Symposium «Molecular Mechanism of Human Pathologies»
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Інститут молекулярної біології і генетики НАН України
2009
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Symposium «Molecular mechanism of human pathologies»
29 May–1 June, 2009
Kyiv, Ukraine
Foreword
The breakthroughs in modern medicine have led to a significant increase in the lifespan and quality of life. This
has also changed the spectrum and frequency of different human diseases. The infectious diseases, the principal
cause of mortality a century ago, do not represent a major threat to humanity these days. At the same time, the
diseases that have been relatively rare at in the XIX and in the first part of the XX century, have emerged as major
challenges for modern medicine. These diseases include cancer, neurodegenerative and autoimmune diseases.
They also represent a challenge to modern society, as their treatment is a heavy financial burden on public health
systems.
A common point between these diseases is the absence of efficient treatment in most cases and the dependence
of outcome on early diagnostics of the disease. Indeed, most cancers develop for several years before appearance of
first clinical symptoms. The same is true for neurodegenerative diseases (Parkinson’s and Alzheimer’s diseases,
hyperprolactinemia) that develop during 20–30 years before attaining a clinical stages, and the clinical symptoms
appear when 80 % of the specific neurons are attained. Therefore, the understanding of early mechanisms of these
diseases is particularly important for the success of treatment and for development of new drugs.
Interestingly, some of the early mechanisms of cancer, autoimmune and neurodegenerative diseases are
similar, eg mutations and translocations in immunoglobulin genes may provoke both autoimmune diseases and
some cancers. Therefore, cooperation between laboratories working on these subjects may provide new insights
into early mechanisms of these diseases.
The meeting «Early mechanisms in human disease» held in Kiev (Ukraine) on May 29 – June 1 has united
Ukrainian, Russian, and French scientists working on cancer, neurodegenerative and autoimmune diseases. This is
the second meeting in the series, the first one was held in Moscow in October, 2007. The quality of the meeting is
reflected in the abstracts published in this issue of «Biopolymers and Cell». I would also thank the local organizing
committee and Iryna Pirozhkova (IGR, Villejuif, France), for the wonderful organization of the meeting.
Yegor Vassetzky
319
ISSN 0233-7657. Biopolymers and Cell. 2009. Ò. 25. ¹ 4
Ó Institute of Molecular Biology and Genetics NAS of Ukraine, 2009
Dynamic microtubules are necessary for the assembly
of YB-1 containing stress granules
K. G. Chernov1, 2, A. Barbet1, L. Hamon1, L. P. Ovchinnikov2, P. A. Curmi1 , D. Pastre1
1Laboratoire Structure-Activite des Biomolecules Normales et Pathologiques INSERM/UEVE U829
EA3637, Evry, 91025 France
2Institute of Protein Research, Russian Academy of Sciences
Pushchino, Moscow Region, Russian Federation, 142290
pcurmi@univ-evry.fr
Following exposure to various stresses (arsenite, UV, hyperthermia, hypoxia), mRNAs assemble
into large cytoplasmic bodies known as «stress granules», in which mRNAs and associated proteins
may be processed by specific enzymes for different purposes like transient storing, sorting,
silencing or other still unknown processes. To limit mRNA damage during stress, the assembly of
micrometric size granules has to be rapid and indeed it takes only about 10–15 min in living cells.
However, such a rapid assembly is against the rules of hindered diffusion in the cytoplasm, which
states that cytoplasmic bodies larger than 100 nm are almost immobile. Consequently, cells must
have developed mechanisms to allow the rapid formation of stress granules. In the present work,
using HeLa cells and YB-1 as a stress granule marker, we identified and explored experimentally
and theoretically three hypotheses to explain how cells overcome the limitation of hindered
diffusion: shuttling small mRNP particles from small to large stress granules, sliding mRNP
particles along microtubules, microtubule-mediated stirring large stress granules. The data favor the
latter hypothesis and underline the necessity of a dynamic microtubules network in the process of
YB-1 rich stress granule formation.
Ubiquitin and UBA domains: new players in
the coordination between transcription and mRNA nuclear
export
C. Dargemont
Institut Jacques Monod
F-75013 Paris, France
dargemont@ijm.jussieu.fr
Con com i tantly to their tran scrip tion, na scent tran scripts are loaded with mRNA bind ing pro teins
im pli cated in the pro cess ing and pack ag ing of mRNA into sta ble and ex port com pe tent mRNPs.
These co-transcriptional re ac tions are tightly cou pled and co or di nated by the RNA poly mer ase II
(RNAPII) C-ter mi nal do main, which acts as a re cruit ment plat form for the dif fer ent RNA pro cess -
ing ma chin er ies. In ad di tion, more and more ev i dence show that chromatin re mod el ling and histone
epigenetic marks play a fun da men tal func tion in RNAPII dy namic and RNA pro cess ing. Fully ma -
ture and cor rectly pack aged yeast mRNPs are then re leased from the tran scrip tion site and trans -
ported into the cy to plasm by the heterodimeric ex port re cep tor Mex67/Mtr2, which pro motes their
translocation through the nu clear pore com plexes. How ever, Mex67/Mtr2 is not di rectly re cruited to
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SYMPOSIUM «MOLECULAR MECHANISM OF HUMAN PATHOLOGIES»
mRNAs but rather re quires spe cific RNA-bind ing ad ap tors that as so ci ate with na scent tran scripts
dur ing the tran scrip tion pro cess. On the other hand, ubiquitin con ju ga tion has been re cently in -
volved in the reg u la tion of nu clear ex port of poly(A) + RNA. In ter est ingly, the yeast mRNA ex port
re cep tor Mex67 as well as its metazoan coun ter part har bours an Ubiquitin-As so ci ated do main
(UBA-Mex67) in its C-ter mi nus that par tic i pates in the in ter ac tion with FG-nucleoporins in nu clear
pores. Our re sults in di cate that UBA-Mex67 not only pro motes in ter ac tion with ubiquitin and
ubiquitylated pro teins but also the co-transcriptional re cruit ment of Mex67. Our stud ies led to the
iden ti fi ca tion of dis tinct UBA-Mex67 in ter act ing pro teins which are im pli cated in tran scrip tion
elon ga tion, histone methylation and 3' end pro cess ing, and chromatin re mod el ing re spec tively. We
now pro pose that the mRNA ex port re cep tor Mex67 could play a ma jor role in the co or di na tion of
dif fer ent steps of tran scrip tion and mRNA trans port through the dy namic in ter ac tion of its UBA do -
main with these var i ous ubiquitylated tar gets.
