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Gliwickie Spotkania Naukowe 2008
Beata
Biesaga1, Joanna Niemiec1, Anna Gasińska1,
Małgorzata Klimek2, Joanna Wysocka3 1Department
of Applied Radiobiology, 2Department
of Gynaecological Oncology, 3Department.
of Pathology Centre of Oncology, Maria Skłodowska-Curie Memorial Center
and Institute of Oncology, Krakow Branch, 31 – 115 Krakow, ul. Garnacrska 11,
Poland Results concerning predictive significance of type of HPV 16 and 18
(Human Papilloma Virus) for concurrent chemoradiotherapy of cervical cancer
are conflicting. Some data indicate
that presence of integrated or episomal HPV DNA in tumour cells can influence
treatment outcome. Also, little is known about biological characteristics of
cervical tumours with different type of HPV infection. Therefore, the aim of
presented study was to assess the influence of HPV 16/18 infection type,
proliferation rate, microvessel density and P53 status on treatment outcome (disease free
survival - DFS) of patients with cervical cancer undergoing concurrent chemoradiotherapy
(CRT). Tumour biopsies were obtained
before treatment from 64 patients with squamous cell carcinoma (SCC) of the
cervix. Median patients` age was 52 years (ranging from 37 to 80). There were
4 tumours at FIGO stage IB, 13 with IIA, 20 with IIB and 19 with IIIB. All
biological parameters were assessed using formalin fixed, paraffin-embedded
tumour samples. HPV 16/18 infection type was detected by in situ hybrydization. To assess the type of HPV infection, two
parameters were analyzed: (1) the percentage of infected tumour cells (%HPV)
and (2) type of HPV signal: diffuse (DS) - representing episomal HPV DNA, and
punctuate (PS) - representing integrated HPV DNA. Proliferation rate (Ki67
labelling index - Ki67 LI), microvessel density (MVD) and P53 proteins status
(P53 labelling index – P53 LI) were assessed on the basis of
immunohistochemical staining. Apoptotic level (apoptotic index - AI) was
evaluated by TUNEL method. In the examined group, the
percentage of HPV positive tumours was 98.4, with mean % HPV value of 44.0 ± 3.0. There
were 45.0% of tumours with both types of HPV signal (punctuate and diffuse)
in tumour cells and 55.0% with only punctuate signal. In the studied group,
no tumours showing diffuse HPV signal only were observed. There were no
correlations between type of HPV infection and clinical features (patients’
age, menopausal status, clinical stage FIGO and histopathological grade). The
mean values of Ki-67 LI, MVD, P53 LI and AI ± SE were: 48.8%±1.5, 80.7 microvessel/mm2 ± 5.4, 11.9% ± 2.0 and
0.8% ± 0.09, respectively. Significantly faster
proliferation rate was found in case of tumours from younger patients (≤
52 years) and a woman before menopause (p = 0.028, p = 0.029, respectively).
No correlations between clinical stage (FIGO) or histopathological grade and
assessed biological features were found. There were also no significant
associations between type of HPV infections and biological parameters.
Significantly higher probability of DSF was found for patients with faster
proliferating tumours (p = 0.005) and those with tumours showing higher
apoptotic index (p = 0.002). Additionally, tumours with lower percentage of
HPV 16/18 infected cells and with both types of HPV signal and higher MVD
showed a trend for better DFS (p = 0.087, p = 0.06, respectively). However,
Cox multivariate analysis showed that only higher apoptotic index was an
independent favourable prognostic factor for DFS. The data presented here
indicate that the apoptosis level may
be used in cervical cancer
clinical practice as a predictor of tumour response to concurrent
chemoradiotherapy. 2. SYMMETRICAL AND UNSYMMETRICAL
AMIDE-CONJUGATED Sławomir
Boncel, Krzysztof Walczak Silesian
University of Technology, Faculty of Chemistry, Department of Organic
Chemistry, Biochemistry and Biotechnology, Krzywoustego 4, 44-100 Gliwice,
Poland Intercalators are molecules
that insert perpendicularly with respect to the axis of DNA, generally
forming all types of weaker bonds than covalent ones. They include van der
Waals, hydrogen bonding, hydrophobic, charge transfer forces and, presumably,
frontier orbital interaction. The intended chemical structures designate the
nature of reversible interactions DNA strand–intercalator. As the
consequence, a large number of diverse classes of compounds is used in
medical treatment, e.g. acridines,
alkaloids, anthracyclines, anthracenediones, arylaminoalcohols, coumarins,
indoles, phenanthridines, quinolines, quinoxalines, etc. [1]. Herein we report a synthesis
of potential intercalators – symmetrical and unsymmetrical a,w-nucleosides based upon aliphatic or aromatic linkages containing one
or two amide bonds. The title compounds (Figure), subdivided into four
groups, were synthesized in the final stage, via condensation of various
units bearing carboxylic and amine groups, in the presence of
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM).
