Abstracts
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Інститут молекулярної біології і генетики НАН України
2012
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| Назва видання: | Вiopolymers and Cell |
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irk-123456789-1568622019-06-20T01:26:31Z Abstracts Initial Scientific Meeting of the COMBIOM partner’s organizations "Molecular Events in Cancer and 2012 Article Abstracts // Вiopolymers and Cell. — 2012. — Т. 28, спецвипуск. — С. 8-24. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/156862 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Initial Scientific Meeting of the COMBIOM partner’s organizations "Molecular Events in Cancer and Initial Scientific Meeting of the COMBIOM partner’s organizations "Molecular Events in Cancer and |
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Initial Scientific Meeting of the COMBIOM partner’s organizations "Molecular Events in Cancer and Initial Scientific Meeting of the COMBIOM partner’s organizations "Molecular Events in Cancer and Abstracts Вiopolymers and Cell |
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Abstracts |
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Abstracts |
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Інститут молекулярної біології і генетики НАН України |
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2012 |
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Initial Scientific Meeting of the COMBIOM partner’s organizations "Molecular Events in Cancer and |
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http://dspace.nbuv.gov.ua/handle/123456789/156862 |
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Abstracts // Вiopolymers and Cell. — 2012. — Т. 28, спецвипуск. — С. 8-24. — англ. |
| series |
Вiopolymers and Cell |
| first_indexed |
2025-07-14T09:10:55Z |
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2025-07-14T09:10:55Z |
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1837612930721382400 |
| fulltext |
8
Gene expression profiling in MCL lymphoma: a focus on
t(11;14)(q13, q32) breakpoints
Barat Ana, Markozashvili Diana, Pichugin Andrei, 1Clayette Valerie,
1Ribrag Vincent, Lipinski Marc and Vassetzky Yegor
UMR 8126, Université Paris Sud, CNRS, Institut de cancérologie Gustave Roussy,
Villejuif, France.
1Institut de cancérologie Gustave Roussy, Villejuif, France.
vassetzky@igr.fr
Mantle cell lymphoma (MCL) is a malignant proliferation of B cells in
the mantle zone of lymphoid follicles. Cytogenetic analyses have revealed
that MCL is closely associated with the t (11; 14) (q13; q32). This
translocation juxtaposes Ig heavy chain gene (IGH, 14q32) sequences,
leading to an overexpression of a number of genes including the cyclin
D1gene (CCND1). This general transcription upregulation might be due to
epigenetic processes. Chromosome 11 is 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) (q13; q32)
translocation induces the transposition of the 11q13 locus from a
heterochromatic to a euchromatic region of the nucleus. This movement
could then cause the overexpression of the genes located on 11q13. We are
currently studying the nuclear dynamics of these regions, using 3D-FISH
on control and MCL derived B-lymphocytes in parallel with chromosome
mapping of MCL gene expression profiles of expression data, originating
from Gene Expression Omnibus (Affymetrix arrays). These transcriptome
studies have revealed that several genes located in the vicinity of the
breakpoint on chromosome 11 are overexpressed in MCL cells.
9
Genes participating in the development of brain tumors and
myeloproliferative diseases and their relation to signaling
pathways.
1Kavsan Vadym and 2Ribrag Vincent.
1Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and
Genetics, NASU, Kyiv, Ukraine.
2UMR 1009 Normals and Pathologics Haematopoiesis, Institut Gustave Roussy,
Villejuif, France.
kavsan@imbg.org.ua
Myeloproliferative and lymphoproliferative disorders (or neoplasms) are
a set of diseases characterized by the abnormal proliferation of myeloid
lineage of cells or lymphocytes, respectivelly. BMP7 is a new gene
involved in secondary drug resistance of mantle cell lymphoma, which was
identified by microarray analysis of clinical samples, and possesses an anti-
apoptotic function. High phosphorylation levels of the MAPK and PI3K
kinases pathway components were shown in primary tumor cell lines,
obtained from patients with increased BMP7 expression level.
