purpose of the present study was to elucidate the
regulation of TFF3 expression by estrogen signaling and its functional role in
development of thyroid cancer. Methods: TFF3 expression was analyzed by
immunohistochemistry using a tissue array comprising 72 specimens representing various
benign, inflammatory and malignant thyroid diseases and normal tissues. The effects of
estradiol stimulation on TFF3 expression were studied in thyroid and breast cancer cells
and by meta-analysis of gene expression datasets. The functional effects of forced TFF3
expression in anaplastic thyroid and breast cancer cell lines were analyzed by assays for
cell proliferation, invasion and gene expression profiling. Results: TFF3 exhibited strong
cytoplasmic staining of thyroid follicular cells and colloid that was increased in
hyperfunctioning thyroid nodules while its expression was decreased in all thyroid cancers
of follicular cell origin. In thyroid cancer cells, on the contrary to the breast cancer, the
expression of TFF3 was downregulated by estrogen signaling and forced expression of
TFF3 resulted in decreased cell proliferation and entry into the S phase. Furthermore, it
induced changes in the cell morphology and expression of the functional differentiation
markers of thyroid follicular cells, such as TPO, TSHR, THRB, SLC26A4 and SLC5A5,
epithelial marker CDH1, and transcription factors implicated in the thyroid
morphogenesis and function FOXE1 and WWTR1. Conclusions: In thyroid cancer, TFF3
expression is decreased by estrogen signaling and its restoration results in the decreased
cell proliferation, and induces the differentiation towards the follicular cell phenotype thus
suggesting that TFF3 may act as a tumor suppressor or an oncogene depending on the
cellular context.
4
CHI3L1 AND TSC22 proteins involvoment in cellular response to
extracellular stress
Bukreieva T. V. and Kavsan V. M.
Institute of Molecular Biology and Genetics of NAS of Ukraine, Kyiv, Ukraine
t.v.bukreieva @ imbg.org.ua
Purpose. High intratumoral heterogeneity and significant chemotherapy resistance to
different agents are distinctive features of glioblastoma, the most aggressive tumor among
all central nervous system neoplasia in adults. Low efficiency of anticancer therapies
using antioxidants to prevent the progress of tumor could be associated with a higher
resistance of malignantly transformed cells to reactive oxygen species. Chitinase-3-like 1
(CHI3L1) gene is extremely overexpressed in glioblastoma cells, while TSC22 gene
encoding the TSC22 protein (transforming growth factor-beta 1 [TGF-β1]-stimulated
clone 22 domain family, member 1) is significantly down-regulated in glioblastoma as
compared to normal human brain. The main purpose of this work was to determine the
contribution of CHI3L1 oncogene and TSC22 tumor suppressor gene to the processes of
survival and apoptosis in human glioblastoma U87 cells under oxidative stress. Methods
and results. We investigated the sensitivity of CHI3L1-producing cells U87 to reactive
oxygen species and identified capabilities of tumor suppressor protein TSC22 to reduce
the viability and to recover the sensitivity of these cells to the action of oxidants. Thus,
U87 cells transfected with a construct containing full-length TSC22 cDNA and treated for
24 h with hydrogen peroxide in a concentration range of 0.05-5 mM showed a significant
reduction of cell viability by 16 % with 0.5 mM hydrogen peroxide according to the MTT
test results, whereas cells without transfection of TSC22 did not undergo cytotoxic effect
within the same concentration of H2O2. The number of metabolically active U87 cells
increased exponentially for 7 days, but the stable production of TSC22 suppressor protein
led to 2.6-fold reduction in number of metabolically active cells during the same period.
In the presence of TSC22 protein, it was observed the increased phosphorylation of p53
protein and reduction of CHI3L1 protein production as it was shown by Western blot
analysis. To elucidate if TSC22 as transcription repressor could regulate the transcription
of CHI3L1 we performed reverse transcription-PCR analysis with primers to CHI3L1
coding sequence, however, overexpression of TSC22 did not inhibit CHI3L1 transcription
in U87 cells. Conclusions. Overproduction of tumor suppressor TSC22 protein inhibits
viability and restores sensitivity of U87 cells to the action of reactive oxygen species,
supposing a new oncogene-dependent mechanism of chemotherapy resistance in
glioblastoma. The presence of TSC22 led to increased p53 phosphorylation and caused
down-regulation of CHI3L1 content in U87 cells.
5
Acceleration of the peroxynitrite decomposition rate, DNA binding and
oxidative stress reduction by 1,4-dihidropiridine derivatives
1,2Buraka Elina, 2Rostoka Evita, 2Duburs Gunars, 1,2Sjakste Nikolajs
1Faculty of Medicine, University of Latvia, Riga, Latvia
2Latvian Institute of Organic Synthesis, Riga, Latvia.
misselina23@gmail.com
Aim. Peroxinitrite is a powerful oxidant exhibiting a wide array of cell damaging effects.
It can cause strand breaks in DNA, introducing base pairs and sugar-phosphate backbone
oxidative modifications. In order to evaluate ability of 1,4-DHP to protect cells and DNA
against peroxynitrite-produced damage the peroxynitrite decomposition rate was
determined in presence of different 1,4-DHP derivatives. The same group of compounds
was tested for ability to protect the cell DNA and to interact with the DNA molecule.
Methods. The peroxynitrite decomposition rate in the presence of different 1,4-DHP had
been studied by means of UV-VIS spectroscopy using the kinetics analysis. The
peroxynitrite decomposition was detected measuring the absorption changes at 302 nm.
Several 1,4-DHP derivatives: AV-153-Na and its analogues, J-8-120 and its analogues,
PP-544- NH4 ,cerebrocrast, etaftorone un fenotorone were tested for ability to accelerate
the peroxynitrite decomposition. Ability of the above compounds to interact with DNA
was determined by measuring fluorescence intensity of the compounds in presence of
increasing concentrations of DNA. Ability of the compounds to protect the HeLa cell
DNA against peroxynitrite-induced DNA single-strand breaks was analysed by means of
single-cell gel electrophoresis in alkaline conditions. Results. Results of the kinetics
analysis did not reveal any influence on peroxynitrite decomposition rate by AV-153-Na,
J-8-120 and PP-544-NH4. Fenoftorone and etaftorone accelerated the decomposition
reaction three times, cerebrocrast – 6 times.AV-153, etaftorone and cerebrocrast can
interact with DNA molecule. Intensity of AV-153-Na fluorescence (λex = 350, λem =
480) increased in presence of increasing DNA concentrations. The binding constant (K) of
AV-153-Na is equal to 6,4 x 103 M-1. The inverse effect was noticed in case of
cerebrocrast (λex = 370, λem = 440), the fluorescence was quenched with increasing the
DNA concentrations. The Stern-Volmer quenching constant of cerebrokrast (Ksv) is equal
to 2,2 x 103 M-1.The single-cell gel electrophoresis results showed, that cerebrocrast and
etaftorone could significantly protect HeLa cells preincubated with these compounds
against peroxynitrite-induced DNA damage. AV-153 produced a similar effect.
Conclusion. Cerebrocrast ,etaftorone, fenoftorone react with peroxynitrite in vitro. 2. AV-
153-Na, cerebrocrasts and etaftorone protect the DNA against peroxynitrite-induced
damage in living cells. 3. Cerebrocrast and AV-153-Na can interact with DNA.
6
Ubiquitin, SUMO, the nuclear pore complex and the DNA damage response
Niño Carlos, Guet David, Hayakawa Akira, Babour Anna, Dargemont Catherine
INSERM U944, CNRS UMR9212, Paris Diderot University, France
catherine.dargemont@inserm.fr
Aim. The Nuclear Pore Complex, a gigantic machine of the nuclear envelope recently
emerged as a “hub” coordinating nuclear transport, gene expression, chromatin
organization and genome integrity. In this context, persistent double-strand breaks,
arrested replication forks and eroded telomeres have been shown to relocate to the yeast
Nuclear Pore Complex (NPC) and some NPCs proteins even influence DNA repair both
in yeast and mammalian cells. However the limited number of NPC per cell imposes a
certain hierarchy of these functions in time and space. Our hypothesis is that dynamic
post-translational modifications control the plasticity of the NPC structural organization
and functions.
Results. Our recent systematic analysis of the ubiquitylation of yeast nuclear pore
complex proteins (Nups) revealed that more than 50 % of them are ubiquitylated, mostly
by mono-ubiquitylation suggesting a non-degradative role of this post-translational
modification. In addition we found that Nups ubiquitylation involves different
components of the ubiquitin conjugation machinery. A detailed analysis of Nup159 mono-
ubiquitylation, a Nup exclusively localized on the cytoplasmic face of the NPC, revealed a
unexpected function in nuclear segregation at the onset of mitosis.
More recently, we focused our attention on modifications of the components of nuclear
basket substructure of the NPC and found that these Nups are extensively modified by
ubiquitylation, sumoylation and phosphorylation. We precisely analyzed the dynamics
and interplay of these modifications during cell cycle progression, their functions in
controlling the plasticity of the nuclear basket as well as the efficiency of the DNA
damage response.
This work is supported by the Ligue Nationale contre le Cancer (Equipe labellisée), the
WhoamI? laboratory of excellence (ANR-11-LABX-0071 to C.D.) funded by the
‘Investments for the Future’ program (ANR-11-IDEX-0005-01), the Fondation pour la
Recherche Medicale.
7
Parameters of nitric oxide metabolism and DNA integrity in patients with
type 1 diabetes mellitus
1Dekante Alise, 1,2Rostoka Evita, 1,2Sokolovska Jelizaveta, 2Baumane Larisa,
1,2Sjakste Nikolajs
1University of Latvia, Faculty of Medicine, Riga, Latvia.
2Latvian Institute of Organic Synthesis, Riga, Latvia.
alise.dekante@gmail.com
Objectives: Hyperglycemia in diabetes mellitus type 1 (DMT1) increases nitric oxide
(NO) and superoxide anion (O2
- ) production resulting in peroxynitrite formation, which
leads to increased DNA damage linked to formation of late -onset complications of
DMT1. Our goal was to determine the parameters of NO metabolism and DNA integrity
in patients with DMT1.
Methods: 88 patients with DMT1 (67 % of them normoalbuminuric, 16 %
microalbuminuric, 17 % macroalbuminuric and with end stage renal disease) and 44
healthy controls matched for age, sex and BMI were involved. Anamnesis and other data
was collected through a questionnaire, blood biochemical analysis was performed in a
clinical laboratory. NO production in blood was detected by the electron paramagnetic
resonance spectroscopy, nitrite and nitrate (NO2
-/NO3
-) concentrations in serum and urine
with the Griess reaction and DNA damage in nucleated blood cells by the alkaline single
cell gel electrophoresis.
Results: NO and DNA damage were significantly higher in DMT1 patients (p < 0.0001),
while NO2
-/NO3
- in serum (p < 0.007) and urine (p < 0.04) were lower compared to the
controls. In patients, NO was inversely proportional to NO2
-/NO3
- in serum (p < 0.04) and
urine (p = 0.05), thus, suggesting NO synthase-independent NO production.
In patients, NO2
-/NO3
- in serum were directly proportional (p < 0.0001) to the stages of
diabetic nephropathy, whilst inversely proportional to glomerular filtration rate (GFR) (p
< 0.001), suggesting decreased output in case of a low GFR. NO2
-/NO3
- in urine were
inversely proportional (p < 0.02) to the stages of diabetic nephropathy, but directly
proportional to glomerular filtration rate (GFR) (p < 0.0001), suggesting decreased output
in case of a low GFR.
We have also revealed a direct correlation between NO and DNA break levels (if
compared to the average of controls) with HbA1c (p < 0,04) and the duration of the illness
(p < 0,05). In comparison to the average level of controls NO and DNA break levels were
high in 74.14 % of patients and only in 16.28 % of controls. But in 26 % of patients NO
was inversely correlated to the DNA break levels (p < 0,0001), thereby NO and DNA
breaks are probably adjacent factors, which are characteristic of DMT1.
Conclusions: Higher levels of NO production and DNA breaks in blood cells are
characteristic of DMT1. These parameters might be used as therapeutic targets for
pathogenetic therapy of DMT1. In future these parameters might be used as therapeutic
targets for pathogenetic therapy of DMT1.
The study was funded by the ESF 1DP/1.1.1.2./13/APIA/VIAA/002 and LSC grant
278/2014.
8
The cooperativity and fractality in the functional genome organization
Ērenpreisa Jekaterina
Latvian Biomedicine Research&Study Centre, Riga, Latvia
katrina@biomed.lu.lv
One of the most intriguing and still unresolved questions of gene regulation is how the
genome coordinates all genes for development and tissue differentiation. While the data
from studies of the genome organisation provide some hints on the hierarchial
cooperativity, starting with Rabl orientation and ending with loop-domains, the gene-
based data were hitherto exlusively elementaristic. However, in recent time, there
appeared the publications (Masa Tsuchyia et al., 2010 and other) based on the whole
transcriptome analysis which revealed on the neutrophil differentiation models the fractal-
like gene ensembles acting as vehicles for differentiation attractor deciding cell fate. It
will be discussed in presentation how it may fit with the known data on the structural and
dynamic genome organisation.