Moreover, for a,w-diaminic aliphatic linkages, a selective aminolysis
was achieved.
The
units containing carboxylic group were obtained by acidic hydrolysis of
Michael N1-adducts of
5-substituted uracil derivatives to methyl acrylate [2]. The main reactant
bearing primary amine group on short aliphatic linkage was synthesized by
reduction of Michael adduct of thymine to acrylonitrile [3]. Additionally, as
the amine reactants 1-(w-aminoalkyl)-5-nitrouracils [2], 5’-amino-5’-deoxy-b-thymidine, 5-aminouracil and adenine have been
applied. The ability of dedicated
molecules to form highly ordered assemblies was indirectly confirmed using
electron microscopy (STM, TEM). The intercalating properties are under
investigation at the Nucleic Acid Centre in Odense, Denmark. References: 1.
R. Martínez, L.
Chacón-García, Curr. Med. Chem., 2006, 12, 127-151 2.
S. Boncel, K.
Walczak, Lett. Org. Chem., 2006, 3, 534-538 3.
S. Boncel, K.
Walczak, Pol. J. Chem., 2007, 81, 2151-2156 4.
E. Węgrzynek,
K. Walczak, Pol. J. Chem., 2009, 3. ON THE GALVANOTAXIS OF PROSTATE CANCER CELLS Przemyslaw
Borys
Department of Physical Chemistry and
Technology of Polymers, Silesian University The Mat-LyLu prostate cancer cells exhibit a galvanotaxis
that is independent of external Ca2+ concentration. This should be
the case in the cathodal galvanotaxis. We explain a possible mechanism of
galvanotaxis in prostate cancer cells which does not depend on extracellular
Ca2+ concentration but relies on the action of sodium channels. 4. Karyometric features
of cell nuclei in papillary thyroid cancer and their correlates in gene
expression profile Mykola Chekan1, Michal Swierniak1, Michal Jarzab3,
Małgorzata Oczko-Wojciechowska1, Dagmara Rusinek1,
Miroslaw Snietura2, Ewa Chmielik2, Anna
Smok-Ragankiewicz2, Dariusz Lange2, Barbara Jarzab1 1Nuclear
Medicine and Endocrine Oncology Department, 2Tumor Pathology Departament, 3Tumor Biology Department, Maria Sklodowska-Curie Memorial Cancer Center
and Institute of Oncology, Branch Gliwice 44-101 Gliwice, ul. Wybrzeże Armii
Krajowej 15, Poland Diagnostic criteria of papillary thyroid carcinoma (PTC) are defined by the presence of
characteristic cell nuclei features. We hypothesize that these features are
driven by specific molecular mechanisms and assess whether karyometric
parameters of PTC nuclei do correlate with gene expression profile assessed
by microarray analysis. Post-operative thyroid frozen samples and respective
paraffin blocks from 41 patients with PTC were analyzed. Images were obtained
by light microscopy with a semiautomatic computer image analyzer. We
calculated parameters directly related to nuclear size, shape and chromatin
distribution. For each karyometric parameter, mean value and its variance
were assessed and correlated with gene expression patterns by
permutation-based test. Global significance was calculated for relation of
each parameter to gene expression profile (evaluated by Affymetrix HG-U133A).
Validation was carried out in the independent group of 36 PTCs by QPCR; in
the same specimens karyometric analysis was carried out as described
previously. We found out that the variability of nuclear size is
the most significantly associated with PTC gene expression profile in PTC.