In an effort to identify genes, which might be used as molecular markers
for glial tumors, we compared gene expression in glioblastoma and normal
adult human brain. Serial Analysis of Gene Expression found Chitinase 3-
like 1 (CHI3L1, HC gp-39, YKL-40) and Chitinase 3-like 2 (CHI3L2, YKL-
39) genes among the most abundant transcripts in glioblastoma. Significant
increase in 293 cells proliferation, correlated with phosphorylation of
extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase
B (AKT1), was shown after stable transfection by CHI3L1 oncogene.
Unexpectedly, dose dependent decrease in total DNA content and
[3H]thymidine incorporation were observed in 293 cells treated with
CHI3L2, probably confirming it’s tumor suppressor function.
The presence of mutual genes overexpressed in glioblastomas and mantle
cell lymphoma was shown by analysis of GEO databases. Analysis of their
expression in clinical samples is needed to evaluate whether they may be
used as markers for mentioned diseases. Investigation of their effect on
signaling pathways in mantle cell lymphomas and mechanisms which drive
their overexpression will provide a new insight into pathogenesis of this
myeloproliferative disorder.
10
Identification of non-canonical targets of mTOR during neuronal
development
Jaworski Jacek
International Institute of Molecular and Cell Biology, Warsaw, Poland
jaworski@iimcb.gov.pl
Mammalian target of rapamycin (mTOR) is a protein kinase that senses
nutrient availability, trophic factors support, cellular energy level, and
cellular stress and adjusts cellular metabolism accordingly. In neuronal
cells, mTOR activity is additionally controlled by neurotransmitters.
mTOR plays several roles in neuronal development and plasticity. It is
involved in the control of neuronal survival, neurite growth, and synapse
formation. It has been believed for long time that all this neuronal
functions of mTOR are attributable to its involvement in translational
regulation. On the other hand, several studies revealed that cellular
functions of yeast mTOR homologs go far beyond this canonical role. In
fact, TOR plays a role in transcription, autophagy, lipid metabolism,
mitochondrial function, cytoskeleton dynamics, and membrane trafficking.
Dendritic arbor development is an important step during neuronal
development as it defines major patterns of connectivity in the brain. We
have previously shown that indeed development of dendritic arbor is
regulated by mTOR and translational control is involved in this process.
However, mTOR forms two functionally different complexes in
mammalian cells: mTORC1 and mTORC2, and our previous studies did
not answer whether only mTORC1, which is responsible for translational
control, is important for dendritic growth. Our current research shows that
mTORC2 acts upstream mTORC1 during neuronal development. What is
more, our shRNA library screen shows that protein translation is not the
only cellular process controlled by an mTORC1 during dendritic arbor
growth. In fact, equally important are mTORC1-dependent control of
microtubule-actin interaction via CLIP-170 protein and mTORC1
involvement in membrane trafficking.
11
Endocytic proteins in control of signaling and transcription
Hupalowska Anna, Pyrzynska Beata, Torun Anna, Szymanska Ewelina,
Maminska Agnieszka, Miaczynska Marta
Laboratory of Cell Biology, International Institute of Molecular and Cell Biology,
Warsaw, Poland
miaczynska@iimcb.gov.pl
Accumulating evidence argues that many proteins governing membrane
sorting during endocytosis participate also in nuclear signaling and
transcriptional regulation. Some clathrin adaptors and endosomal proteins
were shown to undergo nucleocytoplasmic shuttling. Such endocytic
proteins can associate with nuclear molecules, changing their localization
and/or activity and may modulate the levels and specificity of gene
transcription, although in most cases it is not clear how interconnected the
nuclear and cytoplasmic pools of endocytic proteins are.