Evaluation of the PSMB5 (rs11543947) and PSMA3 (rs2348071) gene
polymorphisms on the association with multiple sclerosis in Latvians
1Kalnina Jolanta, 1Paramonova Natalia, 2Sjakste Nikolajs, 1Sjakste Tatjana
1Genomics and Bioinformatics, Institute of Biology of the University of Latvia,
Salaspils, Latvia
2Latvian Institute of Organic Synthesis, Riga, Latvia
tanja@email.lubi.edu.lv
Aim: Functional realization of many signalling proteins and transcription factors
implicated in development and progression of multiple sclerosis are mediated by
proteasomes. This case-control study aimed to evaluate genetic variations in the PSMB5
and PSMA3 genes encoding proteasomal subunits on susceptibility to multiple sclerosis in
Latvians.
Methods: The rs11543947 (PSMB5) and rs2348071 (PSMA3) polymorphisms were
genotyped in 291 multiple sclerosis patients and 305 healthy individuals and analysed on
general, subtype and sex-specific associations with the disease.
Results: Loci rs11543947 and rs2348071 were identified as disease neutral and
susceptible respectively. The rs2348071 heterozygous genotype GA showed strong main
effect (P < 0.001; OR=1.891, 95% CI [1.360 − 2.628]), and moderate (P < 0.01;
OR=1.663, 95% CI [1.152 − 2.402]) and strong (P < 0.001; OR=2.459, 95% CI [1.534 –
3.943]) association with relapsing-remitting and secondary progressive phases of disease
respectively. No genotype-sex interaction associated with multiple sclerosis has been
detected.
Conclusions: Our results suggest susceptibility of the rs2348071 heterozygous genotype
to multiple sclerosis in Latvians.
9
Interaction of BER factors with pleiotropic protein YB-1 in oxidative
clustered DNA lesions repair
1Fomina Elizaveta E., 1Pestryakov Pavel E., 2,3Kretov Dmitry A.,
3Curmi Patrick A., 1,4,5Lavrik Olga I.
1Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk,
Russia
2Institute of Protein Research, RAS, Pushchino, Moscow region, Russia
3INSERM/UMR829; Université Evry-Val d'Essonne, Evry, France
4Novosibirsk State University, Novosibirsk, Russia
5Altai State University, Barnaul, Russia
lisenok.istreb@gmail.com
Aim. Single oxidative DNA lesions (such as abasic sites, single-stranded DNA breaks,
oxidized bases) and their combinations that are referred to as oxidative clustered DNA
lesions – OCDLs are generally corrected by base excision repair pathway (BER). During
BER, the damaged DNA strand is cleaved by AP endonuclease APE1 or bifunctional
DNA glycosylases like NEIL1 that results in nicked intermediate. Due to this fact, repair
of abasic sites located within bistranded OCDLs or single-stranded DNA regions is
closely connected with the risk of high cytotoxic DNA double strand breaks formation.
Pleiotropic protein YB-1 is extensively studied in the context of cellular response to
genotoxic stress, but its unmediated role in DNA repair is still open to question. The aim
of present research was to investigate multifunctional protein YB-1 influence on repair of
abasic sites located within oxidative clustered DNA lesions (OCDLs) or single-stranded
DNA fragments. Methods. In present work bistranded OCDLs in DNA were imitated by
combination of abasic site and 5-formyluracil (potential product of thymine oxidation) in
opposite DNA strand. YB-1 interaction with oxidative DNA lesions was determined by
NaBH4-mediated crosslinking. YB-1 influence on major components of BER machinery
was performed by functional studies and gel retardation assays. Posttranslational
modification of YB-1 was analyzed by Western-blot analysis and Coomassie staining.
Results. The data obtained demonstrated that YB-1 directly interacts with abasic site and
5-formyluracil in DNA via intermediate Schiff base formation. YB-1 was shown to
promote APE1- and NEIL1-depending cleavage of either single abasic sites or AP sites
within OCDLs. YB-1 influence on APE1 occurs during substrate-binding step. Presence
of high YB-1 concentrations resulted in blocking APE1-depending pathway via inhibition
of APE1 activity as well as polβ dRP lyase activity. With AP sites being in the single-
stranded DNA regions, YB-1 inhibited both APE1 and NEIL1 activities. In vitro studies
revealed a possibility of a novel posttranslational modification of YB-1 that could
influence on its interaction with DNA. Conclusions. Pleiotropic protein YB-1 directly
interacts with AP sites and 5-formyluracil within DNA and possesses the capability of
modulating of APE1- and NEIL1-depending AP site cleavage. YB-1 is able to go through
posttranslational modification that can affect the role of YB-1 in DNA repair.
Footnotes. This work was supported by grants from Russian Foundation for Basic
Research (14-04-00268, 12-04-00337), RSCF grant no. 14-14-00501, MCB program,
Ministry of Science and Education and by Scientific School – 420.2014.4.
10
New active compounds derived from molecular diversity.
Isber H., Shahsavarian M., Loussouarn C., Padiolleau S., Avalle B.
& Friboulet A.
Genie Enzymatique et Cellulaire – CNRS Universite de Technologie de Compiegne -
Rue
Roger Couttolenc - CS 60319 - 60203 Compiegne Cedex - France
alain.friboulet@utc.fr
Molecular diversity corresponds to the set of molecules that can be found in various
biological objects or artificially generated. From this diversity can be extracted
compounds able at regulating biological functions. In applied research, drug discovery
more and more involves the use of molecular diversity for screening against targets.
Selection of regulators able to bind targets of all types generally follows iterative
processes leading to the identification of molecules (peptides, nucleic acids…) from a
large randomized pool (library). Among them: *Phage display using filamentous
bacteriophages represents one of the most successful in vitro ligand selection technology.
It is an in vitro process starting from a library of peptides or antibodies. A pool of
degenerated oligonucleotides comprising the required diversity is cloned in fusion with a
coat protein of the phage. In the resulting library, each phage displays a unique random
peptide or antibody. This maintains a physical link between genotype and phenotype:
analysis of selected sequences can thus be performed by DNA sequencing. Each round of
selection is ended by phagepeptide amplification using bacterial infection. In literature,
phage display technology has been wealthily described with versatile applications [1].
*SELEX (Systematic Evolution of Ligands by Exponential enrichment) starts from a
library of synthetic nucleic acids (DNA or RNA) [2] . In this case, the diversity of the
library is not only due to the sequence variability of single-stranded oligonucleotides but
also to the three-dimensional structures they may adopt (loops, hairpins, stems...).SELEX
method relies both on chemistry for library synthesis and nucleic acids modifications and
on molecular biology techniques for the roundamplification of selected entities. It has
been widely used to identify high affinity aptamers against various targets (proteins,
dyes…) [3].
References:
1 Bratkovic, T. (2010) Progress in phage display : evolution of the technique and its
application. Cell. Mol. Life Sci. 67, 749-767.
2 Tuerk C. & Gold L. (1990) Systematic evolution of ligands by exponential enrichment:
RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510.
3 Stoltenburg R., Reinemann C. & Strehlitz B. (2007) SELEX--A (r)evolutionary method
to generate highaffinity nucleic acid ligands Biomol. Eng. 24, 381-403.
11
A RecG helicase involved in the maintenance of the plant organellar
genomes.
Wallet Clementine, Le Ret Monique, Dietrich André and Gualberto José M.
UPR2357 CNRS, Institut de Biologie Moléculaire des Plantes, Strasbourg, France
jose.gualberto@ibmp-cnrs.unistra.fr
Aim. The dynamic state of the plant mtDNA is due to recombination processes involving
repeated sequences that modulate its structure. Large repeats are interconvertible by
homologous recombination (HR) and contribute to the multipartite structure of mtDNA,
while rare ectopic recombination occurs among small repeats and generate alternative
mtDNA configurations at substoechiometric levels, which contribute to the rapid
evolution of the mtDNA structure in plants. In addition, both the mtDNA and the cpDNA
are believed to replicate by recombination-dependent pathways reminiscent of the
mechanisms of replication of certain bacteriophages. HR dependent processes have also a
predominant role in the repair of the plant organellar genomes. Our aim is to identify the
factors implicated in the control of organellar HR or in mechanisms of HR-dependent
repair. Among those, DNA helicases should have a predominant role in recombination
surveillance, and are postulated to be required for rejection of ectopic HR intermediates.
Results. We identified an Arabidopsis DNA helicase homologous to the bacterial DNA
helicase RecG, that we called RECG1. Bacterial RecG acts as a translocase in DNA
recombination, repair and replication. It promotes holliday junctions translocation, the
regression of replication forks and avoiding replication re-initiation when replication forks
collide. RECG1 is found in all plant species, including algae and mosses, but not in
animals or fungi. Transgenic Arabidopsis plants expressing RecG1:GFP fusion showed
that RECG1 is dually targeted to mitochondria and plastids, suggesting that assumes
similar functions in both organelles. Promoter-GUS fusion showed that the gene is
expressed in most plant tissues, in particular in leafs and root vascular systems. The
Arabidopsis RECG1 is able to complement its bacterial orthologue for the repair of UV-
induced DNA repair, but not the bacterial DNA translocase RuvAB. Using T-DNA
insertion mutants we tested the roles of RECG1 in the surveillance of ectopic HR and the
repair of DNA damage induced by genotoxic agents. We found that RECG1 affects the
efficiency of mtDNA repair by recombination. In addition, loss of RECG1 results in
increased ectopic HR of the mtDNA having as consequence specific changes in the
segregation of mtDNA sequences. Surprisingly no effects were observed in cpDNA
replication and segregation, suggesting that compensatory mechanisms exist in
chloroplasts. Plants deficient in RECG1 expression have no remarkable growth
phenotypes, but double mutants of RECG1 and other factors involved in mtDNA repair by
recombination and/or recombination surveillance display exacerbated phenotypes of leaf
distortion and variegation. Analysis of the relative copy numbers of the organellar
genomes sequences by qPCR show that these phenotypes apparently result from
instability of the mtDNA by recombination, resulting in abnormal segregation of mtDNA
subgenomes. Conclusions. Our results show that RECG1 is required for recombination
surveillance, suppressing ectopic HR involving repeated sequences that compromise the
stability and stoichiometric segregation of the plant mtDNA.
This work is supported by funding from the projet d’avenir Labex MitoCross
12
Long chemically modified siRNA provide an efficient and specific gene
silencing
Gvozdeva Olga V., Dovydenko IIya S., Venyaminova Alya G., Zenkova Marina A.,
Vlassov Valentin V., Chernolovskaya Elena L.
Institute of Chemical Biology and Fundamental Medicine SB RAS,
Novosibirsk, Russia,
elena_ch@niboch.nsc.ru
RNA interference (RNAi) is an evolutionarily conserved cellular mechanism of sequence-
specific gene silencing mediated by diverse classes of double-stranded RNAs. Because of
the outstanding potency and specificity in comparison with other loss-of-function
technologies, RNAi has quickly developed into a potent biological tool for the specific
inhibition of gene expression. Synthetic 21 bp RNA duplexes can be introduced into cells
and will function of mimics of the natural siRNAs that result from Dicer processing.
Recent reports have proven that longer duplexes (25-30 bp in length) are processed by
Dicer into 21 bp siRNAs and loaded into RISC more efficiently than canonical siRNAs
and demonstrate better gene silencing. However, it has been shown, that longer synthetic
RNA duplexes could trigger an innate immune response. The chemical modifications
known to prevent the recognition of longer RNA duplexes by the innate immune system
without influencing silencing activity have been assumed. These chemical modifications
can block the immune response and we can suppose that theoretically nothing prevents the
use of partly modified dsRNAs of any length. In our study, we used selectively modified
RNA duplexes 21 bp, 42 bp and 63 bp in length targeted MDR1 mRNA and examined
their ability to silence the expression of the target gene and the specificity of their action.
These siRNAs were found to silence the expression of P-glycoprotein and restore the
sensitivity of drug-resistant cancer cells to vinblastine more efficiently that canonical
siRNA with the same sequence. Our data revealed that 2′-OMe modification of CpA, UpA
and UpG motives in 42 bp and 63 bp siRNAs effectively prevents activation of the innate
immunity response and did not change the expression levels of two key interferon
response genes, PKR and β-actin, in KB-8-5 cells. To our surprise, no substantial
processing of dsRNAs by Dicer was detected inside the cells. Our results remove the
length limits for the design of RNAi effectors, and add another example in the list of
novel RNAi-inducing molecules differing from the classical siRNA. For these reasons, we
believe that these structures have great potential for the development of efficient
therapeutics.
This research was supported by the Russian Academy of Science under the program
“Molecular and Cell Biology” (no. 22-1), and “Science to Medicine” (no. 44), the
Russian Foundation for Basic Research (no. 14-04-00869-a), SB RAS (no. 85).