Nucleus area variance was positively correlated with expression of 55 genes
and showed inverse correlation to 95 genes. Among chromatin distribution
parameters Margination (marg) and Granularity (clump) was correlated with
genes expression prifile. One of the genes related to nuclear size
variability (HDAC1) was validated by QPCR. An observed trend for the relation
of HDAC1 with nuclear size was confirmed. Among karyometric parameters investigated, it is
anisotropy of PTC nuclei which significantly correlate with its gene
expression profile. The association of specific transcripts with karyometric
features of PTC shall be carried out in the extended set of samples. 5. FUNCTIONAL ANALYSIS OF THE HUMAN GSTP1 GENE PROMOTER REGION IN CULTURED CANCER CELLS Agata
Chwieduk1, Andrij Slonchak1,
2, Joanna Rzeszowska-Wolny1,3 1Department
of Experimental and Clinical Radiobiology, Maria Skłodowska-Curie Cancer Center
and Institute of Oncology, Gliwice Branch, Gliwice, Poland, 2Institute of Molecular Biology
and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine, 3Institute of Automation,
Silesian University of Technology, Gliwice, Poland Glutathione S-transferases (GSTs) are a multigene
family of enzymes that play an important part in the second phase of drug
detoxification in cells. One of the most important GST izoenzymes is
glutathione S-transferase P1 (GSTP1), which catalyzes conjugation with a
number of electrophilic hydrophobic compounds such as xenobiotic drugs,
toxins and carcinogens. Expression of this gene is associated with resistance
to some antineoplastic drugs and genotoxic carcinogens. We have examined the
level of GSTP1 gene expression in human normal and neoplastic cells.
The highest level of GSTP1 mRNA was found in the non-tumorigenic
mammary epithelial cell line MCF10a, while human breast adenocarcinoma MCF7
and human hepatocellular carcinoma cell line HepG2 lacked its expression. GSTP1 is transcriptionally silenced by promoter
hypermetylation in several human cancer cells. GSTP1 promoter was
hypermethylated in breast cancer cells MCF7 and partly methylated in BeWo
(human choriocarcinoma cell line). The loss of glutathione S-transferase P1
expression in MCF7 cells resulted from
hypermethylation of CpG sites in the GSTP1 promoter region. Functional analysis of the proximal promoter
indicated that GSTP1 gene is positively regulated by NF-kB element and
negatively regulated by NF-kB-like element and CRE in BeWo, Hbl-100 and Me45
cells. The cytotoxic effects of radiation therapy are
mediated primarily through increased formation of hydroxyl radicals and
reactive oxygen species, which can damage cells, proteins and DNA; the
glutathione S-transferases (GSTs) function to protect against oxidative
stress. Cells of the examined cell lines, irradiated with 2Gy showed reduced
GSTP1 expression from the beginning until 3h after exposure and increased
transcription from 5h to 12h hours
after irradiation. This work was supported by Grant
number N40115732/3043 from the Ministry of Science and Higher Educztion. 6. Structure prediction of a protein channel based on probabilistic
formal grammars and the continuous ion flow model Witold
Dyrka1, Jean-Christophe Nebel2, Małgorzata Kotulska1 1Institute
of Biomedical Engineering and Instrumentation, Wroclaw University of
Technology, 50-370 Wroclaw, Wybrzeze Wyspianskiego 27, Poland, 2Faculty of Computing,
Information Systems and Mathematics, Kingston University, KT1 2EE, Penrhyn
Road, London, UK Structure prediction of
protein channels remains a challenge mostly due to the small number of
already solved structures and conformational space size. The former restricts
homology based methods, while the latter limits de novo techniques. A
promising strategy to make de novo approach capable of dealing with
large channel proteins is to constrain the search space on the basis of
primary and secondary structure features and local spacial homologies. In particular, molecular
interactions between distant residues, which tend to determine the overall
global protein structure, seem to be suitable for this purpose. We propose to
use the framework based on Probabilistic Context Free Grammars (PCFG) to
detect certain characteristic points in order to build a graph of those
interactions and constrain the 3D structure prediction. In our previous work
aimed at detecting protein regions involved in binding sites, the PCFGs were
induced automatically using a genetic algorithm from a set of unrelated
protein sequences that shared a common feature. This framework can be adapted
for robust detection of characteristic points by implementing a priori knowledge
about transmembrane proteins, eg. by appropriate grouping of aminoacids,
introducing specific grammar rules or restricting parsing tree. Prediction of ion channels
structure without known homologous proteins can also be improved by comparing
functional characteristics obtained from the model with experimental data.