Sodium-dependent phosphate transporter NaPi2b
as ovarian cancer marker
R. Kiyamova1, V. Gryshkova1, G. Ritter2, L. Old2 , I. Gout3, V. Filonenko1
1Institute of Molecular Biology and Genetics NAS of Ukraine
150, Academika Zabolotnogo Str., Kyiv, Ukraine, 03680
2Ludwig Institute for Cancer Research
New York, USA
3University College London
London, UK
filonenko@imbg.org.ua
Mouse monoclonal antibody MX35 was developed against ovarian cancer. The antibody showed
homogeneous reactivity with approximately 90 % of human ovarian epithelial cancers and with a
limited number of normal tissues by immunohistochemistry. Although mAb MX35 has been used in
a number of clinical trials in ovarian cancer, it has been difficult to define the molecular identity of
MX35. We report here that mAb MX35 recognizes the sodium-dependent phosphate transport
protein 2b (NaPi2b) in human cancer cells. This conclusion is based on several lines of experimental
evidence, including 1) the identification of SLC34A2, the gene coding for NaPi2b, by
immunoscreening an ovarian cancer cell line cDNA expression library with mAb MX35; 2) mass
spectrometry sequencing of peptides obtained by fragmentation from mAb MX35 affinity-purified
antigen, which show complete sequence homology to amino acid sequences in NaPi2b; 3) selective
down-regulation of SLC34A2 gene expression by RNA interference and the resulting loss of mAb
MX35 binding to MX35-expressing human cancer cells; and 4) demonstration of a specific mAb
MX35 reactivity with recombinant fusion proteins and with synthetic peptides of the putative
largest extracellular loop of NaPi2b. We has further shown that NaPi2b in cancer cells is expressed
on the cell surface as a heavily N-glycosylated protein, with evidence of additional
post-translational modifications such as palmitoylation and formation of disulfide bridges in the
major extracellular loop. The membrane transporter molecules, such as NaPi2b, represent a new
family of potential cell surface targets for the immunotherapy of cancer with monoclonal
antibodies.
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Catalytic antibodies: from a laboratory concept
to physiological entities
A. Friboulet
Universite de Technologie de Compipgne CNRS UMR
6022 – Genie Enzymatique et Cellulaire-BP 20529 – 60205 Compipgne Cedex – France
alain.friboulet@utc.fr
Since the demonstration that catalytic antibodies with catalytic activities may be elicited using
transition state analogues, more and more groups have brought evidence that catalytic antibodies are
not only a chemical curiosity. Antibodies with various catalytic activities are present in the serum of
mammals after a physiological disorder or appear during the evolution of some pathology. We have
exploited the idiotypic network of the immune system to generate antibodies that mimic the
structure and function of different enzymes with esterase, amidase and protease activities. The
appearance of such catalysts in the serum raised the question of their possible beneficial or
deleterious physiological role. Different examples of isolation of catalytic antibodies in human
serum in relation with autoimmune diseases or after physiological perturbations are now available.
In some cases the presence of catalytic antibodies may be indicative for a positive progression of the
pathology or, at the contrary, as a deleterious factor. However, due to their early expression,
catalytic antibodies may be used in some cases as predictive biomarkers for the development of
related diseases.
Catalytic antibodies in pathology
A. Gabibov
Shemyakin&Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
16/10, Miklukho-Maklaya Str., Moscow, Russian Federation, 117984
gabibov@ibch.ru
Discovery of catalytic antibodies (abzymes) was a revolutionary event that created new junctions
between chemistry, biochemistry, immunology and pathology. The general concept of
complementarity introduced in life sciences by Emil Fisher explains the driving force of various
biological processes including genetic machinery, enzyme catalysis, ligand-receptor interaction,
antibody-antigen recognition etc. Creation of abzymes as a new class of biocatalysts is based upon the
intrinsic properties of immunoglobulin superfamily to produce complementary «molecular imprints»
using the hypervariability of CDRs. These «catalytic imprints» could be made from the stable
chemical analogs of transition state (TSA) of the enzyme reaction (Linus Pauling and Bill Jencks).
This approach was successfully developed by Richard Lerner group (Scripps Research Institute,
USA). The alternative way to create abzymes was proposed by author (Schuster et al., Science, 1992).
It is generated on the basis of the immunological network hypothesis of Niels Jerne and declares the
formation of anti-idiotypic antibody repertoire generated against the active site of corresponding
enzyme. This approach allowed us to create abzymes with acetylcholinesterase and protease activities
(Kolesnikov et al. PNAS, 2000, Pillet et al., J. Imm. meth. 2002, Ponomarenko et al., Biochemistry,
2007). In both cases one try to mimic the highly evolved enzymatic function by selection of antibody
catalysts from the vast repertoire of immunoglobulines (Reshetnyak et al, J. Am. Chem. Soc., 2007).
This may give rise to biocatalysts with new functions, previously unknown for common enzymes,
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SYMPOSIUM «MOLECULAR MECHANISM OF HUMAN PATHOLOGIES»
which may be very profitable for fine organic synthesis. This method stimulated our attempts to make
antibody-like acceptors for phosphorus-based poisons. Recombinant antibodies with such functions
have been obtained recently in this lab using chemical selection of «na¿ve» phage-display library. The
second advantage of abzyme field is the opportunity to make «catalytic vaccines». Traditional drugs
including antibiotics and other small-molecule compounds developed in the pre-biotechnology era
showed the limited success in a number of sever bacterial and viral infections. Numerous attempts to
combat HIV infection using drug therapy as well as classical vaccination turned out to be ineffective.
One of the targets for the novel therapeutic approach may be the main surface antigen, viral envelope
protein gp120. The specific cleavage of this protein can lead to the dramatic changes in the immune
response toward virus and decrease binding of HIV to CD4 receptor. This task, impossible to be
solved by enzyme therapy, may have an effective abzyme alternative (Durova et al. Mol. Immunol.,
2009). A novel approach for creating catalytic antibodies against pathogens is described
(Ponomarenko et al., J. Imm. meth; Biochemistry, 2002). This involves utilizing the autoimmune
disorder of SJL mice induced by myelin basic protein as a background for raising a protein-specific
catalytic response toward gp120. Site-specific abzyme-mediated cleavage of gp120 is demonstrated.