13
Etoposide induced dual expression of opposite regulators of self- renewal and
senecence OCT4/P21CIP1 in embryonal carcinoma cells PA1 is modulated
by the serum concentration changes
1Huna Anda, 1Salmina Kristine, 1Krigerts Jekabs, 2Dekthjar Yuri
1Latvian Biomedical Research& Studies Centre, Riga, Latvia
2Riga Technical University, Institute of Biomedical Engineering and
Microtechnologies, Riga, Latvia
anda.huna@gmail.com
Tumour cellular senescence induced by genotoxic treatments was typically considered a
terminal cell fate, however recently it was shown to be to some extent reversible, while
the mechanisms of reversion, in particular in cancer stem cells are unclear [1]. Previously
we found on embryonal teratocarcinoma PA1 serving as a model of cancer stem cells that
the induced by etoposide (ETO) treatment senescence was intimately associated with self-
renewal. We revealed that during the 2-3 days long G2-arrest most cells simultaneously
express in their nuclei the opposite regulators – self-renewal master transcription factor
OCT4A and inhibitor of cell cycle and mediator of senescence P21CIP1. Both depend on
activation of TP53, while its silencing caused divergence for irreversible senescence and
premature aberrant mitoses [2]. Here we report on further exploration of the character of
this duality by studies on the influence of serum concentration in the time course. The
expression of opposite regulators was detected in individual cells by the two-channel
immunofluorescence measurements using flow and image cytometry. Distributions were
characterized by geometrical parameters of cell coordinates on dot plot, showing
p21CIP1/OCT4A expression. In non-treated cells, the two-dimensional distribution of the
intensity of OCT4A and background p21CIP1 staining in individual cells forms a compact
cluster, while in the ETO-treated cells the contents of both factors is enhanced several-
fold (confirmed by Western blotting) and the variability amplitude increases even more.
Nevertheless on day 2-3 the both p53-dependent regulators are correlating positively
indicating to fluctuations of p53 itself. However, a ”swing” of the regulators increases in
the time course, with the opposing outliers (P21CIP1>>OCT4A vs. OCT4A>>P21CIP1)
appearing upon the time of release from the G2-arrest (days 4-5). This is followed by
death of the majority and further dissociation of the remaining minority for proliferating
and terminally senescing cells. P21CIP1>>OCT4A fraction increased by cell starvation,
while the size of regrowing clones was reduced. On the contrary, increased serum
concentration demonstrated OCT4 prevailing over p21CIP1 and larger recovery clones.
Interestingly, however, serum concentrations did not affect the timing of regrowth (from
day 7). Using the hypothesis of ergodicity, we suggest that high amplitude of variation of
both regulators in individual cells reflects the temporal fluctuations of the steady state in
individual cells characterising it as bi-potential and metastable. This state holds a
possibility to keep the both opposite networks potentially functional, however preventing
for the time being each other from accomplishing their destination. Besides, cell vitality
supported by serum concentration is an important factor supporting this bi-potential state
and tumour cell recovery after treatment with Etoposide.
1. Erenpreisa Je, Cragg M. Three steps to cancer cells immortality: senescence, polyploidy, self-
renewal. Cancer Cell Int.13:92. 2. Jackson TR, Salmina K, Huna A, et al DNA damage causes
TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma
cells.- Cell Cycle 12 (3), 430 – 441.
14
Gene expression signature of glial tumors and participation of oncogene
CHI3L1 in malignant transformation
1Iershov A., 1Osadcha L., 1Dmitrenko V., 2Mekler A., 3Lykhovid A., 3Stetsyuk P.,
1Kavsan V.
1Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine;
2The Bonch-Bruevich Saint-Petersburg State University of Telecommunications, Saint-
Petersburg, Russian Federation 3V.M. Glushkov Institute of cybernetics, NASU, Kyiv;
a.yershov@yahoo.com
Glioblastoma is the most aggressive intracranial malignancy characterized by high invasiveness,
recurrence and poor responce to chemo- and radiotherapy. Heterogenous character of glioblastoma
eliminates the value of single molecular markers. High-throughput gene expression analysis
provides huge amounts of data that could be used to identify characteristic gene expression profiles
(signatures), associated with specific tumor properties. Previously, glioblastomas were divided into
three subtypes that correlated with patient survival or into four subtypes using unsupervised cluster
analysis. However, gene expression signatures alone provide limited possibility to understand the
biology of tumor.. Analysis and integration of various signatures, created using different approaches,
can clarify common pathways involved in tumor development, which is not apparent at the level of
individual genes. We used cluster analysis with k-means approach to search for the differences in
expression of 12480 genes in 224 glioblastomas samples obtained earlier, and found two subtypes.
15 genes were upregulated and 401 genes were downregulated two-fold in the first subtype of
glioblastoma (80 samples) as compared to the second subtype (144 samples). 10 from 15 genes with
increased expression in the first subtype encoded proteins involved in the regulation of cell cycle
and proliferation. Significant part of 401 genes were found to be involved in synaptic transduction,
neurogenesis, formation of myelin sheath, and axon development. Kohonen map, built on the basis
of expression of 15 genes with increased expression in the first subtype, and 60 of 401 genes with
increased expression in the second subtype also showed good separation of two determined subtypes
in same glioblastoma samples. «Mesenchymal» subtype of gliobastoma, described earlier, is
characterized by overexpression of the set of genes, and among them the gene, encoding chitinase-3-
like protein 1 (CHI3L1), which involved in abnormal cell proliferation and tumor angiogenesis.
However, it is not amplified in glioblastomas and mechanisms of its overexpression are unknown.
In our previous work we demonstrated that MYC/MAX complex was important but not crucial for
overexpression of CHI3L1 in glioblastoma as single nucleotide mutation in its promoter region,
which disrupted MYC/MAX binding site, led to decreased CHI3L1 expression. As we revealed,
ectopic overexpression of TP53 in glioblastoma U87 cells led to the decreased CHI3L1-level. This
effect was achieved also either by activation of p53 by antitumor agent resveratrol or by inhibiting
p53 degradation mediated by SIRT1, using its specific inhibitor sirtinol. We showed that CHI3L1
level in glioblastoma cells is p53-dependent and could be decreased by p53 activation. To clarify
structural peculiarities of CHI3L1, we compared 3D structures of CHI3L1 and its closest human
homologue CHI3L2 and revealed the cluster of positively charged amino acids 144-RRDKQH-149,
located on the protein surface. Site-directed mutagenesis of this potential heparin-binding region in
CHI3L1 revealed that residues Arg144, Arg145, and Lys147 are crucial for interaction with heparin.
Soft agar assay demonstrated that mutation in heparin-binding site significantly decreased colony
formation efficiency. Thus, we demonstrated that single mutation in the MYC/MAX binding site in
promoter region of CHI3L1 led to decreased CHI3L1 expression in glioblastoma, in the absence of
it expression of CHI3L1 is more variable. Second, the p53 protein is a negative regulator of CHI3L1
expression in glioblastoma U87 cells. Inactivation of p53 in tumors can lead to the overexpression
of CHI3L1, and consequently, increase CHI3L1-mediated pathological effects, such as abnormal
cell proliferation and angiogenesis. CHI3L1 enhanced malignant properties of 293 cells after its
ectopic expression, disruption of its heparin-binding site decreased the cell ability for substrate-
independent growth in soft agar.
15
Antitumor and antimetastatic effect of short immunostimulating dsRNA
against hepatocellular carcinoma G29 and melanoma B16
Kabilova T.O., 1Nikolin V.P., Zenkova M.A., Vlassov V.V., Chernolovskaya E.L.
Institute of Chemical Biology and Fundamental Medicine, SB RAS, Russia.
1 Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia.
kabilova@niboch.nsc.ru
Recently, we designed a series of short double-stranded RNAs that possess pronounced
antiproliferative activity in cancer cells and did not have substantial homology to any
human or mouse mRNAs. These compounds were shown to induce immunostimulatory
effects in culture of human adherent peripheral blood mononuclear cells (PBMCs) and in
mice. The aim was to evaluate the antitumor and antimethatatic potential of selected 19-bp
RNA duplexes with 3-nucleotid overhangs in the 3’-ends (here and after
immunostimulating RNA, or isRNA). Methods. Antiproliferative activity of isRNA in
vitro was analyzed in KB-3-1 (throat), A-549 (lung) and SK-N-MC (neuroblastoma)
cancer cell lines by MTT test. The percentage of apoptotic and dead cells in the
population was assayed by flow cytometry. Selective knock down of different cellular
sensors of dsRNAs was achieved using lentiviral vectors expressing defined shRNAs. The
transplantable hepatocellular carcinoma (HCC) G-29 tumor in CBA/LacSto (CBA) mice
and melanoma B16 in C57BL/6 mice were used to study the effects of isRNA on tumor
growth and metastases spreading. The levels of IFN-α and cytokines in human PBMC
cultural medium and in mouse serum were measured using ELISA. Results. The results of
in vitro experiments demonstrate that isRNAs under study efficiently inhibit growth of
human cancer cells (KB-3-1, A-549, SK-N-MC), and induce synthesis of IFN-α and pro-
inflammatory cytokines IL-6 and TNF-α in the human PBMCs. The examination of
isRNAs sequence/activity relationships revealed that the introduction of substitutions in
the middle part of the isRNA sequence does not alter the antiproliferative and
immunostimulating activities, while substitutions in the 3’-end region of isRNA
substantially reduce the activities. Our research demonstrated that antiproliferative effects
of isRNAs are related to cell growth arrest, rather than the induction of apoptosis. To
define dsRNA-binding receptors involved in stimulation of innate immunity and
inhibition of cancer cell proliferation by isRNAs, we used we used a set of cell lines with
selectively silenced dsRNA sensors. The data showed that the dsRNA-binding protein
kinase (PKR) is essential for the biological activity of isRNA. The results of in vivo
experiments show that single intravenous injection of isRNA complexed with transfection
reagent efficiently increases the level of IFN-α, and to a lesser degree the level of pro-
inflammatory cytokine IL-6 in mice blood serum. We found that isRNA reduces the
metastases area in the liver, kidneys and heart of CBA/LacSto mice with HCC G-29 and
cause a slight, but reliable inhibition of the primary tumor growth. The results of
experiments with C57BL mice with implanted melanoma B16 show that isRNA
efficiently inhibits tumor growth and metastasis spreading in the lungs. Conclusion. The
obtained results revealed the pronounced immunostimulatory, anti-tumor and
antimethastatic properties of the studied isRNAs. So, this short double stranded RNA can
be considered as potential adjuvant for the therapy of oncological diseases.
This research was supported by the RAS under the programs “Molecular and Cell Biology” (no.
22-1), and “Science to Medicine” (no. 44); RFBR (no. 14-04-00869-a); SB RAS (no. 85), and
President's scholarship for young scientists (RF).
16
Mechanisms of heat stress-induced cell senescence.
1,2Petrova N., 1Velichko A., 1Kantidze O.
1Laboratory of Structural and Functional Organization of Chromosomes, Institute of
Gene Biology RAS, Moscow, Russia.
2Department of Molecular Biology, Moscow State University, Moscow, Russia.
kantidze@gmail.com
Aim: The aim of this study was to investigate delayed effects of heat stress on
mammalian cells.
Methods: Comet assay and modified PFGE were used to measure DNA damage. Indirect
immunofluorescence was applied to track DNA damage response events. FISH was used
to quantify the levels of hyper-replication of certain genomic loci. Gene expression
analysis was performed using qRT-PCR and Western blot hybridization.
Results: In the present study we demonstrated that acute heat stress (HS) induces cell
senescence in human cells – HS results in a robust G2/M cell cycle arrest within
approximately one cell cycle. Heat-treated human cells acquire most of the cell
senescence marks (proliferation arrest, senescence-associated (SA) beta-gal activity,
changes in cell size/morphology, etc.) in few days after the treatment. Interestingly, HS
does not lead to formation of SA heterochromatin foci (SAHF) – only ring-like
heterochromatin domains are formed. We demonstrated that HS-induced cell senescence
state is maintained by p21. We found that persistent DNA damage response, which
depends on DNA hyper-replication, is the trigger of HS-induced cell senescence.
Conclusion: Heat stress induces p21-dependent cell senescence.
Funding: The work was supported by RFBR (grants 13-04-00604, 14-04-32169).
17
Complex treatment of chemoresistant glial brain tumors and delivery of
drugs to the tumor cells
Kavsan V.1 Avdieiev S.1, Balynska O.1, Chernolovskaya E.2, Gera L.3, Hodges R.3,
Stepanenko A.1, Zenkova M.2
1Institute of Molecular Biology and Genetics, NASU, Kyiv, Ukraine; 2Institute of
Chemical Biology and Fundamental Medicine, RAS, Novosibirsk, Russian
Federation; 3University of Colorado, Aurora CO, USA
kavsan@imbg.org.ua
Most tumorigenic processes are multi-factorial in nature. They are affected or controlled by multiple
effectors in sequential or parallel form. A dominant effector usually requires the synergy of one or
more secondary effectors. Its loss of function is often compensated for by other secondary effectors.
In order to effectively control a pathological process, it is necessary to modulate multiple related
effectors concomitantly. Glioblastoma, the most common and aggressive malignant tumor of central
nervous system in adults is known to be highly resistant to chemotherapy. The first-line cytotoxic
chemotherapy (temozolomide) is unable to induce long-term remission in the majority of patients.