Two related adaptor proteins APPL1 and APPL2 represent an example of
proteins performing dual functions in endocytosis and transcriptional
regulation. On the one side, APPL proteins are localized to a subpopulation
of Rab5-positive early endosomes termed APPL endosomes. On the other
side, APPL proteins can be released from the endosomal membrane to
undergo nucleocytoplasmic shuttling and to associate with nuclear
proteins. We previously showed that both APPL proteins are activators of
β-catenin/TCF-mediated transcription in the canonical Wnt signaling
pathway. More recently, we have uncovered a novel role of APPL1 as a
positive regulator of the transcriptional activity of NF-κB under basal but
not TNFα-stimulated conditions. APPL1 was found to directly interact
with TRAF2, an adaptor protein known to activate the canonical NF-κB
signaling. Importantly, APPL1 appeared to regulate the proper spatial
distribution of p65 NF-κB subunit. We could show that APPL1 increased
the stability of NF-κB-inducing kinase (NIK), the key component of the
noncanonical pathway, which in turn affected the transcriptional activation
of p65. This places APPL1 as a novel link between the canonical and
noncanonical machineries of NF-κB activation. Furthermore, as no
systematic approaches were undertaken to investigate the involvement of
endocytic proteins in nuclear signaling and transcriptional control, we
initiated small targeted RNAi-based screens of candidate genes to study
this problem. Our initial data indicate that the participation of endocytic
proteins in transcriptional control is a more wide spread phenomenon.
12
Molecular interactions of multifunctional adaptor proteins of
intersectin family
Rynditch A. V., Dergai M. V., Novokhatska O. V., Tsyba L. O.,
Skrypkina I. Ya., Nikolaienko O. V., Kropyvko S. V., Dergai O. V.,
Morderer D. Ye., Gryaznova T. A.
Department of Functional Genomics
Institute of Molecular Biology and Genetics, Kyiv, Ukraine
rynditch@imbg.org.ua
The rich binding capability of the multidomain, adaptor and scaffolding
proteins of intersectin (ITSN) family has linked them to multiple functions
such as clathrin-mediated endocytosis, mitogenic signalling, actin
cytoskeleton rearrangements and apoptosis. Abnormalities of ITSN1
expression were associated with the endocytic anomalies reported in Down
syndrome brains and early stages of Alzheimer’s disease. ITSN2 was
proposed to be a predictive marker for breast cancer. Despite intensive
study of ITSNs, the regulation of their function in different cell processes
and the role of ITSNs in disease development remain unclear.
In order to highlight a role of ITSN genes, we studied their expression in
normal and pathological tissues as well as analyzed the composition of
ITSN-containing protein complexes. We identified 17 alternative splicing
events affecting ITSN1 pre-mRNA and found alternative transcription
initiation site in the fifth intron of ITSN1 gene. ITSN1 isoforms differ in
their domain organization, interaction with protein partners, localization in
different tissues and stages of development. We have found neuron-
specific alternative splicing of microexon 20 that affects binding abilities
of ITSN1 SH3A domain and provides a mechanism for the control of
brain-specific interactions of ITSN1. Using mass spectrometry analysis
and in silico prediction we identified 11 novel protein partners of ITSN1
and ITSN2, among them adaptor proteins Ruk/CIN85, Reps1 and SHB.
ITSN1, and its shortest alternatively spliced isoform ITSN1-22a forms
complex with membrane-deforming proteins SGIP1 and amphyphisin,
respectively. An interaction of ITSN1 with WASP-interacting protein WIP
13
suggests a possible role of ITSN1 in the regulation of protein complexes
during invadopodia formation in cancer cells. We also identified a new
neuron-specific protein partner MAP6/STOP involved in microtubule
stabilization and generation of synaptic plasticity. Our results demonstrated
that ITSNs could be regulated by ubiquitylation and phosphorylation. The
Nedd4-like E3 ubiquitin ligase AIP4 is involved in posttranslational
modification of ITSN1 isoforms. We have also shown that ITSN2
undergoes EGF-dependent tyrosine phosphorylation in HeLa cells.
Moreover, the SH2 domains of signalling proteins Grb2, Crk, Fyn, Fgr,
Abl1, PLCG1 and PI3K differentially interact with ITSN2 in mouse
tissues. These findings expand the role of ITSNs as a scaffolding
molecules bringing together components of endocytic and signalling
complexes as well as highlight the mechanisms of regulations of ITSNs
functions.