This approach developed in this laboratory can be considered as a general strategy to obtain a catalytic
vaccine to proteins of interest (Ponomarenko et al. Biochemistry, 2006). In our studies we firstly
showed that catalytic antibody formation has the strong intrinsic and still enigmatic links with
autoimmune diseases. The existence of DNA-specific abzymes in scleroderma, systemic lupus
erithematosus (SLE), rheumatoid arthritis and AIDS was described in this laboratory (Shuster et al.
Science 1992, Gololobov et al., PNAS,1995, Gololobov et al. Molecular Immunology, 1997). Very
recently the input of abzyme activity in neurodegeneration process has been demonstrated
(Ponomarenko et al. Immunology letters, 2006). Autoantibody-mediated tissue and cell destruction is
among the main features of organ-specific autoimmunity. We have described abzyme contribution to
neural tissue-specific antigens (Ag) degradation. AutoAb to myelin basic protein (MBP) from humans
with multiple sclerosis (MS) and SJL mice with experimental autoimmune encephalomyelitis (EAE)
exhibited site-specific antigen degradation (Ponomarenko et al. PNAS, 2006). AutoAb from patients
with the secondary progressive MS and highest scores on the expanded disability status scale (EDSS)
demonstrated augmented catalysis. An established MS therapeutic Copaxone® inhibited reaction in
vitro. AutoAb catalysis thus appears to be a specific feature associated with MS pathogenesis and
potential marker of disease progression (Belogurov et al. J. Immunol. 2008).
Oncogenic redundancy is a significant obstacle
to the success of target therapy
V. M. Kavsan1, V. V. Dmitrenko1, O. I. Boyko1, T. A. Malisheva2, V. D. Rozumenko2,
Y. A. Zozulya
1Institute of Molecular Biology and Genetics NAS of Ukraine
150, Academika Zabolotnogo Str., Kyiv, Ukraine, 03680
kavsan@imbg.org.ua
2A. P. Romodanov Institute of Neurosurgery
32, Manuilskoho Str., Kyiv, Ukraine, 04050
brain@neuro.kiev.ua
In the 21st century the cancer therapy will be changed from «one size fits all» approach to the
personalized one, in which each patient is treated according to the specific genetic defects in his
tumor. To enhance glioblastoma (GB) marker discovery, using Serial Analysis of Gene Expression
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SYMPOSIUM «MOLECULAR MECHANISM OF HUMAN PATHOLOGIES»
(SAGE), we found 676 genes with 2-fold change of expression in GB, the most aggressive tumor of
human brain. The gene-expression data were used to create a prognostic GB gene-expression
signature. Unfortunately, reviewing available results obtained by microarrays we found that they
were not always reproducible and the lists of detected genes have quite poor overlapping. Besides
possible methodological mistakes, it is more likely that the signatures created by different
laboratories use different genes to monitor the same biological processes. Oncogenic redundancy is
recognized today as a significant obstacle to for a target therapy. A central role that the insulin-like
growth factor system plays in a tumor progression makes it a target for the cancer therapy, but the
enhanced IGF-I gene expression in GB was not found, instead, we revealed CHI3L1 among genes
with the most pronounced changes in expression. Both, YKL-40 and IGF-I, may activate PI3K and
MAPK signaling cascades, which are associated with the control of mitogenesis (Recklies et al.,
2002). The expression of homological YKL-39 and CD74, EGFR, CTGF, IGFBP5, IGFBP7, IGF-II,
also considerably increased in GB, may activate the PI3K and MAPK cascades similarly to the
epidermal growth factor (EGF). Concerning the IGF-II expression in ependimomas and
meningiomas, SAGE revealed extraordinary high content of one particular tag belonging to the
IGF2-associated mRNA. The open reading frame of this mRNA is unusually localized in the 3'UTR
of IGF-II gene and may encode some protein with completely unknown function. Attention of
investigators is directed mostly to genes with overexpression in tumors. However, identification of
genes which are underexpressed in brain tumors is not less important. 85 genes with 5-fold decrease
were found by SAGE, some of them may be considered as potential tumor suppressor genes. The
TSC-22 mRNA level is decreased in astrocytic gliomas. The differential expression of TSC-22 was
confirmed by Northern, RT-PCR and histochemical analysis. In contrast to YKL-40, TSC-22 may
serve as a mediator of TGF-b signaling. TGF-b binds to the receptor type II that leads to the
phosphorylation of the type me receptor, and then activates SMAD2 protein. TSC-22 activates
SMAD4 which in combination with SMAD2 is translocated to the nucleus that leads to arrested
growth and apoptosis, so the decrease in TSC-22 expression has anti-apoptotic consequences in GB.
Therefore, it is possible to see that not only morphological but also molecular heterogeneity of
malignant neoplasms and oncogene redundancy cause a necessity of building «passway
signatures». It might be very useful to create passway signature for each cancer type so that the
optimal treatment can be chosen for each patient.
Detection of key DNA repair proteins responsible
for cell radiosensitivity by chemically reactive
DNA intermediates
S. N. Khodyreva1, E. S. Plekhanova1, M. V. Sukhanova1, S. Marsin2,
J. P. Radicella2, O. I. Lavrik1
1Institute of Chemical Biology and Fundamental Medicine SB RAS
8, pr. Lavrentieva, Novosibirsk, Russian Feredation, 630090
lavrik@niboch.nsc.ru
2Department de Radiobiologie et Radiopathologie UMR 217, CNRS/CEA
F92265 Fontenay aux Roses, France
DNA is continuously damaged by endogenous reactive metabolites and exogenous chemicals or
ionizing radiation. A dedicated network of DNA repair mechanisms safeguards DNA integrity to
prevent the deleterious consequences of genetic degeneration, notably mutations and cell death
leading to cancer and aging. Some key proteins of different DNA repair mechanisms recognizing
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SYMPOSIUM «MOLECULAR MECHANISM OF HUMAN PATHOLOGIES»
DNA damages are known to be responsible for cell radiosensitivity. The most versatile approach to
study interaction of such proteins with damaged DNA is affinity labeling by chemically reactive
DNA with structural peculiarities mimicking proper DNA damages including clustered ones. We
have developed an approach to identify and monitor key DNA repair proteins in cell extracts.