Despite the certain clinical success, only a portion of patients responds and durability of response is
limited, majority of patients exhibit an aggressive clinical course with a median survival time of 1 to
2 years. Glioblastoma is driven by multiple molecular aberrations that cannot be controlled by a
single targeted agent, chromosomal instability and tumor heterogeneity makes every tumor rather
"mobile" than "frozen" target. So, the development of new treatment modalities is desperately
needed for this malignant disease. Although targeting oncogenes becomes a clinical reality, the
therapies aimed at a single target may not be effective: massive biological tumor heterogeneity
dictates the necessity for a therapy directed not at individual genes but rather at the pathological
effects that they cause. One of such pathological effects is angiogenesis, which plays a key role in
tumor vascularization. In anticancer therapy, considerable attention was paid to anti-angiogenic
drugs. Previously, it was shown that CHI3L1 had strong angiogenic properties. We found that
knockdown of CHI3L1 in HeLa_CHI3L1 and glial U87 cells by specific siRNAs led to 90 %
inhibition of CHI3L1 production in cell culture and in the same time suppressed the transformed
cells proliferation and their ability to grow in soft agar, the main feature of malignantly transformed
cells. However, the success of anti-angiogenic drugs in clinic is temporary. Drug resistance, tumor
re-emergence, and rapid appearance of new vessels are manifested at the end of therapy. Angiogenic
(oncogenic) redundancy is a significant obstacle to the success of targeted therapy. It is supposed
that simultaneous treatment with anti-CHI3L1 together with other anti-angiogenic drugs as for
example bradykinin antagonists should give better positive result. Bradykinin antagonists were
shown to be quite promising as new potential anti-cancer compounds for complex therapy. Several
mechanisms of resistance have already been proposed, however changes in chromosome stability of
glioblastoma cells after acquisition of resistance to temozolomide have not been elucidated. The
project was focused on the characterization of karyotype and phenotype changes in 293
immortalized cells and U373 glial cells after acquisition of resistance to this drug. This project
provides a simultaneous application of new bioactive chemical reagents of different types, specific
siRNAs or new antisense strategies, namely stable in bloodstream morpholino oligonucleotides
together with conventional chemotherapeutics used in clinics. Anti-tumor properties of different
combinatorial drug schemes were investigated in vitro in several malignant cell lines of different
origin and in vivo in mouse and rat tumor models. It is the goal to develop the highly effective
multitargeted pharmaceuticals, which could modulate multiple related effectors (drug targets)
concurrently. It is possible to expect that the combinatorial anti-cancer therapy aimed
simultaneously at different elements of tumor formation mechanisms will be more effective and will
promote the extension of patients’ life. For multiple-target drugs delivery to gliomas we are going to
use a specific nanovector Polycefin (Ljubimova et al. 2012), to one molecule of which inhibitors of
multiple molecular targets may be attached easily.
18
Timing characteristics of degeneration of nigrostriatal dopaminergic neurons
in mice at the model of the early clinical stage of Parkinson’s disease
Kolacheva A. A., Ugrumov M. V.
Institute of Developmental Biology RAS, Moscow, Russia
Degeneration of nigrostriatal dopaminergic (DA-ergic) neurons leads to a dopamine (DA)
deficit in the striatum and development of Parkinson’s disease (PD). Long-term
asymptomatic development of PD is explained by activation of compensatory processes in
the brain. Motor dysfunctions of PD, resting tremor, rigidity and bradykinesia, appear
after a threshold degradation of the DA-ergic neurons (a loss of 50-60 % of nigral cell
bodies and 70 % of striatal axons) and depletion of the brain compensatory reserves. The
aim of this study was to examine the timing characteristics of degeneration of nigrostriatal
DA-ergic neurons in mice with modelled early clinical stage of PD for a subsequent use of
this model for searching potential neuroprotectors. Materials and Methods. The model
of the early clinical stage of PD was obtained by 4 injections of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP), a precursor of DA-ergic neurons neurotoxin, at the
dose of 12 mg/kg with a 2 h interval between injections (Ugrumov et al., 2011). The
timing characteristics of nigrostriatal DA-ergic neurons degeneration in MPTP-treated
mice were evaluated by quantification of surviving neurons, nigral cell bodies and striatal
axons. Surviving DA-ergic neurons were identified by immunostaining of tyrosine
hydroxylase (TH), the rate-limiting enzyme of DA synthesis. Moreover, contents of DA
and TH in cell bodies and axonal terminals of DA-ergic neurons were estimated as an
index of the functional state of surviving DA-ergic neurons. Results. An initial loss of
DA-ergic cell bodies in the SN was observed 3 h after the last MPTP injection. At that
time, the number of DA-ergic axonal terminals in the striatum was reduced by 65 %
compared to the control. A loss of DA-ergic cell bodies and axons continued for the next
3 h, whereas their number was not changed for the next 18 h. The TH concentration in
nigral cell bodies remained at the control level during the whole studied period, 24 h after
the last injection of MPTP. The DA content in the SN decreased by 70 % 3 h after the last
injection of MPTP, followed by its continuous increase in subsequent 21 h up to 70 % of
the control value. Obtained data suggest that TH activity decreased in first few hours after
the last injection of MPTP. The concentration of DA in the striatum was reduced by 90 %
compared to the control over the period from 3 to 24 h after the last injection of MPTP.
The TH concentration in the axonal terminals decreased by approximately 25 % and 40 %
after 3 and 6 h after the last injection of MPTP, respectively, without any change over
next 6 h, and increased up to 82 % of the control value 24 h. A partial loss of DA in the
striatum may be either result of decrease of its synthesis caused by reduced TH activity or
a compensatory increase of DA release in the striatum. The availability of our
parkinsonian model for searching and testing of potential neuroprotectors was evaluated
by using nomifensine, the inhibitor of the membrane transporter of DA and MPTP
(MPP+). It was shown that the number of axonal terminals was reduced only by 10 %
without any loss of DA in the striatum 12 h after their simultaneous treatment with MPTP
and nomifensine. By contrast, the mice treatment with only MPTP resulted in a loss of 50
% of striatal DA-ergic axons. Conclusions. Timing characteristics of degeneration of the
nigrostriatal dopaminergic neurons in MPTP-treated mice were evaluated in this study
showing availability of this model for searching potential neuroprotectors.
19
Alternative mechanisms account for plant mitochondrial competence
1Klimenko E.S., 1Koulintchenko M.V., 1Grebnev P.V., 2Weber-Lotfi F.,
2Dietrich A., 1Konstantinov Y.M.
1Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch, Russian
Academy of Sciences, Irkutsk, ul. Lermontova, 132, 664033 Russia
2Institut de Biologie Moléculaire des Plantes, CNRS/Université de Strasbourg, 12 rue du
Général Zimmer, 67084 Strasbourg, France
yukon@sifibr.irk.ru
Aim. Up to now, such question as how DNA molecules of different size traverse outer and inner
mitochondrial membranes is still unclear. However, the resolution of this problem is the critical
prerequisite for the development of efficient gene transfer system in mitochondria of plants and
mammals. Earlier it was shown that mitochondria, isolated from various plant species (potato,
maize, cauliflower, tobacco cell suspension culture) and from mammals (rat liver, human cell
cultures) are capable to import double-stranded linear DNA molecules of a reasonable size ( < 10
kb) via an active mechanism, nonspecific to DNA sequence and involving the permeability
transition pore complex in plant mitochondria (Koulintchenko et al., 2003; 2006). The main
substrate for mitochondrial DNA import in such a studies was the PCR-product corresponding to the
sequence of mitochondrial plasmid of 2.3 kb from maize. It was shown that (i) DNA import into
plant mitochondria does not depend on nucleotide sequence of the imported molecule; (ii) the
import efficiency decreases with increasing of imported DNA molecule size; (iii) import of circular
DNA molecules is not as effective as that of linear molecules; (iv) single-stranded DNA is not
imported into mitochondria. But in further study (Ibrahim et al., 2011) it was established that the
DNA import into plant mitochondria is specific with respect to large DNA substrates. For DNA
import into plant mitochondria and into mitochondria of human cell culture the linear plasmid from
rapeseed mitochondria (Brassica napus L.) with the size 11.6 kb was used. This plasmid, like the
2.3 kb plasmid from maize is characterized by the terminal inverted repeats (TIRs) present at each
end of the molecules. It’s known that inside the mitochondria TIRs are covalently bound to proteins
involved in replication and maintaining of the plasmids. It was shown that (i) the efficiency of large
DNA molecules import into plant mitochondria depends on molecule sequence; (ii) the specificity
of DNA import is mediated by the presence of TIRs. The aim of the present study was to clarify the
question, there is one or multiple paths for different size DNA molecules specific transport into
plant mitochondria. Methods. To study different size DNA molecules import efficiency the freshly
isolated intact mitochondria from potato tubers (Solanum tuberosum) were used. The respiration
control value of mitochondria used in experiments was ca 4.0 with succinate as respiratory substrate.
To estimate the efficiency of exogenous DNA uptake by mitochondria two methods were used
(evaluation of isotopic labeling DNA transport into mitochondria and quantitative PCR). Results.
We had showed that the efficiency of 9 kb DNA molecules import into isolated potato mitochondria
is increased substantially in the case of incorporation in their structure of terminal inverted repeats
(327 bp) of the linear 11.6 kb plasmid from Brassica napus mitochondria. Another important
finding is the different sensitivity of import activity in case of using different size DNA substrates
(109 bp, 269 bp, 717 bp, 1540 bp, 3700 bp, 6000 bp, 9000 bp) to the action of such an inhibitor of
porin (VDAC) in the outer mitochondrial membrane – Ruthenium Red and inhibitors of ADP/ATP
carrier in the inner mitochondrial membrane – atractyloside and carboxyatractyloside. We revealed
that the treatment of potato mitochondria by Ruthenium Red even increased substantially the import
efficacy for definite size of DNA molecules. Conclusions. The DNA import into plant mitochondria
is presumably specific with respect to substrate sequence context and size. Therefore, we propose
the alternative biochemical pathways for transfer of different size DNA molecules into plant
mitochondria.
This work is supported by funding of Russian Fund for Basic Research (12-04-01400).
20
Haplotype HLA-DQ frequency distribution in Latvian patients with Lyme
disease
1Kovalchuka Lilija, 1Eglite Jelena, 4Lucenko Irina, 5Logina Inara, 2.3Zalite Mara,
2.3Viksna Ludmila, 2Krumina Angelika
1Riga Stradiņš University, Clinical Immunology and Immunogenetic laboratory
2Riga Stradiņš University, Infectology and Dermatology department
3 Riga Eastern Clinical University Hospital, Infectology Center of Latvia
4 Centre for Disease Prevention and Control of Latvia, Riga
5Riga Stradiņš University, Neurology and Neurosurgery Department
Lilija.Kovalcuka@rsu.lv
Introduction. Lyme disease (Lyme borreliosis) is caused by infection with the tick- borne
bacterium Borrelia burgdorferi. Human pathogen, B. burgdorferi causes a multisystem
disease that may affect the skin, nervous system, heart, or joints. The disease incidence in
Latvia is one of the highest in Europe. The genes encoding the HLA-DQ heterodimer
molecules, DQA1 and DQB1, have been found to have the association with Lyme
borreliosis risk, although there is cumulative evidence for the effect of other gene loci
within the major histocompatibility complex gene region. Objective. The purpose of this
study was to determine of HLA -DQ haplotypes in patients with clinical, epidemiological
and laboratory approved Lyme borreliosis diagnosis. Materials and methods. The study
included 91 patients with clinical stage – erythema migrans and 100 control (healthy)
persons. The diagnosis was confirmed and imposed Latvian Infectology Center.
Immunogenetic examinations were performed RSU Clinical Immunology and
Immunogenetic Laboratory with PCR-SSP method. Results. The frequency of haplotypes:
DQA1*02:01:01/DQB1*03:02:01 (OR 3.27; p < 0,040);
HLA- DQA1*05:01:01/DQB1*02:01:01 (OR 2.54; p < 0,048)
and -DQA1*01:01:01/DQB1*02:01:01 (OR 2.11; p < 0,031) were significantly increased
in the Lyme disease patients compared with the control groups.But, the haplotypes of
DQA1*01:02:01/DQB1*06:02:01(OR 0.24; p < 0.036); DQA1*01:03:01/DQB1*06:02:01
(OR 0.27; p < 0.046) and DQA1*01:02:01/DQB1*06:02:01 (OR 0.31; p < 0.029) were
smaller in Borreliosis patients and significantly higher in controls. Conclusions. This data
suggest that HLA-DQ haplotypes may have a considerable effect on susceptibility/or
protection to Lyme borreliosis. In particular, HLA -DQA1*02:01:01/DQB1*03:02:01; -
DQA1*05:01:01/DQB1*02:01:01; and -DQA1*01:01:01/DQB1*02:01:01 haplotypes
definitely contributes to a genetic predisposition to Borrelia burgdorferi infection in
Latvian population, which may have implications in our understanding of pathogenesis of
this disease.
This work was supported by the European Social Fund (ESF) project “Support for
doctoral study program and scientific degree receiving in Riga Stradiņš University,
agreement No 2009/0147/1DP/1.1.2.1.2/09/IPIA/VIAA/009.”