14
Potential implication of leucyl-tRNA synthetase in
tumourigenesis
Tukalo M., Boyarshyn K.,Yaremchuk A., Kovalenko O., Gudzera O.,
Kondratov A.
Department of Protein Synthesis Enzymology
Institute of Molecular Biology and Genetics, Kyiv, Ukraine
mtukalo@imbg.org.ua
Recent screening the novel differentially expressed genes (DEG) in A549
lung cancer line has revealed the over-expression of leucyl-tRNA
synthetase (LRS) and its up-regulation has been confirmed by other
methods (Shin at al., 2008). The oncogenetic potential of LRS gene
(LARS1) was validated by siRNA knockdown and various transformation
assays. On the other hand, two resent studies (Han at al., 2012 and Bonfils
at al., 2012) have shown that leucyl-tRNA synthetase is a leucine sensor
for TORC1, in both yeast and mammalian cells. This finding suggests a
potential implication of LRS in tumorigenesis and opens a new dimension
for cancer therapy. Since mTORC1 inhibitors are used for cancer therapy,
the inhibitors of LRS could be used as new therapeutic regiments for
cancer.
We have investigated the level of LARS1expression in A549 cell line,
normal pulmonary epithelial cell line, tissues of kidney cancer and tissues
of normal kidney. The level of LARS1 mRNA expression of in cancer
cells A549 was more than 2 times higher than in cells of normal
epithelium. In the case of kidney cancer in 16 samples of tumor tissue an
increased expression of LARS1 was observed in 13, and only in two its
slight decrease was noted. However, only three samples showed a more
than twofold increased expression, and one – more than three times. Thus,
the data obtained on the samples of kidney tumors studied show a tendency
of increasing LARS1 expression. At the same time the level of expression
does not allow us to consider this gene as a biomarker of tumor growth in
the kidney cancer.
15
Multimolecular translation elongation factor eEF1H in human
carcinomas.
Negrutskii B. S., Veremieva M. V., Shalak V. F., Trosyuk T. V. and
El’skaya A. V.
Laboratory of Protein Biosynthesis Department of Translation Mechanisms of
Genetic Information,
Institute of Molecular Biology and Genetics, Kyiv, Ukraine
negrutskii@imbg.org.ua
Multisubunit complex eEF1H comprises eEF1A and eEF1B entities.
eEF1B complex serves as a GDP/GTP exchanging device for eEF1A
which interacts with GTP and aminoacyl-tRNA transporting the latter to
the 80S ribosome. The eEF1B complex of unknown stoichiometry consists
of the eEF1Bα, eEF1Bβ and eEF1Bγ subunits. Recently, a variety of
multiprotein complexes, including translation ones, were shown to release
their individual components to fulfill novel functions under unfavorable
circumstances. We aimed to check if independent functioning of the
eEF1B subunits in human organism is also possible. Manifestation of non-
coordinated changes in the subunits expression levels would be an essential
evidence for the possibility of independent from complex appearance of
the eEF1B constituents in cancer tissues.
Indeed, we have found non-coordinated changes in the expression level
of all eEF1B subunits in cardioesophageal cancer and lung carcinomas
strongly suggesting a lack of synchronized regulation of the eEF1B
constituents in cancer tissues. Those findings were confirmed by
immunohistochemical studies.
Importantly, cancer-related increase in the level of the eEF1B subunits
was observed in the majority of cardioesophageal and lung tumor samples.
The purpose of planned research is to investigate the organization of the
eEF1B complex in vitro, to establish a stoichiometry of the subunits in the
complex, to examine a possibility of the existence of mini-complexes
including not all of the eEF1B subunits. Expressed in E. coli the individual
eEF1B subunits will be investigated by physical methods. The perspective
of the project is to approach crystallization and subsequent X-ray analysis
of the individual eEF1B subunits and the complex as a whole.