The most accurate Molecular Dynamics technique is currently limited to
nanoseconds time scale due to the high computational cost. The ion flux
phenomenon requires longer time and therefore other frameworks are needed,
e.g. Poisson-Nernst-Planck (PNP) model. The PNP, although an averaged theory,
is capable of reproducing biologically valid characteristics. We have already
optimised the PNP method in the terms of computational cost and prepared the
pipeline for obtaining the current-voltage and conductance-concentration
characteristics for a given channel structure. The criterion of compatibility
of the experimental and simulated characteristics can be then used to choose
the structural model of the channel or even to provide some feedback into the
3D modelling process. 7. Carriers of BRCA1
mutations have elevated level of oxidative DNA damage; reversal of this
effect in adnexectomised patients due to selenium supplementation Tomasz Dziaman1,
Marek Foksinski1, Jolanta Guz1, Daniel Gackowski1,
Bartłomiej Kalinowski1, Rafał Rozalski1, Agnieszka
Siomek1, Anna Szpila1, Ewelina Zarakowska1,
Karol Białkowski1, Tomasz Huzarski2, Ryszard Olinski1,Jan
Lubinski2 1Department of Clinical Biochemistry, Collegium
Medicum, Nicolaus Copernicus University, 85-092 Bydgoszcz, ul. Karlowicza 24,
Poland, 2International
Hereditary Cancer Center, Department of Genetics and Pathology, 70-115
Szczecin, ul.Polabska 4, Poland A mutation in the BRCA1 gene predispose women to a
high risk of breast and ovarian cancers. Although the precise biological
functions of the BRCA1 tumor suppressor are still unknown it is widely
accepted that the proteins encoded by the gene participate in the monitoring
and repair of DNA damage. Selenium has several anticancer properties which are
linked with protection against oxidative stress. Namely, Se is required to
maintain the activity of some antioxidant enzymes and was found to scavenge
free radicals Therefore, to asses a involvement of BRCA in oxidative damage to DNA
and to
have a further insight into the issue concerning a role of the damage
in cancer development in the present study all the above mentioned parameters
reflecting oxidative DNA damage were analyzed in three groups of subjects; i/
the group of healthy subjects; ii/ patients with BRCA1 mutation without symptoms of the
disease and iii/ patients with breast or ovarian cancer with the mutations. The aim of the present study was two-fold; aside
from aforementioned aim we also wanted to know whether supplementation of BRCA1 carriers with selenium have
beneficial effect concerning oxidative stress/DNA damage. Collectively the results of our study demonstrate
that BRCA1 carriers have elevated
level of promutagenic 8-oxodG in cellular DNA. Moreover, the results
demonstrate that supplementation of supranutritional selenium doses to high
risk group of subjects with BRCA1
mutations resulted in significant decrease of oxidative damage to DNA.
However, this effect was restricted only
to the patients who underwent adnexectomy. Therefore selenium supplementation may be
recommended to the carriers with adnexectomy without symptoms of the disease.
Our results also demonstrated that selenium supplementation of the breast
cancer patients is responsible for decreased oxidative damage to DNA what in
turn may slow down the disease
progression. This work was financed by grant from
the State Committee for Scientific Research (No. N40105532/1380). KB, TD, MF, JG, DG, BK, RO, RR, AS, ASz,
EZ are partners of ECNIS (Environmental Cancer Risk, Nutrition and Individual
Susceptibility), a network of excellence operating within the European
Union 6th Framework Program, Priority 5: "Food Quality and Safety"
(Contract No 513943). 8. Staphylokinase
production in plant systems Aneta
Gerszberg1, Katarzyna Hnatuszko-Konka1, Piotr Łuchniak1,
Aneta Wiktorek-Smagur2,
Andrzej K. Kononowicz1 1Department
of Genetics, Plant Molecular Biology and Biotechnology, University of Lodz, 12/16
Banacha St, 90-237 Lodz, Poland 2Department of Audiology and
Phoniatrics, Nofer Institute of Occupational Medicine 8 Sw Teresy St, 91-348 Lodz, Poland Recombinant protein production in planta is recognized as a promising alternative to microbiological
synthesis and human or mammalian cell systems. During the past decades,
several efficient methods of plant transformation have been developed. This
resulted in construction of numerous transgenic plant systems for in planta production of plant antibodies,
vaccines and various recombinant proteins of medical and industrial value.