21
Selenoproteins in health and disease. Towards understanding their peculiar
mechanism of biosynthesis
Krol Alain
Architecture and Reactivity of RNA – Institute of Molecular and Cellular Biology
CNRS – University of Strasbourg
a.krol@ibmc-cnrs.unistra.fr
Aim. Selenoproteins harbor the selenium-containing amino acid selenocysteine (Sec) in
the active site. These proteins fulfill as varied functions as defence against reactive
oxygen species, sperm and thyroid hormone maturation, muscle biogenesis. Selenoprotein
biosynthesis is not a standard mechanism because UGA, otherwise a stop codon for
translation, encodes selenocysteine. Reading selenocysteine instead of stop at the UGA
Sec codon is a complex event that requires a dedicated machinery with specialized RNAs
and proteins, including the SECIS stem-loop in the 3’UTR of selenoprotein mRNAs and
the SECIS binding protein SBP2. Mutations in these or other components lead to
embryonic lethality or various pathologies, attesting the importance of selenoproteins in
health and disease. At the mechanistic level, the central question is to understand how
assembly of a selenoprotein mRNA-multicomponent complex occurs in an orderly and
temporal fashion so that approaching ribosomes are cleared at the UGA Sec codon. As
little was known regarding the issue, our goal was to understand where and when
selenoprotein mRNAs and components of the machinery interact with the ribosome in the
course of translation. Methods. We used synthetic mini-selenoprotein mRNAs carrying
photoactivatable groups in the SECIS motif for UV cross-linking to ribosomes; hydroxyl
radical footprinting of SBP2-human ribosomes complexes; bi-functional reagents to cross-
link SBP2 to purified human ribosomes. RNA-protein cross-links were revealed by gel
electrophoresis. Results. We showed that SBP2 is bound to the SECIS at the pre-initiation
step of translation; it then binds the ribosome at the pre-translocation step to bring the
Sec-tRNASec to the UGA Sec codon; subsequently, SBP2 returns to the SECIS after the
transpeptidation step. We also identified the binding site of SBP2 on the human ribosome.
Conclusion. Altogther, our findings led for the first time to broaden our understanding
about the unique mechanism of selenoprotein incorporation in mammals. Also, our study
established that SBP2 makes direct contacts with a discrete region of the human 28S
ribosomal RNA.
22
PARP1 as a mediator of the catalytic activity of the enzymes processing
apurinic/apyrimidinic site
Khodyreva S.N., Sukhanova M.V., Lebedeva N.A., Rechkunova N.I.,
Anarbaev R.O., Kutuzov M.M., Ilina E.S., 1Amé J.-C., 1Schreiber V., Lavrik O.I.
Institute of Chemical Biology and Fundamental Medicine,
Novosibirsk State University, Novosibirsk, Russia.
1UMR7242, Biotechnology and Cell Signaling, Université de Strasbourg,
CNRS, France
lavrik@niboch.nsc.ru
The apurinic/apyrimidinic (AP) site is considered to be a common lesion in genomic
DNA. If unrepaired, AP sites present mutagenic and cytotoxic consequences to the cell
[1]. The capacity of human poly( ADP-ribose) polymerase 1 (PARP1) to interact with the
intact AP sites in DNA has been demonstrated by us earlier [2].The specificity of PARP1
interaction with AP sites was confirmed by the mutual tests. The identity of PARP1 cross-
linked to AP sites in DNA in crude cell extracts was confirmed by mass spectrometry.
PARP1 was weakly activated to conduct poly(ADP-ribose) synthesis upon binding to the
AP site containing DNA. PARP1 interacting with AP sites catalyzes DNA strand incision
by its internal AP lyase activity. PARP1 demonstrates also 5’dRP lyase activity. The
interaction of PARP1 with AP sites modulates activity of the enzymes which catalyze the
AP-site cleavage during the base excision repair (BER). The major enzyme which is
responsible for the AP site cleavage activity is apurinic/apyrimidinic endonuclease 1
(APE1). The activity of this enzyme was influenced by PARP1 in the process of cleavage
of DNA containing single or clustered AP sites. The capacity of the other enzyme, human
tyrosyl-DNA phosphodieasterase (Tdp1), to the AP site cleavage was detected by us [3].
Tdp1 catalyzes the cleavage of an AP site and its synthetic analog, 3-hydroxy-
2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester
bond between the substituent and 5'-adjacent phosphate. The product of the Tdp1 cleavage
in the case of the AP site is unstable and is hydrolyzed with the formation of 3'- and 5'-
margin phosphates. The following repair demands the ordered action of polynucleotide
kinase phosphatase, with XRCC1, DNA polymerase β and DNA ligase. In the case of
THF, Tdp1 generates break with the 5'-THF and the 3'-phosphate termini. Tdp1 is also
able to effectively cleave the non-nucleotide insertions in DNA, decanediol and
diethyleneglycol moieties, by the same mechanism as in the case of the THF cleavage.
The efficiency of the Tdp1 catalyzed hydrolysis of the AP-site analog correlates with the
DNA helix distortion induced by the substituent. The following repair of 5'-THF and the
other AP site analogs can be processed by the long-patch base excision repair pathway.
PARP1 stimulates activity of Tdp1 in the AP site cleavage. The protein–protein
interactions of PARP1 with APE1 and Tdp1 were detected by various techniques. These
interactions were suggested as a basis of the modulation of APE1 and Tdp1 activities by
PARP1 in the AP site processing.
This work was supported by the RFBR (13-04-93107, 13-04-40197-comfi and 14-04-
00268), by the Program of RAS Presidium on Molecular and Cellular Biology, by the
grant from the President of the RF for the leading scientific schools (420.2014.4), and by
the Ministry of Education and Science of the RF.
1.Lindahl T (1993) Nature, 362, 709-15.; 2. Hodyreva SN et al (2010) PNAS, 107, 22090-5.
3.Lebedeva NA et al (2013) DNA Repair, 12, 1037-42.
23
Tyrosyl-DNA phosphodiesterase 1 initiates repair of apurinic/apyrimidinic
sites in single stranded DNA
Lebedeva Natalia A., Rechkunova Nadejda I., Lavrik Olga I.
Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
Novosibirsk State University, Novosibirsk, Russia
nataleb@niboch.nsc.ru
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester
linkage between the DNA 3′ phosphate and a tyrosine residue as well as a variety of other
DNA 3′ damaged termini. Recently we determined that human Tdp1 interacts with AP
sites and catalyzes the AP-site-cleavage reaction to generate breaks with the 3′- and 5′-
phosphate termini. Tdp1, unlike APE1, much more efficiently cleaves the AP-site, located
in the single-stranded DNA regions or opposite to bulky DNA damages in the regular
DNA duplexes. This property of Tdp1 suggesting that this enzyme has the potential to be
involved in AP site repair in the single-stranded regions of the genomic DNA, resulting in
its metabolic processes, including during replication, DNA repair and transcription. It
should be noted that the destabilization of the double helix DNA and, consequently, the
occurrence of single-stranded regions in it significantly increases the likelihood of AP
sites, as a result of spontaneous apurination / apyrimidination of DNA and under the
action of DNA glycosylases, removing the modified (oxidized or alkylated) base. The
introduction of bulky DNA damage does not affect the binding of Tdp1 with DNA
containing a "bubble", but reduces the efficiency of the AP-site cleavage relative to DNA
structure without a bulky lesion, possibly due to steric hindrance created by the bulky
lesions. The observed effect may have biological significance, because such DNA
structure represents the intermediate of nucleotide excision repair (NER) process, formed
as a result of the partial unwinding of the DNA duplex around the bulky lesion by the
NER factors.
This work was supported by RSCF grant no. 14-14-00501; the Program of the President
of the Russian Federation for state support of leading scientific schools (project no. NSh-
420.2014.4), the Program of the Presidium of RAS ‘Molecular and Cellular Biology’.
24
Effects of Wnt/β-catenin pathway and RET/PTC oncogene on tumour
progression and treatment of papillary thyroid carcinoma
Massade Liliane
UMR 8203 CNRS, Institut Gustave Roussy, France
Liliane.massade@igr.fr
Junction oncogenes are the consequence of genomic rearrangements, leading to intragenic
gene fusion; they represent around 20 % of total cancers. In fact, deregulation of gene
expression is a common feature in cancers. The possibility to inhibit the expression of an
oncogene at the mRNA level, instead of blocking the function of the gene product, has elicited
for a long time a great interest as the potential therapeutic applications are obvious. Therefore,
RNA interference offers promising new opportunities to target very specifically genes
deregulated in cancers carrying junction oncogenes. We aimed in one hand to inhibit
dedifferentiation due to RET/PTC junction oncogene by siRNA and in the other hand to
investigate the effect of the inhibition on the thyroid transcription factor 1 (TTF-1), a tissue-
specific transcription factor essential for the differentiation of the thyroid. Methods. First, we
designed siRNA against RET/PTCs (siRNARET/PTC1 and RET/PTC3) and tested their
efficiencies and specificities by RT-qPCR and by western blot in cell lines expressing
RET/PTCs junction oncogenes. The effects on cell cycle growth (MTT tests), cell cycle (flow
cytometry), and apoptosis (caspase-3 clivage and TUNEL method) were studied. Then the
siRNA RET/PTCs were conjugated to squalene (SQ) and the corresponding nanoparticles
(NPs) were prepared by nanoprecipitation and characterised for their size and zeta potential. In
vivo gene silencing efficiency of NPs was assessed by administration in nude mice either via
i.v. or intra tumoral (i.t.) routes. Tumour growth was followed during the course of experiment
while gene and protein inhibitions were assessed by RT-qPCR and Western blotting. To
investigated if the Wnt/β-catenin pathway might regulate TTF-1 expression in a human PTC
model we knock-down the Wnt/β-catenin components by siRNAs and mimicking the
activation of Wnt signaling by lithium chloride. Functional promoter studies and ChIP analysis
were also performed to localize the TCF/LEF binding site into TTF-1 promoter. Results.
Concerning RET/PTCs junction oncogenes, the designed siRNA against RET/PTC1 and
RET/PTC3 were found to decrease oncogene expression > 80 %. This inhibition of mRNA
levels is paralleled with a reduction of protein content that persists for at least 72h.
Furthermore, the siRNAs are able to decrease cell proliferation of about 50 % and to increase
apoptotic index and to cleave caspase-3. Interestingly, in vivo, on a mice xenografted
RET/PTC experimental model, RET/PTCs-SQ NPs were found to inhibit tumor growth and
RET/PTCs oncogene and oncoprotein expression. Then, we investigated whether the Wnt/β-
catenin pathway regulate TTF-1 expression. Knocking-down the Wnt/β-catenin components by
siRNAs inhibited both TTF-1 transcript and protein expression. Activation of Wnt signalling
by lithium chloride induced TTF-1 gene and protein expression. Functional promoter studies
and ChIP analysis showed that the Wnt/β-catenin pathway exerts its effect by means of the
binding of β-catenin to TCF/LEF transcription factors on the level of an active TCF/LEF
response element at [-798, -792 bp] in TTF-1 promoter. Moreover, immunohistological studies
performed on human normal thyroid and PTC tissues revealed that TTF-1 and transcription
factor 4 (TCF4) proteins were expressed in the same areas. Conclusions. These results showed
that we success to establish siRNA efficient to RET/PTC able to inhibit tumour growth in vivo.
Moreover, we demonstrated that the Wnt/β-catenin pathway is a direct and forward driver of
the TTF-1 expression.
This work is supported by funding of ANR-11-NANO-003, Programme P2N, Nanosqualonc and by
the European Research Council.
25
Differential Regulation by ppGpp vs pppGpp
1,2Potrykus Katarzyna, 2Murphy Helen, 3Murakami Katsuhiko S., 2Cashel Michael
and 4, 2Mechold Undine
1Department of Molecular Biology, University of Gdansk, Gdansk, Poland
2Laboratory of Molecular Genetics, Eunice Kennedy Schriver National Institute of Child
Health and Human Development, National Institutes of Health, Bethesda, Maryland,
USA
3Department of Biochemistry and Molecular Biology, The Center for RNA Molecular
Biology, The Pennsylvania State University, University Park, PA, USA
4Institut Pasteur, UMR 3528, Unité de Biochimie des Interactions Macromoléculaires,
Département de Biologie Structurale et Chimie, Paris, France
undine.mechold@pasteur.fr
Background: Multidrug resistance caused by bacterial persisters poses a serious problem
in the treatment of reoccurring or chronically bacterial infections. Bacterial persisters are
transiently drug tolerant bacteria that enter a physiological state allowing the escape from
lethal action of drugs in a mechanism different from antibiotic resistance. The bacterial
stress alarmone (p)ppGpp plays an important role in pathogenesis and in particular in
persistence. Binding of (p)ppGpp to RNA polymerase results in reprogramming of
bacteria by affecting the expression of hundreds of genes. Both ppGpp and pppGpp are
thought to function collectively as second messenger. There are few indications that their
regulatory effects might be different, however this question has been largely unexplored
for lack of an ability to experimentally manipulate the relative abundance of ppGpp and
pppGpp.
Aim: In order to understand the molecular mechanism underlying (p)ppGpp controlled
mechanisms better we wanted compare the effects of ppGpp and pppGpp on stringently
controlled processes.
Methods: Preferential accumulation of either ppGpp or pppGpp was achieved in E. coli
strains through induction of different Streptococcal (p)ppGpp synthetase fragments. In
addition, expression of E. coli GppA, a pppGpp 5’-gamma phosphate hydrolase that
converts pppGpp to ppGpp, was manipulated to fine tune differential accumulation of
ppGpp and pppGpp.