16
DNA methylation, chromatin modifications and SSLP
polymorphism in the 4q35 chromosomal region in
facioscapulohumeral dystrophy myoblasts and cervical
carcinoma cells
1,2Kisseljova Natalia P., 2Dmitriev Petr, 1Katargin Alexey, 2Kim Elena,
1,2Ezerina Daria, 2Planche Emmeline, 4Lemmers Richard J. L. F.,
van der 4Maarel Silvère M., 3Laoudj Dalila, 2Lipinski Marc, and
2Vassetzky Yegor S.
1NN Blokhin Russian Cancer Research Center, RAMS, Russia,
2 UMR 8126, Université Paris Sud, CNRS, Institut de cancérologie Gustave Roussy,
F-94805, Villejuif, France.
3 INSERM EA 4202 ERI25, 371 Avenue du Doyen Gaston Giraud F-34295
Montpellier, France.
4Department of Human Genetics, Leiden University Medical Center, Leiden, the
Netherlands
vassetzky@igr.fr
Facioscapulohumeral dystrophy (FSHD) is a hereditary disease with a
prevalence of 1 in 20,000 linked to a partial deletion of the D4Z4 repeat
array on chromosome 4q. The nuclear matrix attachment region (FR-
MAR) present in the vicinity of the repeats loses its efficiency in myoblasts
from FSHD patients. The FR-MAR coincides with a region of sequence
variation (SSLP) and contains an enhancer-blocking element. Here, we
have analyzed DNA methylation patterns and histone post-translational
modifications in the FR-MAR of cervical carcinoma and primary
myoblasts from FSHD patients. A specific DNA methylation pattern
combined with lack of histone H3 acetylation was found to be required for
binding of the MeCP2 protein, a known component of the nuclear matrix,
and for association of the FR-MAR with the nuclear matrix. An 8-
nucleotide insert present in the SSLP appeared to enhance the association
of the FR/MAR with the nuclear matrix. Together, these results constitute
the first evidence that DNA and histone modifications affect MeCP2-
mediated FR/MAR attachment to the nuclear matrix in normal and
pathological cells. In FSHD myoblasts, release of the FR/MAR from the
nuclear matrix is favored by the acetylation of the amino-terminal tail of
histone H3 and the absence of the 8-nucleotide insert.
17
Identification and characterization of medullary breast
carcinoma-associated antigens as a potential targets for breast
cancer diagnostics and therapy
1Kiyamova R., 1Kostianets O., 1Shyian M., 2Gout I., 1Filonenko V.
1Department of Cell Signaling,
Institute of Molecular Biology and Genetics, Kyiv, Ukraine
2University College London
filonenko@imbg.org.ua;
i.gout@ucl.ac.uk
Medullary breast carcinoma (MBC) despite anaplastic features and high
grade has a good prognosis that can be related to prominent lymphocytic
infiltration. We applied SEREX technology (serological recombinant
expression cloning) to search MBC antigens as a potential targets for
diagnostics and therapy of breast cancer. In a course of MBC cDNA
screening by autologous MBC patient sera we have identified 41 potential
MBC antigens. Analysis of frequency of antibody responses toward these
41 antigens in sera of allogenic MBC patients and healthy individuals
using plaque-spot serological assay allowed us to select 18 autoantigens
which were exclusively reactive with sera of medullary breast carcinoma
patients. qPCR analysis of mRNA expression for 6 of these 18 antigens
(RAD50, FAM50A, PABPC4, RBPJ, HMGN2, DEK) represented by more
than one independent cDNA clone isolated from the MBC cDNA library
showed their differential expression profile in MBC tissue samples.
However, we didn’t reveal any correlation between the MBC antigens
mRNA expression profiles and the level of specific autoantibodies in
patient sera.
Antigens which showed cancer-related serological profile will be further
investigated in a large cohorts of patients with different types of breast
cancer using ELISA assay with recombinant analogues of identified
antigens to clarify whether they are candidates for breast cancer
diagnostics or therapy.