However, the recombinant protein expression level still appears to be too low
from industrial (biotechnology) point of view. For this reason, the
optimization of transgene expression has recently become one of the major
goals of molecular biotechnology research. Our current research is focused on optimizing the
production of recombinant proteins in plants and on developing efficient
methods of extraction and purification of these proteins. As a model protein
we used a bacterial anticoagulant factor – staphylokinase, which is an
effective activator of human plasminogen. It is well documented that high
level expression of bacterial genes in eukaryotic cells depends on a large number
of factors. So far, we attempted to obtain a high level of staphylokinase
expression in three model plants: Nicotiana
tabacum, Solanum tuberosum and Arabidopsis thaliana. For genetic
transformation we used a gene construct consisting of either constitutive
(CaMV 35S) or facultative (Pphas)
promoter and various regulatory elements and signal sequences (KDEL, ss, acr5-I or TMV Ω leader sequence). These experiments resulted in constructing
transgenic plants characterized by whole plant or seed specific expression of
staphylokinase transgene. However, our system requires further optimization
to become economically viable. 9. 5-(4-NITROIMIDAZOL-1-YL- AND
1,8-DICARBOXYNAPHTHALIMID-2-YL)-2’DEOXYURIDINE AS SPECIFIC GUANOSINE SINGLE
NUCLEOTIDE POLYMORPHISM PROBE A. Gondela1,*,
T. Santhosh Kumar1, E. B. Pedersen1, K. Walczak2
J. Wengel1 1Nucleic Acid Center, Department of
Physics and Chemistry, University of Southern Denmark, Campusvej 55, 5230
Odense M, Denmark. 2Department of Organic Chemistry,
Biochemistry and Biotechnology, Selisian University of Technology,
Krzywoustego 4, 44-100 Gliwice, Poland. * Currently: Department of Organic Chemistry,
Biochemistry and Biotechnology, Selisian University of Technology, Krzywoustego
4, 44-100 Gliwice, Poland Single nucleotide polymorphism (SNPs), where
one of the bases is substituted by other, is recently one of the major
interests in the oligonucleotide chemistry [1]. Base pairing
mutations of a gene sequence can occur resulting in switched-off gene
activity or induction of disease [2]. There are two main approaches of
determining SNPs. Most available methods are based on differences in
hybridization efficiency of the matched and mismatched oligonucleotide probe
and the target sequence [3]. Second group of SNPs probes contains the
nucleotide bearing the fluorescent reporter group. The reporter group can be
attached to the sugar or nucleobase moiety or flurophore which can mimic the
base [4]. Single nucleotide polymorphism (SNPs)
oligonucleotide probes containing the C-5 substitued 2’-deoxyuridine modified
by 4-nitroimidazol-1-yl (X modification) and
1,8-dicarboxynaphthalamid-2-yl (Y modification) moiety were
synthesized and the effect on duplex stability
upon incorporation of monomers X
or Y into mixed 21-mer sequence (ON11 and ON12) was evaluated. Fluorescent incorporation of Y opposite to G in mismatched duplex displays significant hipsochromic shift of
fluorescence spectra. References: 1.
D.N. Cooper, M. Krawczak,
S.E. Antonarakis, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic and Molecular Bases of
Inherited Disease, vol. 1, McGraw-Hill, New York, 1995, p. 259.; A. Chakravarti, Nat. Genet. Suppl. 1999, 21,
56.; D.G. Wang, J.-B. Fan, C.-J. Siao, A. Berno, P. Young, R. Sapolsky, G.
Ghandour, N. Perkins, E. Winchester, J. Spencer, L. Kruglyak, L. Stein, L.
Hsie, T. Topaloglou, E. Hubbell, E. Robinson, M. Mittmann, M.S. Morris, N.
Shen, D. Kilburn, J. Rioux, C. Nusbaum, S. Rozen, T.J. Hudson, R. Lipshutz,
M. Chee, E.S. Lander, Science 1998,
280, 1077.; H. Haga, Y. Yamada, Y.
Ohnishi, Y. Nakamura, T. Tanaka, J.
Hum. Genet. 2002, 47, 605. 2.
Saenger, W. Principles of
Nucleic Acid Structure; Springer-Verlag: New York, 1984 3.
Tyagi, D.P.