Results: In vivo and in vitro experiments show that pppGpp is less potent than ppGpp
with respect to regulation of growth rate, RNA/DNA ratios, ribosomal RNA P1 promoter
transcription inhibition, threonine operon promoter activation and RpoS induction. To
provide further insights into regulation by (p)ppGpp, we have also determined crystal
structures of E. coli RNA polymerase-σ70 holoenzyme with ppGpp and pppGpp. We find
that both nucleotides bind to a site at the interface between β’ and ω subunits.
26
Endocytic scaffold ITSN1: novel connections and way of regulation in
neurons
1Morderer D., 1Nikolaienko O., 1Skrypkina I., 1Rymarenko O., 2Cherkas V.,
2Belan P., 1Rynditch A.
1State Key Laboratory of Molecular and Cellular Biology,
Institute of Molecular Biology and Genetics NAS of Ukraine, Kyiv, Ukraine.
2Bogomoletz Institute of Physiology NAS of Ukraine, Kyiv, Ukraine
dmytromorderer@gmail.com
ITSN1 is an endocytic scaffold that has a considerable impact in functioning of a nervous
system. Changes in its expression level or disruption of its function were reported to affect
synaptic vesicle endocytosis and dendritic spine morphology. Overexpression of Itsn1
gene is associated with development of Down syndrome and Alzheimer’s disease. Thus, a
role of ITSN1 in neuronal development and functioning is an important theme for
research.
It is widely accepted that many cellular events in neurons are regulated by change in
concentration of Ca2+ ions. ITSN1 molecule possesses two EF-hand motifs that can
potentially bind Ca2+ ions within its EH domains. However, we have shown that presence
of Ca2+ does not affect binding of ITSN1 EH domains with their interactor epsin 1.
Additionally, bioinformatic predictions indicate that ITSN1 can potentially be
phosphorylated by Ca2+/calmodulin-dependent kinases. In order to test this hypothesis we
purified fraction of Ca/calmodulin-binding proteins from mouse brain lysate by affinity
chromatography and used them for in vitro kinase reaction with recombinant fragments of
ITSN1. We have shown that at least two fragments of ITSN1 containing coiled-coil and
SH3 domain-containing regions are phosphorylated in presence of Ca2+ ions and
calmodulin. Using tandem mass spectrometry we have identified 9 sites of
Ca2+/calmodulin-dependent phosphorylation of ITSN1, suggesting existence of Ca2+-
dependent regulation of ITSN1 functioning.
Using in vitro-binding assay and MALDI-TOF mass spectrometry we have identified
novel neuronal ITSN1-binding protein STOP (stable tubule-only polypeptide) — a
microtubule-associated protein that is required for formation of cold-stable subpopulations
of microtubules in neurons and some other cell types. Complexes of ITSN1 and STOP
were precipitated from mouse brain lysate by both antibodies against STOP and ITSN1.
Moreover, we found that ITSN1 and STOP are partially co-localized in primary rat
hippocampal neurons. Functional role of interaction between ITSN1 and STOP remains
unknown. Considering the role of microtubules in formation of dendritic outgrowth, we
used shRNA-mediated knock-down assay in order to reveal the effect of ITSN1 level
decrease on morphology of cultivated hippocampal neurons. We found that total dendritic
length is decreased in shRNA-transfected cells compared to cells transfected with empty
vector. Further studies will be aimed to uncover the mechanism of ITSN1 influence on
dendritic growth and its possible role in formation of alterations in Down syndrome brains
that were reported previously.
27
Charge-dependent mechanism for retention of proteins in the nucleoli via
dynamic interaction with nucleolar RNAs
1Musinova Y.R., 1Kananykhina E.Y., 2Potashnikova D.M., 1Lysitsina O.M.,
1Sheval E.V.
1A.N. Belozersky Institute of Physico-Chemical Biology,
Moscow State University, Russia;
2Moscow State University, Dept. Cell Biology and Histology, Russia
musinova@libro.genebee.msu.ru
Aim. The majority of known nucleolar proteins examined thus far are highly mobile:
these proteins diffuse rapidly in the nucleoplasm and typically exchange quickly with their
binding sites. The key questions are how macromolecules find their target sites inside the
nucleus, and how they accumulate in these target sites. One way of protein retention in the
nucleolus is associated with the presence of specific short amino acid sequences –
nucleolar localization signals (NoLSs). The aim of the present study was to investigate
mechanism by which NoLSs may lead to the nucleolar accumulation of the proteins.
Methods. There are several ways for NoLS identification and investigation. We have used
the approach based of analysis of tested sequence as factor for nucleolar accumulation of
marker fluorescent protein (EGFP). To quantitatively estimate the effect of tested
sequence on the nucleolar accumulation of GFP, the NoLS activity (concentration of
EGFP in the nucleoli/concentration of EGFP in the nucleoplasm) was measured for all
fusions obtained. Results. Three groups of observations are in agreement with the
hypothesis of that charge-dependent (electrostatic) interactions of NoLSs with nucleolar
components lead to nucleolar accumulation. (i) Known NoLSs are enriched in positively
charged amino acids, but the structure of NoLSs is highly heterogeneous, and it is not
possible to identify a consensus sequence for this type of signal. (ii) In two analyzed
proteins (NF-κB inducing kinase and HIV Tat), the NoLS coincide with the boundaries of
the region enriched with positively charged amino acids. Using the method of quantitative
estimation of nucleolar accumulation, after substitution of charged amino acids to non-
charged we were able to demonstrate that there is a strong correlation between the charge
of protein fragment tested as a NoLS and its ability to accumulate marker protein (EGFP)
in the nucleoli. (iii) The sequences containing only lysine or arginine (which were referred
to as imitative NoLSs, iNoLSs) were accumulated in the nucleoli in a charge-dependent
manner. We investigated localization of the strongest iNoLS, containing 19 agrinines
(EGFP-R19), and found that the localization and some of properties were closely similar
to that of B23, the protein of late rRNA processing. Using this iNoLS, we demonstrated
that charge-dependent accumulation inside nucleoli was dependent on interaction with
nucleolar RNAs. Conclusions. These results are in agreement with the hypothesis that the
accumulation of proteins in the nucleolus by NoLSs can be determined by the electrostatic
interaction of positively charged signals with nucleolar RNAs.
28
PUB-NChIP – “in vivo biotinylation” approach to study chromatin in
proximity to a protein of interest
1Shoaib M., 1Kulyyassov A., 1Robin C., 2Mechold U., 1Winczura K., 1Tarlykov P.,
and 1Ogryzko Vasily V.
1Institut Gustave Roussy, CNRS UMR 8126, Unité de Signaling, Nuclei and
Innovations in Oncology, Villejuif, France
2Institut Pasteur, CNRS URA3528, Unité de Biochimie des Interactions
macromoléculaires, Département de Biologie Structurale et Chimie, Paris, France
vogryzko@gmail.com
Background : Chromatin Immunoprecipitation (ChIP) has been widely used for over a
decade to study the in vivo association of a particular DNA sequence with regulatory
proteins. However, due to the need to use chemical crosslinking, the protein part of
chromatin is typically discarded in this approach. On the other hand, the analysis of
histone modifications and replacement histone variants could provide valuable
information about the state of chromatin around the particular protein of interest.
Aims : Our aim was to develop an alternative approach to ChIP that would allow us to
preserve the protein part of chromatin associated with a protein of interest, in order to
study it with proteomics methodology.
Methods : Our method is based on coexpression of a) a protein of interest, fused with the
bacterial biotin ligase BirA together with b) a histone fused to BAP biotin acceptor
peptide, which is specifically biotinylated by BirA fusion in the proximity of the protein
of interest.
Results : Using RAD18 protein as a model, we demonstrate that the RAD18-proximal
chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal
chromatin containing a replacement histone H2AZ has a different pattern of H4
acetylation. Finally, biotin pulse-chase experiments show that H4 acetylation pattern starts
to resemble acetylation pattern of total H4 after the proximity of chromatin to RAD18 has
been lost.
Conclusions : We hope that the new method will find a wide use in the fields of
chromatin studies and epigenetics.
29
Changes in bone marrow mesenchymal stem cell count during
osteoreflextherapy
Osina K., Harju L., Freivalds T.
Institute of Experimental and Clinical Medicine, University of Latvia
kristine.osina@gmail.com
Aim. The human organism ages over its lifetime, and that may lead to different diseases
including Alzheimer's disease, atherosclerosis, osteoporosis etc. Many age-related
illnesses can be treated with osteoreflextherapy (ORT), which is based on stimulation of
the bone sensory system. Osteoreceptors are in close contact with the central nervous
system and thus different organs and organ systems can be affected through reflex arcs.
ORT entails the injection of physiological solution into the marrow of porous bone,
thereby stimulating osteoreceptors. Among the cell types in bone marrow are multipotent
mesenchymal stem cells (MSCs), which can differentiate into osteoblasts, chondrocytes,
muscle cells and other cell types. MSCs migrate to sites of damage in the body and can
regenerate multiple tissues. Since MSCs do not have one specific marker, different
markers can be used to identify MSCs, including CD105 (endoglin), CD29 (integrin-�)
and CD54 (intracellular adhesion molecule-1). The aim of the present study was to
determine how osteoreflextherapy affects mesenchymal stem cell count in bone marrow.
Methods. Patients (n = 21) undergoing ORT once a week had bone marrow harvested
from different bones, depending on their illness. Females (n = 12) ranged from 50 to 87
years of age while males (n = 9) were 17 to 59 years of age. Prior approval was obtained
from the Scientific Research Ethics Committee of the University of Latvia Institute of
Experimental and Clinical Medicine. Marrow samples from each patient were obtained
twice: prior to ORT and four weeks following the initiation of ORT. The counts of
CD105, CD29 and CD54 antigen-expressing cells were determined by
immunocytochemistry and light microscopy. Results. Prior to ORT, CD105+, CD29+ and
CD54+ cell counts were 0.08 ± 0.02 %, 0.08 ± 0.02 % and 0.08 ± 0.03 %, respectively, of
mononuclear bone marrow cell count. Four weeks after the initiation of ORT, CD105+,
CD29+ and CD54+ cell counts were 0.12 ± 0.03 %, 0.10 ± 0.03 % and 0.11 ± 0.03 %,
respectively, of mononuclear bone marrow cell count. This increase in antigen-expressing
cell counts after initiation of ORT was statistically significant (p < 0.001) in all three
groups, while the difference among the three groups of antigen-expressing cells was not
significant (p > 0.05). Statistically significant (p < 0.05) Pearson's correlations were
between CD105+ and CD54+ cell counts before ORT and after initiation of ORT (r = -
0.47 and r = -0.49, respectively) and also between increase in CD105+ cell count after
ORT initiation and patient age (r = -0.47). Conclusions. The variable increase in CD105,
CD29 and CD54 antigen-expressing cell count following initiation of ORT could be
attributable to the limited number of mesenchymal stem cells in human bone marrow.
Minimal increases observed in MSC count following ORT initiation could be due to an
initial MSC count close to the upper limit in bone marrow, while greater increases in MSC
count could be due to low pre-ORT MSC levels. This difference seemed to be age-
dependent, as increases in MSC count in older patients were not as pronounced as those
observed in younger patients. Increased MSC counts in bone marrow through
osteoreflextherapy could be one of successful therapy factors.
30
Association of the PSMB5 (rs11543947), PSMC6 (rs2295826, rs2295827)
genes polymorphismswith obesity in the Latvian population
1Paramonova N., 1Sjakste T., 2,3Rumba-Rozenfelde I, 1,4Sjakste N.
1Genomics and Bioinformatics, Institute of Biology of the University of Latvia,
Salaspils, Latvia
2Faculty of Medicine, University of Latvia, Riga, Latvia
3University Children Hospital, Riga, Latvia
4Latvian Institute of Organic Synthesis, Riga,
Latvianatasa@email.lubi.edu.lv
Aim. The ubiquitin-proteasome system (UPS) is the major nonlysosomal proteolytic
pathway that affects crucial intracellular processes including the cell cycle, the "quality
control" of newly synthesized proteins, transcription factors machinery, gene expression,
cell differentiation, homeostasis and apoptosis. Multiple data have proved that UPS has
the potential to be a therapeutic target for treatment of some diseases (vascular disorders,
diabetes mellitus, autoimmune diseases, cancer and its outcome). Importance of
proteasomes in both normal and pathological processes triggers interest for search of
sequence variations in the proteasome genes to be associated with human pathologies. The
genetic variations of the PSMA6, PSMC6 and PSMA3 proteasome genes were previously
shown to be associated with JIA, T2DM, BA and cardiovascular disorders. Aim of the
present study is to investigate polymorphisms in the PSMB5 and PSMC6 genes for
association with children obesity in Latvian population.
Methods. The rs11543947 (PSMB5), rs2295826 and rs2295827 (PSMC6) loci were
genotyped in 94 overweight children versus 191 controls. Stratification was performed by
sex and family history of obesity. Both the two-tailed Fisher’s exact test and the χ2 test
were applied. Only 2< OR < 0.5 was considered to be clinically significant.
Results. In both case and control cohorts the genotyping call rate was 100% and markers
were found to be in Hardy-Weinberg equilibrium. The rs2295826 and rs2295827 were
found to be in linkage in Latvians (D’ =1; r2=1). In obesity and family obesity groups
nominal risk effect for rare allele’s variations G/T (P<0.05) was detected. Risk 2-loci
genotype AG/CT showed nominal association (P<0.01) with obesity and obesity with
family history.