Photoreactive branch point DNA intermediate of base excision repair (BER) has been used in
cellular extracts of mouse embryonic fibroblasts to identify composition of protein ensembles
interacting with damaged DNA and with the BER pathway intermediates. The main target proteins
covalently linked to photoreactive BER intermediate formed in cellular extracts were identified by
immunoprecipitation assay and by MALDI-MS as poly(ADP-ribose)polymerase 1 (PARP1), flap
endonuclease 1 (FEN1), DNA polymerase b (Polb), apurinic/apyrimidinic endonuclease 1 (APE1)
and the high mobility group box 1 protein (HMGB1). The functions of these proteins in BER were
analysed. Natural apurinic/apyrimidinic (AP) sites in DNAs were used as groups reactive to
proteins. PARP1, Ku80 subunit of Ku antigen and XRCC1 were shown to efficiently interact with
AP sites via Schiff base formation. PARP1 and Ku80 were identified in the cell extract as targets of
cross-linking to AP site by immunochemical methods and/or MALDI-TOF-MS. The specificity of
Ku antigen interaction with AP sites was proven by more efficient competition of DNA duplexes
with an analogue of abasic site than non-AP DNA. Ku80 was cross-linked to AP DNAs with
different efficiencies depending on the size and position of strand interruptions opposite to AP sites.
Ku antigen was shown to inhibit AP site cleavage by APE 1. We compared the results of dot-ELISA
based on anti-Ku80 antibodies and the levels of Ku80 cross-linking to AP DNA in the extracts
derived from HeLa and several melanoma cell lines. The efficiency of Ku80 trapping varies
considerably depending on the type of cell extract and correlates with the amount of Ku80 in the
extracts. Thus, AP site containing DNA can be used as an efficient tool to test the content of Ku
antigen in cell extracts. XRCC1 was shown to interact with AP site more efficiently in DNA
duplexes containing strand breaks opposite AP site. These proteins participate in repair of single-
and double-strand breaks in DNA and control sensitivity of cells to ionizing radiation. A new
unknown protein was shown to specifically interact with AP site closely opposed to analog of AP
site. The level of these proteins in cancer cells is considered as a relevant factor for the prediction of
tumor radiosensitivity and, therefore, AP DNA can be used as a test system for determining the
content of these proteins in tumor cells to estimate appropriateness of radiotherapy and treatment
outcome. This work was partially supported by RFBR, project N 07-04-00178; Siberian Branch of
the RAS, project N 104; FASI, contract N 02.512.11.224 and by RAS, Program «Molecular and
Cellular Biology».
Thyroid transcription factor-1 could be a target gene
of RET/PTC and canonical Wnt pathways in papillary thyroid
carcinoma
L. Massade
UMR 8121 Laboratoire d’Enzymologie et Biochimie Structurales – CNRS
IGR, 39 rue C Desmoulins, 94805 Villejuif Cedex
mirande@lebs.cnrs-gif.fr
Background and purpose: The RET/PTC1 oncoprotein, exclusively expressed in tumour cells, is an
early event in the development of Papillary Thyroid Carcinoma (PTC) but would not be sufficient
for the tumour progression. We hypothesize that Wnt/b-catenin signalling pathway may cooperate
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SYMPOSIUM «MOLECULAR MECHANISM OF HUMAN PATHOLOGIES»
with ret/PTC oncogene to promote the disease. Therefore, we assessed the cross-talk between the
RET/PTC and the canonical Wnt pathways on the thyroid transcription factor-1 (TTF-1) involved in
PTC relapse. Methods: LiCl and SB216763, inhibitors of GSK-3, were used to activate the Wnt
signalling. SiRNAs sequences were designed to knockdown RET/PTC1 oncogene. The cross-talk
between RET/PTC and Wnt pathways was assessed by combining LiCl or SB216763 with the
siRNA RET/PTC1. TTF-1, RET and b-catenin expressions were analysed by Q-RTPCR and western
blot in the TPC-1 cell line that constitutively express ret/PTC1. Results: Both LiCl, SB216763 and
siRNA ret/PTC1 induced the TTF-1 gene expression. The b-catenin protein was induced by LiCl and
SB216763; the RET gene and protein expression were inhibited by siRNA RET/PTC1. Combined
treatments leads to the preservation of ret/PTC1 gene inhibition and minimization of the TTF-1 gene
induction. Discussion and conclusion: TTF-1 would be a target gene of the Wnt/b-catenin and
RET/PTC pathways. Treatment with siRNA RET/PTC1 inhibits the oncogene expression but
induces a major gene involved in tumour aggressiveness. In order to use siRNA RET/PTC1 as a
targeted therapeutic against PTC, we need to define them to inhibit the molecular components of
Wnt pathway responsible for the TTF-1 activation.
Viral hijacking of mitochondrial lysyl-tRNA synthetase
L. Kobbi1, G. Octobre1, R. Freguja1, M. Kaminska1, V. Shalak1,2, M. Mirande1
1Laboratoire d’Enzymologie et Biochimie Structurales – CNRS
1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France
2Institute of Molecular Biology and Genetics NAS of Ukraine
150, Academika Zabolotnogo Str., Kyiv, Ukraine, 03680
mirande@lebs.cnrs-gif.fr
Human immunodeficiency virus type 1, HIV-1, is a retrovirus. Its genome is made of two
single-stranded RNA molecules. Reverse transcriptase encoded by HIV-1 uses tRNA3
Lys of the host
cell to start the reverse transcription of its RNA genome into proviral DNA. During assembly of
HIV-1 particles, tRNA3
Lys is taken up along with lysyl-tRNA synthetase (LysRS) from the host cell,
the tRNA binding protein that specifically aminoacylates the different tRNALys isoacceptors. In
human, the cytoplasmic and mitochondrial species of LysRS are encoded by a single gene by means
of alternative splicing. We showed that polyclonal antibodies directed to the full-length cytoplasmic
enzyme equally recognized the two enzyme species. We raised antibodies against synthetic
peptides, that allowed to discriminate between the two enzymes, and found that mitochondrial
LysRS is the only cellular source of viral LysRS. Mitochondrial localization of LysRS in HeLa cells
is altered after addition of the auxiliary viral protein Vpr into the culture medium. These results open
new routes toward the understanding of the molecular mechanisms involved in the specific
packaging of tRNA3
Lys into viral particles. Molecular mechanisms involved in viral hijacking of
molecules of the host cell will be described.