Bratu, F.R. Kramer, Nat. Biotechnol. 1998, 16, 49.; D. Whitcombe, J. Brownie,
H.L. Gillard, D. McKechnie, J. Theaker, C.R. Newton, S. Little, Clin. Chem. 1998, 44, 918.; W.M.
Howell, M. Jobs, U. Gyllensten, A.J. Brookes, Nat. Biotechnol. 1999, 17, 87.; J.G. Hacia, Nat. Genet. 1999, 21, 42.; D.
Whitcombe, J. Theaker, S.P. Guy, T. Brow, S. Little, Nat. Biotechnol. 1999,
17, 804. 4.
A. Okamoto, Y. Saito and I.
Saito, Photochem. Photobiol. C: Photochem. Rev., 2005, 6, 108 10. ANALYSIS OF EXPRESSION OF
GAS41 AND RELATED PROTEINS IN HUMAN CANCERS Jakub
Hanus, Katarzyna Szołtysek, Michał Jarząb, Piotr Widłak Maria Skłodowska-Curie Memorial Center
and Institute of Oncology, Gliwice Branch Yaf9 protein is a S.
cerevisiae
protein involved in remodeling of chromatin structure as well as in
controlling stability of the yeast genome. Human ortholog of Yaf9 is called
GAS41 and contain a structural domain called YEATS. This domain has been
identified in several other human proteins, which comprised YEATS protein
family: MLLT1/ENL, MLLT3/AF9 and BRDT. YEATS protein family members are
involved in regulation of gene expression, yet their exact functions are not
clear at the moment. However, their similarity to yeast Yaf9 suggested involvement
in controlling genomic stability and possible importance for cancer
development. Here we aimed to analyze
the levels of GAS41 and other YEATS protein family transcripts in human
cancer tissues. Data regarding levels of YEATS protein family transcripts
have been extracted from global gene expression profiles obtained by means of
expression microarrays. We have examined datasets from Affymetrix microarrays
analyzed at the Institute of Oncology in Gliwice or present in publicly available databases. Analyses were performed on material
from breast cancer, papillary thyroid cancer, bladder cancer, lung cancer,
mesothelioma and melanoma, matched with corresponding non-malignant tissues.
We observed that expression of MLLT3/AF9 gene was two-fold lower in thyroid
cancer as compared to non-cancerous thyroid. The change has a high
statistical significance (p<0.000001). Some other cancer/control
differences were also detected, however they didn’t have a high statistical
significance. Additionally, we have analyzed levels of YEATS protein family
transcripts in a dataset from ovary cancer samples. Higher levels of MLLT1/ENL and BRDT gene transcripts correlated with
better responses to chemotherapy, yet statistical significance of the
difference was only moderate (p=0.015 and p=0.029, respectively). Considering
impact of studies that suggested possible interactions between GAS41 and
N-MYC oncoprotein, we have additionally analyzed putative correlation between the levels of GAS41 and N-MYC gene
transcripts in human brain tumor tissues. Obtained data suggest collectively
that members of YEATS protein family might be involved in processes related
to cancer. This work was supported by the
Ministry of Science and Higher Education, Grant PBZ-MIN-015/PO5/2004. 11. Molecular
Switch of enzymatic activities: The case of topoisomerase I Takao
Ishikawa, Alicja Czubaty, Barbara Kowalska-Loth, Agnieszka Girstun, Institute of Biochemistry, Faculty of Biology, University of Warsaw,
ul. Miecznikowa 1, Human DNA topoisomerase I (topoI)
exhibits two different enzymatic activities depending on the substrate.