18
Influence of hypoxic stress on anti-tumor rresponse
Chouaib Salem
INSERM U753Institut Gustave Roussy
Villejuif 98805- France
salem.chouaib@igr.fr
The identification of tumor-specific epitopes as targets for antitumor
cytotoxic effector cells has made possible their use in vaccination trials.
However, positive clinical results have been scarce most likely because of
the weak immunogenicity of these peptides, the low frequency of tumor-
specific T lymphocyte precursors and the resistance of tumor cells to
cytotoxic effector cells. Large established tumors, which are associated
with the acquisition of tumor resistance to specific lysis, are usually not
fully controlled by the immune system. It is obvious that the evasion of
immunosurveillance by tumor cells is under the control of the tumor
microenvironment complexity and plasticity. Hypoxia, a key component of
the tumor microenvironment, is a common characteristic of locally
advanced solid tumors that has been associated with diminished therapeutic
responses, malignant progression, increased probability of recurrence,
locoregional spread and distant metastasis. It occurs in the majority of solid
tumors and is strongly correlated with advanced disease stages and poor
clinical outcome. This is, in part, due to increased genomic instability in
hypoxic tumors cells and enhanced resistance to cytotoxic treatments. We
have made significant contributions in the understanding of the impact of
hypoxic stress on the anti-tumor immune response.The influence of tumor
microenvironment in particular hypoxic stress in shaping the quality of
CTL response and the susceptibility of tumor target cells to CTL-induced
cell death will be discussed.
19
Identification and characterization of a tumor-associated antigen
for the development of a novel cancer vaccine approach in lung
cancer
Durgeau A., El Hage F., Vergnon I., Chouaib S.
and Mami-Chouaib F.
Inserm U753, Gustave Roussy Institute
Villejuif, France
cfathia@igr.fr
We identified an antigen recognized in a human lung carcinoma by a
cytotoxic T lymphocyte (CTL) clone derived from autologous tumor-
infiltrating lymphocytes (TIL). The antigenic peptide is presented by HLA-
A2 and is encoded by the CALCA gene, which codes for calcitonin and
which is overexpressed in several lung tumors compared to normal tissues.
This peptide (ppCT16-25) is derived from the C-terminal region of the
preprocalcitonin (ppCT) signal peptide, and is processed independently of
proteasomes and the transporter associated with antigen processing (TAP).
Processing occurs within the endoplasmic reticulum of all tumoral and
normal cells tested by a novel mechanism involving signal peptidase (SP)
and signal peptide peptidase (SPP). Lung cancer cells bearing this epitope
displayed low levels of TAP, but restoration of their expression by IFN
treatment or TAP1 and TAP2 gene transfer inhibited ppCT antigen
presentation. In contrast, TAP up-regulation in the same tumor cells
increased their recognition by proteasome/TAP-dependent peptide-specific
CTL, namely anti-mutated -actinin-4 epitope-specific CTL. Thus,
ppCT16-25 is the first human tumor epitope whose surface expression
requires down-regulation of TAP. Lung tumors frequently display low
levels of TAP and might thus be ignored by the immune system. Our
results indicate that emerging SP-generated peptides represent alternative T
cell targets, which permit CTL to destroy TAP-impaired tumors and thus
overcome tumor escape from CD8+ T cell immunity. This new epitope is
therefore a promising candidate for cancer immunotherapy. Current studies
aim is to identify antigenic peptides derived from ppCT and processed by
proteasome/TAP pathway to target both antigen processing-efficient and -
deficient tumors. Our objective is to set up an immunotherapy strategy
more adapted for the treatment of patients suffering from lung cancer.
20
Transcription signature of FSHD myoblasts
Dmitriev Petr, 1Laoudj Dalila, Lipinski Marc, and Vassetzky Yegor S
1 UMR 8126, Université Paris Sud, CNRS, Institut de cancérologie Gustave Roussy,
F-94805
Villejuif, France.