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SYMPOSIUM «MOLECULAR MECHANISM OF HUMAN PATHOLOGIES»
Two tissue-specific isoforms of translation elongation factor
1A: why one of them is oncogenic?
O. Novosylna1, T. Lukash1, P. Futernyk1, A. Pogribna1, S. Gavrylenko1, I. Serdyuk2,
A. Timchenko2, E. Tiktopulo2, V. Blizhyuk3, G. Panasyuk1, B. Negrutskii1
1Institute of Molecular Biology and Genetics NAS of Ukraine
150, Academika Zabolotnogo Str., Kyiv, Ukraine, 03680
2Institute of Protein Research
Pushchino, Russian Federation
3Western Michigan University
Kalamazoo, Michigan, USA
negrutskii@imbg.org.ua
eEF1A is the main component of eukaryotic translation elongation machinery. It is responsible for
correct binding of aminoacyl-tRNA to A site of ribosome. In tRNA channeling cycle eEF1A forms
the complex with GTP and aminoacyl-tRNA and delivers the latter to the 80S ribosome. After the
translocation and transpeptidation steps are finished deacylated tRNA stays in the ribosomal E site.
Thus, other role of eEF1A in elongation cycle was suggested to pick up deacylated tRNA from E site
and to deliver it back to aminoacyl-tRNA synthetase for recharging. Multienzyme complex of
aminoacyl-tRNA synthetases associates in cell with multicomponent eEF1H complex containing
GDP/GTP exchanging subunits for eEF1A, thus aminoacylation of tRNA and GDP/GTP exchange
in eEF1A molecule occur simultaneously. As a result, the ternary complex aminoacyl-
tRNA*eEF1A*GTP is formed and ready to go to A site of the ribosome. There are two 97 % similar
isoforms of eEF1A (A1 and A2) which are tissue and development-specific. A1 is expressed in all
tissues during development but is absent in adult muscles, heart and neurons. A2 is found instead in
these tissues. A2 reveals oncogenic properties when appears in non-specific tissue (ovary, pancreas
etc.). A2 was found to be highly expressed in ovary, pancreas and breast tumors. Rodent fibroblasts
overexpressing A2 demonstrated enhanced focus formation, anchorage-independent growth and
decreased doubling time. In addition, A2 expression made NIH3T3 fibroblasts tumorigenic and
increased the growth rate of ES-2 ovarian carcinoma cells xenografted in nude mice. Importantly,
the copy number at the EEF1A2 locus does not always correlate with expression level of the gene.
No functional mutations were found and the EEF1A2 gene is unmethylated in both normal and
tumour DNA, showing that overexpression is not always dependent on genetic or epigenetic
modifications at the EEF1A2 locus. Thus, we deal with two highly similar proteins both of which are
routinely expressed in different tissues and apparently possess the same translation function.
However, appearance of A2 in non-specific for that isoform tissue leads to carcinogenesis. There
should be an abnormal competition between the isoforms that results in unfavorable outcome. What
is the molecular background for such a competition between two very similar proteins? No profound
difference between the isoforms was found in different translation tests (GDP/GTP exchange,
intrinsic GTPase activity, kinetics of poly(U) translation, tRNA binding). A1 and A2 were also
observed to possess similar chaperon-like activity which rules out the involvement of these kinds of
activity in providing cancer-related properties of A2. However, we have found more pronounced
ability of the A2 isoform to interact with SH2 and SH3 domains of different molecules involved in
phosphotyrosine-mediated signaling pathways. Vise versa, A2 isoform completely lost the ability
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SYMPOSIUM «MOLECULAR MECHANISM OF HUMAN PATHOLOGIES»
to interact with calmodulin in the presence of Ca2+ while A1 demonstrated a specific complex
formation in different tests. These findings indicate a possibility of non-similar involvement of A1
and A2 in cell regulation which could be important for cancer development. To decipher structural
features which might contribute to the distinct signaling abilities we have analyzed spatial
structures of A1 and A2. Striking conformational dissimilarity of the isoforms was found by X-ray
scattering and confirmed by scanning microcalorimetry. A2 isoform behaves like a globular protein
in solution while A1 possesses an extended conformation. Moreover, atomic force microscopy
revealed the pronounced hydrophobicity of the eEF1A2 surface but hydrophilic nature of eEF1A1
one. Since, apart from translation, eEF1A is apparently involved in cell signaling, actin and tubulin
cytoskeleton rearrangement, apoptosis, nuclear transport, ubiquitin-dependent proteolytic system,
heat shock, mRNA transport, interaction with viral genomic RNA etc. it is difficult at present to
determine exactly which regular cellular process is influenced by unusual appearance of A2 un some
tissue. We have shown the background for the competition between A1 and A2 isoforms might not
be limited by the minor dissimilarity of primary structures but apparently includes the difference in
their spatial organization and surface hydrophobicity. All those factors could have an impact on the
oncogenic properties of A2 isoform facilitating interactions which are not present in the tissues
containing generally A1 protein only, or excluding contacts which are regular for A1 protein.
Development of surface plasmon resonance biosensor
for diagnostics of Ph+ leukemia
A. E. Rachkov, Yu. Holodova, G. D. Telegeev, A. P. Soldatkin
Institute of Molecular Biology and Genetics NAS of Ukraine
150, Academika Zabolotnogo Str., Kyiv, Ukraine, 03680
a_soldatkin@imbg.org.ua
This presentation describes important steps for the development of optical biosensor based on
Surface Plasmon Resonance (SPR) for detection of the bcr-abl gene related to Ph+ leukemia. A
bioselective element (a specific recognition layer on the sensor surface) of SPR spectrometer for
label-free detection of DNA sequence of the hybrid bcr-abl gene is firstly developed using
thiol-modified oligonucleotides. Preparation of the bioselective element and subsequent
hybridization events were monitored in real time by SPR spectrometer «Plasmon SPR-4m». The
thiol-modified single-stranded oligonucleotide mod-Ph is shown to be immobilized efficiently on a
gold sensor surface of the SPR spectrometer. Specific hybridization between immobilized mod-Ph
and complementary oligonucleotide P1 was detected, whereas no sensor response to non-
complementary target was observed. The experimental results on interactions between the
oligonucleotides under study are in good agreement with theoretical calculations of thermodynamic
parameters of these interactions.