For rs11543947 rare allele T detected nominal risk effect (P<0.01) for obesity and obesity
with family history. The heterozygous genotype CT showed strong association (P<0.001)
with obesity and family obesity (OR = 2.641 [95% CI 1.484 − 4.699] and (OR = 2.966
[95% CI 1.542 −5.706] respectively. In cases for obesity without family history all
markers were considered to be the diseases neutral (P > 0.05).
Conclutions: Genetic variation of PSMB5 (rs11543947) and PSMC6 (rs2295826 and
rs2295827) 14q proteasomal genes may influence children obesity in Latvian population.
The work was supported from the European Social Foundation project The work was
supported the project of the European Social Foundation
2013/0043/1DP/1.1.1.2.0/13/APIA/VIAA/002. "Establishment of a new interdisciplinary
group to an effective treatment for diabetic nephropathy finding ways"
31
Prolonged culture of skin-derived mesenchymal stem cells changes their cell
cycle and proliferation marker expression
1Popena I., 1,2Saulite L., 1Parfejevs V., 1Beitnere U., 1Riekstina U.
Faculty of Medicine, University of Latvia
Faculty of Biology, University of Latvia
ineta.popena@gmail.com
Aim. Mesenchymal stem cells (MSCs) are present in many tissues, including bone
marrow, adipose tissues, skin and dental pulp. Skin-derived MSCs can be induced to
differentiate into mesodermal lineages as well as neuronal and glial cells which make
them suitable candidates for regenerative therapies, however, there is a substantial need
for evidence that cultured MSCs would be safe for patients if used as therapeutic agents.
The aim of this study was to analyse changes of different cell cycle and proliferation
marker (TERT, Ki-67, polyploidy) expression during prolonged culture of human skin
derived mesenchymal stem cells. Methods. Skin derived MSC cultures were propagated
in DMEM/F12 medium supplemented with fetal calf serum. Cultured MSC were
characterized for expression of MSC markers (CD90, CD73, CD105) by flow cytometry.
For TERT expression analysis, cells were seeded on coverslips and propagated for 48
hours followed by immunofluorescence analysis. Results were confirmed by Western blot
analysis. Cell cycle marker Ki-67 expression and polyploidy was analysed by flow
cytometry. Results. Prolonged culture of skin-derived mesencymal stem cells changes
their cell cycle and proliferation marker expression: later passages of MSC tend to show
higher TERT expression, but Ki-67 expression decreases with a later passage number.
Flow cytometry data also indicate that later passage number is associated with an increase
of polyploidy in cultured MSC. These common trends remained to show up regardless of
donor-to-donor differences in cell cycle marker expression between different MSC
lineages. Conclusions. There is a strong correlation between MSC culture passage
number and increased expression of telomerase activity marker TERT and decreased
expression of Ki-67. Between TERT and Ki-67, there is a strong inverse relation, i.e.,
higher TERT expression is associated with lower Ki-67 expression, and vice versa.
Prolonged propagation of MSC causes increase of polyploidy. However, the possible
effect of these findings on potential therapeutic applications of skin-derived MSC still
remains to be elucidated.
32
Interactions of the NER factors with DNA are modulated by PARP1 activity
1Rechkunova N.I., Maltseva E.A., Krasikova Y.S.,. Sukhanova M.V, 1Lavrik O.I.
Institute of Chemical Biology and Fundamental Medicine
1Department of Natural Sciences, Novosibirsk State University
nadyarec@niboch.nsc.ru
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 and the sequential
individual reactions is achieved through multiple DNA-protein and protein-protein
interactions. The interactions of key protein factors of the NER process, XPC-RAD23B,
XPA, and RPA with DNA structures mimicking NER intermediates and their modulation
by PARP1 activity 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. Western blotting
analysis was used to determine ADP-ribosylated proteins. 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 that is reduced in the following order: damaged bubble>undamaged
bubble>damaged duplex>undamaged duplex. The affinity of XPC/Rad4 for various DNA
structures correlates with DNA bending angle. 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.
Poly(ADP-ribose)polymerase-1 (PARP-1) is one of the candidate to participate in NER
regulation. PARP-1 is rapidly activated in response to DNA damage using NAD+ as a
substrate to form poly(ADP-ribose) (PAR) subunits. Using EMSA, the influence of
PARP1 on binding of XPC-RAD23B and XPA to model DNA-structures modulated by
PAR synthesis was revealed. Both XPC and RAD23B subunits of DNA damage
recognition complex were found to be ADP-ribosylated by PARP1. 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 no. 13-04-00538, 13-04-93107 and 14-04-
31363); the Russian Ministry of Education and Science (grant for leading scientific
schools 420.2014.4); the Program of the RAS Presidium ‘Molecular and Cellular
Biology’.
33
Mobility of broken proto-oncogenes within nuclear space of cells treated with
DNA topoisomerase poisons.
1,2Rubtsov M.A., 1,2Lomov N.A., 3Alexeyevsky D.A., 3,5,6Alexeevski A.V.,
1,2,4Razin S.V. and 2,4Iarovaia O.V.
1Department of Molecular Biology, Lomonosov Moscow State University, Moscow,
Russia
2LIA 1066 French-Russian Joint Cancer Research Laboratory, Villejuif, France–
Moscow, Russia
3Department of Bioengineering and Bioinformatics, Lomonosov Moscow State
University, Moscow, Russia
4Institute of Gene Biology RAS, Moscow, Russia
5A.N.Belozersky Institute for Physical and Chemical Biology, Lomonosov Moscow
State University, Moscow, Russia
6Scientific Research Institute for System Studies (NIISI RAN), Moscow, Russia
ma_rubtsov@mail.ru
Aim: Today it is known that treatment of cells with topoisomerase poisons leads to the
formation of double stranded DNA breaks in breakpoint cluster regions (BCRs) of known
proto-oncogenes such as MLL or AML1. And that incorrect repair of these breaks may
cause chromosomal rearrangements which in turn may induce the so-called treatment-
related leukaemias. But the exact mechanisms of these rearrangements remain unclear.
Methods: We have treated cultured human lymphoid cells (Jurkat) with etoposide or
camptothecin and then visualized the chromosomal territories and proto-oncogenes with
using 3D-FISH technique. 3D images have been processed by using software which
detects differently colored parts of proto-oncogenes (upstream and downstream of BCR)
and labeled chromosomal territories.
Results: We have found that exposure of Jurkat cells to etoposide resulted in frequent
cleavage of MLL and AML1 genes, with the flanks of the break located distant from each
other and are often found outside of chromosomal territories. Therewith it was no
differences between 5'- and 3'- flank of the breaks. It was also observed that spatially
separated flanks of proto-oncogenes are more frequently located outside of chromosomal
territory than non-separated ones. Treatment of cells with camptothecin does not affect the
integrity of MLL and AML1 genes.
Conclusion: Our findings demonstrate that flanks of the breaks introduced in BCR of MLL
or AML1 gene under inhibition of DNA topoisomerase II acquire additional mobility
inside the nucleus and tends to be on the edge or even outside of corresponding
chromosomal territory. And this may in turn contribute to the more probable meeting and
incorrect joining of translocation partners.
The work was supported by RFBR (grant 14-04-93105_CNRS_а)
34
Poly(ADP-ribose) glycohydrolase in DNA repair and replication: towards
potential applications in anticancer strategies ?
Schreiber Valérie
IREBS-Biotechnology and Cell Signalling, UMR7242-CNRS, Université de
Strasbourg, Illkirch, France.
valerie.schreiber@unistra.fr
AIM: 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 and replication stress. Replication stress is a source of DNA
damage, leading to transient stalling of replication forks or to their collapse followed by
the generation of double strand breaks (DSB). The involvement of PARP-1 in replicative
stress response has been described, whereas the consequences of a deregulated PAR
catabolism are not yet well established. RESULTS: Here, we show that wheFreas PARG
appeared dispensable for normal replication, PARG-deficient cells showed increased
sensitivity to the replication inhibitor hydroxyurea (HU). PARG is dispensable to recover
from transient replicative stress but is necessary to avoid massive PAR production upon
prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR
accumulation impairs replication protein A (RPA) association with collapsed forks
resulting in compromised DSB repair via homologous recombination. Our results
highlight the critical role of PARG in tightly controlling PAR levels produced upon
genotoxic stress to prevent the detrimental effects of PAR over-accumulation
Project funded by CNRS, Université de Strasbourg, Labex ANR-10-LABX-0034_Medalis,
Ligue contre le Cancer (Equipe Labellisée), EDF, ANR-13-BSV8-0003-01.
35
Immunoregulatory role of Hsp60 at thyrod cancer progression
Sidorik L., Chornyy S., Tsisarenko A., Yakovenko L., Kroupskaya I., Vigontina O.,
Tykhonkova I., 1Pogribnyy P.,2Gurtovyy V., 2Usenko V.
Institute of Molecular Biology and Genetics NAS of Ukraine, Kyiv, Ukraine
1Kavetskiy Institute of Experimental Pathology, Oncology and Radiobiology NAS of
Ukraine, Kyiv, Ukraine
2Morphological Laboratory BIONTEK, Dnipropetrovsk, Ukraine
sidorik@imbg.org.ua
The molecular chaperones (HSPs) are involved in numerous diseases, including cancer,
revealing changes of their expression and cellular localization. The expression of Hsp60
in neoplasia has been implicated in the regulation of apoptosis, as a modulator of p53
function, in the immune response against tumors, and in multidrug resistance. A wide
range of tumor cells or tissues have been shown to express atypical level or localization of
HSPs. For example, Hsp60 expression in breast or gastric cancer is associated with poor
prognosis and resistance to chemotherapy or radiation therapy. Such observations have led
to suggestions that HSPs could be used as biomarkers. Molecular chaperon (chaperonin)
Hsp60 is a special interest due to recently observed pro-inflammation potential of Hsp60
and anti-Hsp60 autoantibodies which have been detected in circulation of diseased and
healthy persons as well. 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 using
immunohistochemical studies. 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.
36
Ki-67 and Oct3/4 expression in two types of breast cancer histological
samples
1Simsone Z., 1Freivalds T., 1Harju L., 2Gudrā D., 3,4Kudaba I., 4,5Liepniece-Karele
I., 1Bērziņš J., 1Buiķis I.
1Institute of Experimental and Clinical Medicine, University of Latvia, Riga, Latvia.,
2University of Luxembourg, Faculty of Science, Communication and Technology,
Luxembourg, Luxembourg,
3Oncology Centre of Latvia, Riga East University Hospital, Riga, Latvia,
4Riga Stradins University, Riga, Latvia,
5Centre of Pathology, Riga East University Hospital, Riga, Latvia
z.simsone@gmail.com
Breast cancer is the most common oncological disease in women. Improvement in
treatment efficiency is a major priority. There is known more types of breast cancer, and
each of them are characterised by biological markers who describe cancer malignancy.
Aim. To determine cancer cell resistance, cell population heterogeneity in luminal and
triple negative breast cancer cell population using paraffin sections.
Methods. Women breast cancer primary operation material in paraffin sections. Two
groups of breast cancer samples, luminal (n = 32) and triple negative breast cancer (n =
11). For each case were five cuts (1.5µm) from paraffin section. Biological markers
(CD44, CD24, ALDH, Oct3/4) expression were detected by semi-quantitative method,
where 0- expression was not detected, 1- low expression, 2- expression was detected in
average low, 3- high expression, but not in whole sample, 4- strong expression in whole
sample. In this study patient clinical data was summarized: age, tumour size and
histological grade, and metastases in axillar lymph nodes, Ki67, ER, PgR and HER2.
Results. In both cancer types we observed very strong CD44 expression in different cell
types but not in all histological structures. In triple negative breast cancer, CD44 is
negatively correlated with lymph node metastases and positively correlated with
proliferation marker Ki-67. In luminal breast cancer, type CD44 is positively correlated
with histological grade.
In triple negative breast cancer, proliferation marker Ki-67 correlates with embryonic
stem cell marker Oct3/4 and lymph node metastases. In luminal breast cancer, however,
Ki-67 is negatively correlated with hormone receptors (oestrogen receptors and
progesterone receptor).
Conclusions. Marker CD44 overexpression was observed in different cell types, among
them, microcells, which possibly can be cancer stem cells. In triple negative breast cancer,
proliferation marker Ki-67 correlates with embryonic stem cell marker Oct3/4 and lymph
node metastases. The embryonic cell marker Oct3/4 was observed to be dominant in two
cell types: polyploid cells and microcells.
Acknowledgement: Our research was accomplished with support from the University of
Latvia, Faculty of Biology Student Governing. We wish to thank Riga East University
Hospital Oncology Centre of Latvia and Pathology Centre for cooperation.
37
Detection at the single molecule level of interaction of poly(ADP-ribose)
polymerases 1 and 2 with base excision repair DNA intermediates by Atomic
Force Microscopy
1,2Sukhanova Maria V., 2Hamon Loic, 2Abrakhi Sanae, 2Joshi Vandana, 1Kutuzov
Mikhail M., 2Pastré David, 2Curmi Patrick and 1,3Lavrik Olga I.