2 INSERM EA 4202 ERI25, Montpellier, France.
vassetzky@igr.fr
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal
dominant neuromuscular disease with a prevalence of 1 in 20,000. FSHD
is characterized by progressive weakness and atrophy of the facial muscles
and shoulder girdle. Recently our group has tested the structure of
chromatin loops in the locus 4q35 affected in the disease. We demonstrated
that the chromatin loops are reorganized in FSHD myoblasts compared to
healthy myoblasts (Petrov et al., 2006; Pirozhkova et al., 2008). However,
the etiology of FSHD remains unknown. To elucidate the mechanism of
the disease and propose strategies of treatment, we have established the
transcription signature of FSHD myoblasts and found numerous pathways
deregulated in FSHD, which opens a new way in treatment of FSHD.
21
A search for transcription signature of FSHD myoblasts
1Kavsan Vadym and 2Vassetzky Yegor
1Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and
Genetics, Kyiv, Ukraine
2 UMR 8126, Université Paris Sud, CNRS, Institut de cancérologie Gustave Roussy,
F-94805 Villejuif, France.
kavsan@imbg.org.ua
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal
dominant disorder resulting from an unusual genetic mechanism. The
mutation, a deletion of 3.3 kb subtelomeric repeats, appears to disrupt the
regional regulation of 4q35 gene expression. The specific gene(s)
responsible for facioscapulohumeral muscular dystrophy has(ve) not been
identified. However, the "vacuolar/necrotic" phenotype exhibited by
facioscapulohumeral muscular dystrophy myoblasts suggests that aberrant
gene expression occurs early in facioscapulohumeral muscular dystrophy
muscle development. In order to test this hypothesis, global gene
expression profiling and in vitro characterization of facioscapulohumeral
muscular dystrophy and control myoblasts were carried out. The genes
involved in several cellular processes such as oxidative stress,
MAPK/PI3K signaling, immune response were found to be dysregulated.
Serial Analysis of Gene Expression (SAGE) revealed a set of genes with
the most pronounced changes in expression in human glioma tumor cells.
The most corresponding proteins were involved in angiogenesis, host-
tumor immune interplay, multidrug resistance, extracellular matrix
formation, IGF-signaling, or MAP-kinase pathway. CHI3L1 and CHI3L2
were identified as the most upregulated genes in glioblastomas. Next, it
was shown that CHI3L1 or CHI3L2 expression is increased significantly
under pathological conditions such as inflammation or tumors. Moreover,
recent research suggests that CHI3L1 and CHI3L2 acts as proliferative or
proapoptotic agents, correspondently. These activities are mediated by
signaling through the MAPK and PI3K cascades.
The fact that the expression profiles of two types of human disorders
such as FSHD and glial tumors, have pools of genes characterized by
similar functions, may indicate that the same genes or gene signatures
could participate in the development of these diseases. Especially, it can be
applied to the genes which are involved in the regulation of mitogenesis
and cell proliferation.
22
MGMT repair enzyme and its protective role in the response of
tumor and normal cells to alkylating compounds
Lukash L. L., Iatsyshyna A. P.
Department of Human Genetics
Institute of Molecular Biology and Genetics, Kyiv, Ukraine
lukash@imbg.org.ua
Tumor therapy with alkylating agents is limited by the development
of resistance of tumor cells and undesired systemic side effects.
Understanding and overcoming these limitations might be possible
on the basis of our knowledge how these anticancer drugs are acting
and the cells are protecting their DNA. Regarding alkylating
anticancer drugs belonging to the groups of methylating and
chloroethylating agents a critical site for their attack is the O6-
position of guanine in the DNA, and as a result of this interaction the
adduct O6-alkylguanine is formed. This lesion is considered to be a
major cause of mutations and malignant transformation induced by
alkylating agents. It also gives rise to genotoxicity and cell death
through the stimulation of apoptosis.