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BMP7 is involved in Mantle cell lymphoma secondary drug
resistance
V. Camara-Clayette1,2, S. Koscielny3, Ribrag2-4
1Institut Federatif de Recherche (IFR54), Institut Gustave Roussy
Villejuif, France
2Institut National de la Sante et de la Recherche Medicale (INSERM) unite (U)790; Universite Paris Sud; Institut Gustave Roussy
Villejuif, France
3Service of Biostatistics and Epidemiology, Department of Translational Research; Institut Gustave Roussy
Villejuif, France
4Departement de Medecine; Institut Gustave Roussy
Villejuif, France
ribrag@igr.fr
The identification of genes involved in the mechanisms of secondary drug resistance is a challenge.
In order to select such genes in mantle cell lymphomas (MCL), we designed a gene profiling
experiment based on the use of paired tissue samples obtained before and after treatment of 5
patients. For each gene of each patient, the variation in gene expression was estimated as a ratio
between the expression after treatment and the expression before. For each gene, the mean variation
was estimated for patients initially resistant and for responders who developed a secondary drug
resistance. Genes were considered relevant when the ratio between the variation in expression
measured in initially resistant patients and the variation in secondary resistant patients was greater
than 2, with an associated P value less than 0.01. Nine relevant genes were selected, BMP7 was the
only one with fold change > 5 and the only one with a significantly increased expression at relapse in
patients who developed a secondary resistance. The validation of BMP7 as a key gene involved in
secondary resistance was performed using cell line cultures. In the MCL cell line expressing BMP7,
BMP7 RNA interference markedly increased apoptosis (P < 0.01) after exposure to Bortezomib and
Cytarabine.
Alternative splicing regulation of intersectin functional
interactions in humans
L. Tsyba1, M. Dergai1, I. Skrypkina1, O. Nikolaienko1, O. Dergai1, S. Kropyvko1,
T. Gryaznova1, O. Novokhatska1, D. Morderer1, V. Gorchev2, L. Drobot2,
L. Matskova3, G. Winberg3, J. Moreau4, O. Boldyryev5, M. Veselovskiy5,
S. de la Luna6, A. Rynditch1
1Institute of Molecular Biology and Genetics NAS of Ukraine
150, Academika Zabolotnogo Str., Kyiv, Ukraine, 03680
2Palladin Institute of Biochemistry NAS of Ukraine
9 Leontovicha Str., Kyiv, Ukraine, 01601
3Karolinsk a Institute, Stockholm, Sweden
4Institut Jacques Monod, Paris, France
5Bogomolets Institute of Physiology NAS Ukraine
4 Bogomoltsa Str., Kyiv, Ukraine, 01024
6Center for Genomic Regulation, Barcelona, Spain
a.v.rynditch@imbg.org.ua
Neurodegenerative diseases are linked to abnormalities in endocytosis and vesicle-trafficking
disorders. The enlarged early endosomes were shown in mouse model of Down Syndrom (DS).
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Neurons of individuals with DS demonstrate similar defects as in Alzheimer Disease (AD) patients.
Intersectin1 (Itsn1) gene is located on human chromosome 21 in so-called critical region for DS and
is over expressed in DS individuals. Moreover, over expression of ITSN1 enhances huntingtin
aggregation and neurodegeneration at Huntington’s disease. ITSN1 is a membrane-associated
adaptor protein involved in clathrin-mediated endocytosis, mitogenic signalling, and actin
cytoskeleton rearrangements. Structural and functional diversity of multidomain intersectin in
vertebrates is generated by alternative splicing, by usage of alternative promoters and presence of
paralogous gene Itsn2. We identified an alternative transcription initiation site in the fifth intron of
Itsn1. We revealed 17 alternative splicing events for pre-mRNA of Itsn1 gene. Eleven of them
presumably are subjected to degradation whereas six affect different functional domains of ITSN1.
Among novel isoforms the shortest one, ITSN1-22a, with C-terminus presented by mobile element
LINE2C has been localized in endomembrane compartments. It forms a complex with ubiquitously
expressed ITSN1-s isoform. Comparing to the essential inhibition by ITSN1-s and ITSN1-22a,
C-terminus of ITSN1-22a has only slight effect on endocytosis. Nevertheless, this C-terminus is
turned out to bind the SH3 domain of amphiphysin and the SH3A domain of intersectin suggesting
participation of C-terminus in regulation of dynamin-amphiphysin and dynamin-ITSN interactions.
An expression analysis of alternatively spliced transcripts revealed brain- and development-specific
ITSN1 isoform with exon 20 inclusion affecting the SH3A domain. This exon encodes five
additional amino acids in the n-Src loop of the SH3A domain and altered its binding properties. The
protein partners were divided into three groups: with higher affinity to the brain-specific isoform,
with lower and unchanged ones. Changes in n-Src loop due to insertion of additional amino acids are
assumed to evoke rearrangement of charged groups engaged in interaction interface affecting the
interaction with PRD containing charged side chains. Set of pull-down experiments with mutants of
the SH3A domain confirmed it. Transcripts with exon 20 are detectable at the early stages of brain
development. We investigated in details and compared interaction interfaces of ITSN1 and ITSN2
for their binding partners. Some differences were observed in binding for c-Cbl and Sema6a, no
differences were found for Sos1 and dynamin 1, Ruk/CIN85 is a specific partner for ITSN1.
Furthermore, ITSN1-s, ITSN2 and ITSN1-22a were co-localized significantly. Thus, ITSN proteins
provide complex interfaces for interaction between basic endocytic machinery and the members of
different cell processes such as signaling, sorting and cytoskeleton rearrangements which are
largely regulated by alternative splicing.