1Institute of Chemical Biology and Fundamental Medicine, Russia
2 Université Evry-Val d’Essonne, INSERM U829, France.
3NovosibirskState University, Russia
sukhanovamv@mail.ru
Aim. Atomic force microscopy (AFM) is considered as one of the unique instrument for
studying direct DNA-protein interactions. Here we designed synthesis of long 1400 bp
DNA fragments containing single DNA damaged site, uracil or synthetic analog of
apurinic/apirimidinic (AP) site –tetrahydrofuran (THF). Both AP site and uracil are
common DNA lesions, which are repaired via base excision repair (BER) process. The
repair process can be initiated by DNA glycosylase (UDG) action to produce an AP site,
and then apurinic/apyrimidinic endonuclease (APE1) cleaves the phosphodiester bond 5′
to the AP site, generating a single strand break. The present biochemical studies indicate
that the initial stage of BER may be modulated by other proteins involved in the process,
among them PARP1 and PARP2. Although PARP1 (PARP2) do not have BER-related
enzymatic activities, but the proteins interact with DNA intermediates containing AP site
or single-strand breaks. To estimate the efficiency of interaction of these proteins with
BER intermediates at single molecule level we tested the interaction of PARP1 and
PARP2 with 1400 bp DNA fragment containing AP site or with one nucleotide gap using
imaging of PARP1(PARP2)-DNA complexes with AFM. Methods. We prepared the
1400 bp DNA fragment by ligation of 30-mer DNA duplex with THF or uracil at a
defined position into the pEGFP plasmid with followed by digestion of the plasmid with
restriction enzymes (AseI and Not I) to generate 1400 bp fragment. Obtained DNA
fragments have single THF or uracil in precise localization on DNA. We shown that the
1400 bp DNAs are processed by BER enzymes in system reconstituted from purified
proteins, like UDG, APE1, DNA polymerase β and DNA ligase III. We developed
experimental conditions for adsorption of PARP1 (PARP2) complexes with the 1400 bp
DNA on Mica surface using putrescine (Pu2+) in AFM deposition buffer. Such conditions
enable to visualize the PARP1(PARP2)-DNA complexes and compare binding specificity
of PARP-1 and PARP-2 with AP site or single nucleotide gap generated after AP site
cleavage by APE1. Results. AFM-based quantitative data analysis shows that PARP1
interacts with similar efficiency both with TFH-site and with one nucleotide gap on 1400
bp DNA . The amount of PARP1-DNA complexes is slightly increased at damaged site
after incision of AP site with APE1. In the contrast to PARP1, PARP2 shows low
efficiency in interaction both with TFH-site and with single nucleotide gap. Conclusion.
Thus, AFM data presented here support the notion that PARP1 has important role in
modulation of BER process after forming of AP site and single nucleotide gap, while
PARP2 do not play a critical role at these early stages of BER.
The work is supported by INSERM, RFBR projects (13-04-40197-COMFI, 13-04-93107
and 14-04-00268), by RSCF grant no. 14-24-00038, MESRF State Contract number IR
14-20, the grant for leading scientific schools 420.2014.4.
38
Potential implication of aminoacyl-tRNA synthetases in tumourigenesis
Tukalo M., Kondratov A.,Yaremchuk A., Gudzera O.
Institute of Molecular Biology and Genetics, National Academy of Science of
Ukraine, Kyiv, Ukraine.
mtukalo@imbg.org.ua
Aminoacyl-tRNA synthetases (aaRSs) are ancient ubiquitous “house-keeping” enzymes
that are responsible for cellular protein synthesis and cell viability. Also nontranslational
functions of vertebrate aaRSs have recently been discovered. Taken together, new finding
suggest that aaRSs have critical mechanistic roles in a variety of cellular processes which
are relevant for disease development and pathology, and these roles may be used as one
possible avenue for improvement of diagnostics and open a new dimension for cancer
therapy.
To uncover the biomarkers related with tumorigenesis and behavior of cancer we have
studied of differently expressed genes of some aaRSs in tissues of kidney and colon
cancers by the quantitative polymerase chain reaction (qPCR) method.
Firstly, we have investigated the level of gene expression of cytoplasmic leucyl-tRNA
synthetase (LARS) in A549 cell line and normal pulmonary epithelial cell line, and in
tissues of kidney cancer and tissues of normal kidney. The level of mRNA expression of
LARS in cancer cells A549 was more than 2 times higher than in cells of normal
epithelium. In the case of kidney cancer from 16 samples of tumor tissue, increased
expression of LARS was observed in 13, and only two were noted its slight decrease.
However, only three samples showed a more than twofold increased expression, and one –
more than three times. Thus, from a data on the samples of tissues of kidney tumors we
used one can see a tendency for increasing of LARS expression.
In the case of human colon cancer we have observed the expression profile for LARS,
histidyl-tRNA synthetase (HARS), seryl-tRNA synthetase (SARS) and lysyl-tRNA
synthetase (KARS) in 18 primary cancer samples. We have found that genes of HARS and
KARS underwent most changes. These genes might be used for diagnosis of colon tumors.
39
Optimization of cell motility evaluation in scratch assay
Gotsulyak N. Ya., Kosach V. R., Cherednyk O. V., Tykhonkova I. O.,
Khoruzhenko A. I.
Institute of Molecular Biology and Genetics, NAS of Ukraine
Kyiv, Ukraine
i.o.tykhonkova@imbg.org.ua
Migration is one of the most important cell functions that includes any directed movement
and results in change of cell position within the body. It plays a key role in embryonic
development, wound healing, immune response and a number of others fundamental
biological processes. A scratch test is one of the most popular methods of classical cell
migration assay in monolayer culture. At the same time, the scratch assay has some
disadvantages that can be easily corrected.
Aim: The aim of the study is to justify and demonstrate that the detection of migration
distance calculated from experimental wound surface area is more objective and
minimizes the main drawbacks of scratch assay in contrast to the classical width
measuring.
Methods: The cell migration activity was estimated using a scratch assay.
Results: The scratch assay results expressed as routine detected width and mean distance
between opposite cell layers calculated from the area of experimental wound does not
significantly differ.
Conclusions: The procedure of wound width measure calculated from the area between
the edges of an experimental wound in scratch assay is more effective. Theoretically this
approach gives more accurate results, practically such detection is much more simple and
removes the main disadvantage of the method, namely human factor.
40
A role of SDF1/CXCR4 signaling and mesenchymal stem cells in
pathophysiology of facioscapulohumeral dystrophy
1,2,4* Dmitriev Petr, 2,3*Kiseleva Ekaterina, 2,3Kharchenko Olga,
2,3Ivashkin Evgeny, 4Carnac Gilles, 1,2Lipinski Marc, 4Laoudj-Chenivesse Dalila,
3Vasiliev Andrei and 1,2Vassetzky Yegor S.
1 UMR 8126, Univ. Paris-Sud, CNRS, Institut de Cancérologie Gustave-Roussy,
Villejuif, France
2 LIA1066 Laboratoire Franco-Russe de Recherches en Oncologie, Villejuif, France
3 N.K. Koltzov Institute of Developmental Biology, RAS, Mosocw, Russia
4 INSERM U1046, 371 Avenue du Doyen Gaston Giraud F-34295 Montpellier,
France,
vassetzky@igr.fr
Background: 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. Several genes identified in this region are overexpressed in FSHD
myoblasts including the double homeobox genes DUX4 and DUX4c.
Methods: We used immunostaining and cell mobility assays to assess the role of
SDF1/CXCR4 signaling in FSHD.
Results: We have shown that DUX4 induces an overexpression of the SDF1 ligand and
its receptor CXCR4 in FSHD muscle. This results in adipocytes and mesenchymal stem
cells being attracted to damaged muscles, and subsequent muscle invasion contributing to
the disease phenotype.
Conclusion: SDF1/CXCR4 signaling contributes to FSHD phenotype.
41
The role of resveratrol and miRNAs in regulation of proto-oncogenic
eukaryotic translation elongation factor 1A2 expression.
1Vislovukh A., 2,3,4Porto E., 1Gralievska N., 2,3,4Beldiman C., 2,3,4Harel-Bellan A.,
2,3,4Groisman I., 1El’skaya A., 1Negrutskii B.
1State Key Laboratory on Molecular and Cell biology, Institute of Molecular Biology
and Genetics, National Academy of Sciences of Ukraine, Kiev, Ukraine.
a.a.vislovukh@imbg.org.ua
2Université Paris Sud, Laboratoire Epigenetique et Cancer, Formation de Recherche en
Evolution 3377, Gif-Sur-Yvette, France.
3Centre National de la Recherche Scientifique (CNRS), Gif-Sur-Yvette, France.
4Commissariat à l’Energie Atomique (CEA), Saclay, F-91191 Gif-sur-Yvette, France.
Aim: The eukaryotic translation elongation factor 1A2 (eEF1A2), besides functioning in
translation, is a known proto-oncogene which is detected in human tumors of different
localization. The overexpression of the gene is not related to the genetic or epigenetic
modifications in the eef1a2 locus suggesting a possibility of posttranscriptional regulation.
We proposed that the eEF1A2 expression in cancer tissues is controlled by miRNAs.
We aimed to discover miRNAs that can regulate eEF1A2 expression and to investigate
the effect of the miRNA-mediated eEF1A2 down-regulation on the MCF7 breast cancer
cell line proliferation.
Methods: Changes in the miRNAs and EEF1A2 mRNA levels were quantified by qPCR.
Dual-luciferase assay was applied to detect the influence of miRNAs on the EEF1A2
expression. For proliferation assay, the stained cells were counted using the Operetta®
High Content Imaging System (Perkin Elmer).
Results: We found that eEF1A2 is a direct target of two oncosuppressor miRNAs miR-
663 and miR-744. Both miRNAs were able to downregulate the expression of luciferase
gene attached to the 3’UTR of EEF1A2. Moreover, overexpression of miR-663 and miR-
744 in MCF7 cells reduced the endogenous EEF1A2 mRNA level by 45 % and 70 % and
decreased the eEF1A2 protein level by 20 and 40 % respectively. In line with that,
overexpression of both miR-663 and miR-744 negatively influenced the proliferation of
MCF7 cancer cells.
miR-663 is well-known oncosuppressor which is up-regulated during treatment of THP-1
and SW-480 cells with resveratrol. Resveratrol also inhibits the eEF1A2 expression in
serum- or insulin-stimulated PA-1 cells. We observed direct correlation between the up-
regulation of miR-663 and miR-744 and decrease in the eEF1A2 amount in resveratrol-
treated MCF7 cells. Transfection of MCF7 cells with miR-663 and miR-744 inhibitors
eliminated inhibitory effect of the drug, suggesting that resveratrol may influence the
EEF1A2 expression through miRNA-dependent pathway.
Conclusion: Our findings demonstrate that expression of proto-oncogenic eEF1A2 is
controlled by miR-663 and miR-744. Thus, essential increase in the eEF1A2 amount in
tumor tissues may be caused by the loss of microRNA-mediated post-transcriptional
control.
42
Rearrangement of intracellular pathways in tumor cells caused by RNase A
treatment: full transcriptome analysis
Mironova N.L., Patutina O.A., Brenner E.V., Kurilshikov A.M., Vlassov V.V.,
Zenkova M.A.
Institute of Chemical Biology and Fundamental Medicine Siberian Branch of Russian
Academy of Sciences,
Lavrentieva ave. 8, Novosibirsk, Russia
marzen@niboch.nsc.ru
Recently, we have shown that the pancreatic RNase A is capable of inhibiting tumor and
metastasis growth and one of the mechanisms is associated with the change of miRNA
profiles in the blood and tumor cells (Mironova et al, 2013). Here, in order to find
intracellular targets of RNase A, we performed an analysis of whole transcriptome of
Lewis lung carcinoma tissue after treatment of tumor-bearing mice with RNase A by
high-throughput sequencing using SOLiD 5.5 platform.
Analysis of sequencing data revealed that inhibition of tumor and metastasis growth by
RNase A is accompanied by up-regulation of 320 genes and down-regulation of 645 genes
in tumor cells. Top 20 of the mostly down-regulated genes includes snoRNA class (both
C/D and H/ACA boxes) elevated in tumor cells; genes encoding proteins with cell-growth
promoting and transforming activity (PARK7), genes encoding proteins functioned as
negatively regulators of MAP kinase superfamily and tumor suppressor (DUSP6), which
is associated with cellular proliferation and differentiation, anti-apoptotic genes (LCN2).
Among top 20 of the mostly up-regulated genes are negative regulators (FAM89B) of
TGF-beta signaling known to exert metastasis-promoting activity associated with
epithelial-to-mesenchymal transition, modulation of cancer microenvironment and
extracellular matrix components, inflammation and immune suppression at the later stage
of tumor progression. Also we observed the increase in expression of genes encoding
phosphatidylserine receptors (JMJD6) involved in phagocytosis of apoptotic cells, p53-
inducible proteins (STEAP3), regulators of p53/TP53 stability (USP10).
Obtained data give the evidence that RNase A caused in tumor cells rearrangement of
intracellular pathways associated with malignant transformation and tumor escape from
immunological surveillance towards normalization.
This work was supported by RAS programs “Molecular and cellular biology” and
“Fundamental sciences to medicine 2012-24”, Scientific schools №1350.2014.4,
Fellowship 2307.2012.4 and by RFBR grant 14-04-0107a.
43
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