O6-alkylguanine is corrected by the DNA repair enzyme O(6)-
methyguanine-DNA methyltransferase (MGMT) which transfers the
alkyl group to the own active center cysteine and is inactivated due to
a suicide mechanism. The guanine in DNA is restored despite a cell
origin, normal or tumor one. The capacity of cells to repair the O6-
alkylguanine depends on levels of expression and activity of the
MGMT in cell or the rate at which a cell can synthesize this enzyme.
MGMT has a strong protective effect on the proliferation capacity,
survival and apoptosis after the treatment of cell populations with
antineoplastic O6-alkylating agents. Thus, MGMT is the most
important determinant in the alkylating drug resistance.
The MGMT is ubiquitously expressed in Mammals, but levels of its
expression widely vary depending on the type of cell or tissue, the
cell cycle phase, the developmental stage of organism. Because its
expression may also differ dramatically in individual tumors,
determination of MGMT activity would be important as a
predictive indicator on the one side, and inactivation of MGMT in
23
tumors to sensitize them to antineoplastic agents is realized in
experimental works and in clinical trials on the other side. Various
highly efficient MGMT inhibitory compounds have been
developed. However, despite effective in vitro studies and lack of
systemic side effects in patients, a recent phase II trials did not
reveal a significant increase in the efficacy of alkylating agents in
the treatment of malignant glioblastomas. That is a reason why it
would be desirable to develop new more efficient inhibitors and
strategies of inhibitor targeting to inactivate MGMT preferably in
tumors. We are going to develop highly active and specific
inhibitors of human MGMT enzyme by using an approach of
molecular modeling, biological screening and combinatorial
synthesis jointly with Prof. Sergey Yarmoluk.
It has been shown that various environmental factors influence
MGMT expression. MGMT was found to be inducible in human, rat
and mouse tissues with genotoxic drugs, including O6-alkylating
agents and X-rays, glucocorticoids and cytokines. Observed
variations of the MGMT expression indicate a complicated regulation
of this gene, but molecular basis of intra- and inter-individual
variation in the MGMT expression levels are still not fully defined. It
has been shown that epigenetic and genetic factors are involved in
the regulation of the MGMT expression.
In a result of the in silico search of regulatory sequences within
mouse and human MGMT promoters we revealed many novel
potential sites for inducible and tissue-specific transcription factors,
but their functional activity has to be subjected to experimental
confirmation. The epigenetic regulation could explain a variation of
the MGMT gene expression during embryogenesis. The mouse Mgmt
gene is an accessible model for study of a development- and a tissue-
specific regulation of the MGMT gene transcription. Therefore, we
propose to study status of methylation of the mouse Mgmt gene
promoter and the gene body at different stages of embryogenesis and
the regulation of the transcription of this gene in different mouse
tissues in collaboration with Prof. M. Bochtler, Laboratory of
Structural Biology, IIMCB.
24
The link between CG methylation and depletion across the
kingdoms of life
Wojciechowski Marek, Czapinska Honorata and Bochtler Matthias
International Institute of Molecular and Cell Biology,
Warsaw, Poland
mail
DNA methylation occurs in prokaryotes and eukaryotes, but in different
forms and with different functions. In prokaryotes, methylation is very
diverse. Mechanistically, the modification can affect the N4 or C5 of
cytosine or N6 of adenine. Sequence context is variable. Functionally,
methylation plays a role in restriction modification systems, in DNA repair
for the distinction of parental and daughter strand, and also in the control
of bacterial lifestyle. Some of this is conserved in primitive eukaryotes, but
in higher eukaryotes, particularly vertebrates, methylation is predominantly
reduced to C5 methylation in a single sequence context (CG, more
traditionally CpG), and serves to control the epigenetic state of DNA, in
crosstalk with appropriate histone modifications. For eukaryotic organisms
with DNA methylation, it is known that the CG sequence is not only
important, but also rare: in fact, the actual number of CGs is about fourfold
lower than statistically expected. In my talk, I will discuss the mechanistic
link between methylation and the depletion of target sequences and I will
address the question about the generality of the link across all kingdoms of
life.
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