Poly(ADP-ribosyl)ation reactions in the control
of the balance between life and death
E. Fouquerel, A. Hakme, D. Quenet, C. Boehler, H. K. Wong, R. El-Ramy, V. Gasser,
J. C. Ame, A. Bresson, F. Dantzer, V. Schreiber
Departement Integrite du Genome, FRE3211, CNRS, Universite de Strasbourg ESBS
Bd S. Brant, BP 10413, 67412 Illkirch, France
schreiber@esbs.u-strasbg.fr
Poly(ADP-ribosyl)ation is a post-translational modification of proteins mediated by poly(ADP-
ribose) polymerases (PARPs), involved in DNA repair, transcription, mitotic segregation, and
telomere homeostasis and cell death. PARP-1 is a molecular sensor of DNA breaks, which uses
NAD+ to synthesize poly(ADP-ribose), a signal molecule involved in the recruitment of base
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excision/single-strand break repair factors at the damaged site. We have identified another DNA
damage dependent PARP, PARP-2. Cellular and animal models deficient in PARP-1 and PARP-2
developed in the laboratory revealed the redundant but also complementary functions of these two
proteins in the surveillance of the genome integrity. More recently, we identified specific and
essential roles of PARP-2 in cell differentiation. Our laboratory has established in silico the
sequence of novel PARPs, bringing to 17 the number of PARP family members. The diversity of
functional domains associated to the PARP domain, the variety of subcellular localizations and the
novel biological functions discovered, extend considerably the field of poly(ADP-ribosyl)ation
reactions to various aspects of the cell biology. Many studies revealed the importance of PARPs and
PARG, the poly(ADP-ribose) degrading enzyme poly(ADP-ribose) glycohydrolase, to control
poly(ADP-ribose) levels regulating the balance between life-and-death in response to DNA
damage. The expression of PARG was knocked down in HeLa cells, leading to poly(ADP-ribose)
accumulation. PARG deficient cells showed increased radiosensitivity caused by single and double
strand breaks repair defect and alteration of mitosis progression due to mitotic spindle checkpoint
defect. Irradiated PARG deficient cells displayed centrosome amplification leading to mitotic
supernumerary spindle poles, and accumulated aberrant mitotic figures, which induced either
polyploidy or cell death by mitotic catastrophe. Our results suggest that PARG could be a novel
potential therapeutic target for radiotherapy.
Mechanisms of the brain plasticity in Parkinson’s
disease and hyperprolactinemia
M. Ugrumov
Koltzov institute of Developmental Biology
26, Vavilova Str., Moscow, Russian Federation, 119991
P. K. Anokhin Institute of Normal Physiology RAMS
6 bd 4, Bolshaya Nikitskaya Str., Moscow, Russian Federation, 103008
michael.ugrumov@mail.ru
In addition to the monoaminergic (MA-ergic) neurons possessing the whole set of enzymes of MA
synthesis from the precursor amino acid and the MA membrane transporter, the neurons partly
expressing the monoaminergic phenotype have been first discovered almost twenty years ago. Most
of the neurons express individual enzymes of MA synthesis having no MA transporter. These
so-called monoenzymatic neurons are widely distributed throughout the brain in adult mammals
being even more numerous than MA-ergic neurons. Individual enzymes of MA synthesis are
expressed continuously or transiently over certain periods of ontogenesis and in adulthood under
functional insufficiency of the MA-ergic neurons, e. g. under their chronic stimulation or in certain
neurodegenerative diseases. The above data suggest an important functional role of monoenzymatic
neurons. Some monoenzymatic neurons appear to co-express the monoamine transporters. Most
numerous monoenzymatic neurons possess enzymes of dopamine (DA) synthesis, tyrosine
hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC). TH and AADC are
enzymatically active in most monoenzymatic neurons being capable to convert L-tyrosine to
L-DOPA and L-DOPA to DA or serotonin, respectively. L-DOPA produced in monoenzymatic
TH-neurons is supposed to play a role of a neurotransmitter or a neuromodulator providing its action
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on the target neurons via catecholamine receptors. Moreover, L-DOPA released from the
monoenzymatic TH-neurons can be captured by monoenzymatic AADC-neurons or dopaminergic
and serotoninergic neurons for DA synthesis (Kannari et al., 2006). Such cooperative synthesis of
MAs is considered as a compensatory reaction under the failure of MA-ergic neurons, e. g. in
neurodegenerative diseases like hyperprolactinemia and Parkinson’s disease which are developed
primarily because of degeneration of DA-ergic neurons of the tuberoinfundibular system and the
nigrostriatal system, respectively. Noteworthy, hyperprolactinemia is followed with time by the
normalization of prolactin secretion due to stimulation of DA synthesis by the neurons of the
tuberoinfundibular system, most probably because of the turning on cooperative synthesis of DA by
monoenzymatic neurons. The same compensatory mechanism is supposed to be used under the
failure of the nigrostriatal DA-ergic system that is manifested by the increased number of
monoenzymatic neurons in the striatum of animals with pharmacologically-induced parkinsonism
and in humans with Parkinson’s disease. Expression of the enzymes of MA synthesis in
non-monoaminergic neurons is controlled by intercellular signals such as classical
neurotransmitters (catecholamines), neurotrophic factors (brain-derived neurotrophic factor,
glia-derived neurotrophic factor), and perhaps hormones (prolactin, estrogens, progesterone). Thus,
a substantial number of the brain neurons express partly the monoaminergic phenotype, mostly
individual complementary enzymes of MA synthesis, serving to produce MAs in cooperation that is
considered as a compensatory reaction under the failure of MA-ergic neurons.
Intranuclear relocalization of translocated regions:
a common mechanism of oncogene activation in lymphomas?
J. Allinne1, O. V. Iarovaia1, 2, A. Pichugin1, N. Petrova2, V. Clayette3, V. Ribrag3,
S. V. Razin2, M. Lipinski1, Y. Vassetzky1
1UMR8126, CNRS, Institut de Cancerologie Gustave Roussy; Universite Paris XI Sud
Villejuif, France
2Institute of Gene Biology RAS
34/5 Vavilov Str., Moscow, Russia, 119334
3Institut de Cancerologie Gustave Roussy
Villejuif, France
vassetzky@igr.fr
Lymphoma is a cancer involving cells of the immune system; it represents over 35 different subtypes.
We are particularly interested in two lymphoma types, Burkitt's lymphoma (BL) and mantle cell
lymphoma (MCL). Most BLs carry a translocation of the c-myc oncogene from chromosome 8 to
either the immunoglobulin (Ig) heavy-chain region on chromosome 14 [t(8;14)] or one of the
light-chain loci on chromosome 2 (kappa light chain) [t(8;2)], or chromosome 22 (lambda light chain)
[t(8;22)]. This translocation juxtaposes Ig heavy chain gene (/IGH/, 14q32) sequences with the c-myc
locus, leading to an overexpression of a number of genes, including the c-myc gene. The translocation
site may be as far as 500 kb from the translocation point, which makes direct regulation unlikely. MCL
is closely associated with the translocation (11;14)(q13;q32). This translocation juxtaposes Ig heavy
chain gene (/IGH/, 14q32) sequences with the /BCL-1/locus, leading to an overexpression of a
number of genes, including the cyclin D1 gene (CCND1). However, CCND1 overexpression alone is
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not sufficient for hematopoietic transformation since mice transgenic for cyclin D1 do not develop
MCL. Recent transcriptome studies have revealed that several genes located in the vicinity of the
breakpoint on chromosome 11 are overexpressed in MCL cells. This general transcription
upregulation might be due to epigenetic processes as in the BL. Chromosomes 8 and 11 are located in a
largely heterochromatic region of the nucleus, while chromosome 14 is found in a more euchromatic
context. We propose that the t(11; 14) and t(8; 14) translocations induce the transposition of the 11q13
or 8q22 loci from an heterochromatic to an euchromatic region of the nucleus. This movement could
then cause the overexpression of the genes located on 11q13 and 8q22. We have studied the
localization of the rearranged (11;14)(q13;q32) locus in MCL and (8;14) locus in BL using 3D FISH
and have found that the translocated loci are relocalized within the nucleus towards the nuclear center
form the peripheral regions. The biochemical and epigenetic mechanisms which may be activated by
such translocations are currently under study and will be discussed.
Activation of p53 by MDM2 antagonists has differential
apoptotic effects on EBV-positive and EBV-negative Burkitt’s
lymphoma cells
B. Renouf, E. Hollville, A. Pujals, C. Tetaud, J. Garibal, J. Wiels
Interactions Moleculaires et Cancer, UMR 8126 CNRS, Univ Paris-Sud, Institut Gustave Roussy
Villejuif, France
wiels@igr.fr
P53 inactivation is often observed in Burkitt’s lymphoma (BL) cells, due either to mutations in p53
gene or to overexpression of the p53-negative regulator, MDM2. BL is closely associated with
Epstein-Barr virus (EBV) infection although not all cases are EBV(+). In EBV-infected cells, viral
DNA persists in cells, leading to the production of a limited number of viral proteins. Three patterns
of EBV latency have been defined, based on the differential production of these proteins. Most cases
of BL express the latency I programme (only EBNA1 protein produced), but some express the
latency III (all viral proteins produced). We have shown previously that in EBV(–) BL cells,
reactivation of p53, induced by reducing MDM2 protein level, led to apoptosis. We show here that
nutlin-3, a potent antagonist of MDM2, activates the p53 pathway in all BL cell lines harboring
wild-type p53, regardless of EBV status. However, nutlin-3 strongly induced apoptosis in EBV(–)
or latency I EBV(+) cells, whereas latency III EBV(+) cells were much more resistant. Furthermore,
we also show that prior treatment with sublethal doses of nutlin-3 sensitizes EBV(–) or latency I
EBV(+) cells to apoptosis induced by etoposide or melphalan, but protects latency III EBV(+) cells.
P21WAF1 which is overexpressed in the latter, is involved in this protective effect, as siRNA-me-
diated inhibition of p21WAF1 restored sensitivity to etoposide. Nutlin-3 protects latency III BL cells
by inducing a p21WAF1-mediated G1 arrest. Most BL patients with wild-type p53 tumors could
therefore benefit from treatment with nutlin-3 following a careful determination of the latency
pattern of EBV in infected patients.
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Gene repositioning upon genotoxic stress
M. Rubtsov1–3, J. Allinne3, Y.S. Vassetzky3, S.V. Razin1, 2, O. V. Iarovaia1
1Laboratory of Structural and Functional Organization of Chromosomes; Institute of Gene Biology RAS
34/5 Vavilov Str., Moscow, Russia, 119334
2Department of Molecular Biology, Lomonosov Moscow State University
GSP-1, Leninskie Gory, Moscow, Russian Federation, 119991
3UMR8126, CNRS, Institut de Cancerologie Gustave Roussy, Universite Paris XI Sud
Villejuif, France
iarovaia@inbox.ru
The translocation t(8;21)(q22;q22) affecting AML1 and ETO genes is known to be one of the
frequent chromosome translocations in childhood and treatment related acute myeloid leukemia. Up
to now, no data are available concerning mutual positioning of these particular genes in the nucleus
of a living cell as well as the mechanism of their realignment. Here we show that there is no marked
proximity between these two genes in the nuclei of normal human male Wbroblasts. Moreover,
these genes are located in different nuclear layers. Treatment of cells with VP-16 (etoposide), an
inhibitor of DNA topoisomerase II (Topo II) widely used in anticancer chemotherapy, causes the
ETO gene repositioning which allows AML1 and ETO genes to be localized in the same nuclear
layer close to the nucleoli. Inhibitor studies demonstrate that such an effect is likely to be connected
with the formation of stalled cleavable complexes Topo II–DNA. Finally, inhibition of ETO gene
repositioning by 2,3-butanedionemonoxime (BDM) suggests that this process depends on nuclear
myosin. Together, our data corroborate the so called «breakage Wrst» model of the origins of
recurrent reciprocal translocation. CHIP assay demonstrated that etoposide treatment and
consequent ETO gene relocalization to the perinucleolar space stimulated enrichment of nucleolar
form of DNA Topo II beta and nucleolin at Topo II cleavage site. A possible role of nucleoli in the
DNA rearrangement and maintaining genome stability is discussed.
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|
| id | nasplib_isofts_kiev_ua-123456789-5683 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-01T13:47:16Z |
| publishDate | 2009 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | 2010-02-02T11:27:27Z 2010-02-02T11:27:27Z 2009 Symposium «Molecular Mechanism of Human Pathologies» // Біополімери і клітина. — 2009. — Т. 25, № 4. — С. 319-334. — англ. 0233-7657 https://nasplib.isofts.kiev.ua/handle/123456789/5683 en Інститут молекулярної біології і генетики НАН України Хроніка та інформація Symposium «Molecular Mechanism of Human Pathologies» Article published earlier |
| spellingShingle | Symposium «Molecular Mechanism of Human Pathologies» Хроніка та інформація |
| title | Symposium «Molecular Mechanism of Human Pathologies» |
| title_full | Symposium «Molecular Mechanism of Human Pathologies» |
| title_fullStr | Symposium «Molecular Mechanism of Human Pathologies» |
| title_full_unstemmed | Symposium «Molecular Mechanism of Human Pathologies» |
| title_short | Symposium «Molecular Mechanism of Human Pathologies» |
| title_sort | symposium «molecular mechanism of human pathologies» |
| topic | Хроніка та інформація |
| topic_facet | Хроніка та інформація |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/5683 |