Interaction with DNA facilitates the relaxation activity of topoI, whereas
contact with SF2/ASF protein results in its phosphorylation catalyzed by
topoI. Primarily, SF2/ASF protein acts in spliceosomes influencing the
determination of splicing sites. It works antagonistically to hnRNP A1
protein that, similarly to SF2/ASF protein, has two RRM domains. For this
reason, both proteins interact with topoI in the same manner. Interestingly,
the in vitro tests carried out in
our study confirm that the interaction of hnRNP A1 protein with topoI
promotes its DNA relaxation activity even in the presence of SF2/ASF. What is responsible for switching
of the topoI activity? We looked for structural differences in both SF2/ASF
and hnRNP A1 proteins. The most obvious dissimilarity is the linker region
located between two RRM domains. The former is equipped with a flexible
linker consisting of 9 glycine residues, while the latter has a comparatively
short and rigid linker that seems to prevent unrestricted movements of RRM
domains. In order to answer the above
question, we constructed two recombinant proteins with swapped linkers:
SF2/ASFhard and UP1flex that have native linkers from
hnRNP A1 and SF2/ASF proteins, respectively (UP1 stands for the shortened
hnRNP A1 protein commonly used in the in
vitro studies). Unlike the native UP1 protein, the UP1flex was
not able to fully promote the DNA relaxation activity of topoI, which
continued to phosphorylate SF2/ASF. On the other hand, using SF2/ASFhard
protein, we confirmed that the linker region has no impact on the interaction
of SF2/ASF with topoI nor on its phosphorylation efficiency. Moreover, presence of SF2/ASFhard
protein left the DNA relaxation activity of topoI unaffected. Taking together
these results, we suggest that the linker region of SF2/ASF protein is
predominantly responsible for inhibition of the relaxation activity of
topoI. Further, the antagonistic
action of SF2/ASF and hnRNP A1 in the determination of splicing sites
possibly may result from the different structure of the linker region between
two RRM domains. 12. OPTIMISATION OF PROCEDURES OF ISOLATION AND CULTURE OF MURINE
HEART MICROVASCULAR ENDOTHELIAL CELLS Karol
Jelonek1 and Chryso Kanthou2 1Department of Experimental and Clinical Radiobiology, Maria
Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice,
Poland, 2Cancer Research UK Tumour Microcirculation Group, School of Medicine
and Biomedical Sciences, University of Sheffield, Sheffield, UK The aim of the study was to optimise previously
published methods for isolation of endothelial cells from mouse hearts to
establish pure cell cultures. The isolation of microvascular endothelial
cells was achieved by releasing cells from hearts by mechanical mincing of
the tissue and incubating with proteolytic enzymes, followed by selection with
antibodies targeting endothelial-specific cell-surface antigens CD31,
magnetic selection and culturing in specialised media. Optimal tissue
digestion and release of single cells was achieved by incubation with
collagenase followed by mechanical treatment (syringing). Although
dissociation of tissue into single cell suspension was improved after
trypsinization, this treatment was found to compromise subsequent binding of
CD31 antibodies and reduced cell yields. Two different commercial systems
employing magnetic cell sorting of antibody-labelled endothelial cells were
evaluated: MACS and Dynal system. The endothelial cell recovery by MACS technique was
very low and therefore subsequently the Dynal system was evaluated. This
system employs large visible magnetic beads, which are first coated with
specific antibodies and then applied to tissue digests. Selection of
antibody-bound cells was achieved by applying the cell suspension into a tube
placed in a magnet followed by removal of unbound cells by aspirating the
supernatant. Primary endothelial cells were found to adhere efficiently on 1%
gelatin coated dishes and endothelial colony expansion was optimal in the
presence of 100 mg/ml endothelial cell growth supplement (ECGS). To reduce fibroblast
contamination we used media in which L-valine was substituted for D-valine,
which is reported to be poorly metabolised by fibroblasts. A second magnetic
selection procedure was also employed once the original primary cells reached
confluence. The resulting microvascular endothelial cells were positively
identified by their morphology and expression of specific antigens.
Successful isolation of endothelial cells was achieved from hearts of animals
up to 16 weeks of age. This work was supported by the
SP5-Euratom 221403 Grant CARDIORISK. 13. EXPRESSION OF
ELP-GUSPLUS FUSION PROTEIN IN
TRANSGENIC TOBACCO PLANTS Tomasz
Kowalczyk, Piotr Łuchniak,
Katarzyna Hnatuszko-Konka, Andrzej K. Kononowicz Department
of Genetics and Plant Molecular Biology and Biotechnology, University of
Lodz, S. Banacha 12/16, 90-237 Lodz, Poland, contact: akononow@biol.uni.lodz.pl Elastin-Like Polypeptides (ELP) are synthetic
polypeptides composed of repeatig pentapeptide Val-Pro-Gly-Xaa-Gly where Xaa
can by any amino acid except proline. This polypeptides are characterized by
reversible inverse phase transition in response to the changes of
environmental parameters such as temperature, pH or ionic strength. This
transition from a soluble form to insoluble coacervates is fully reversible
by changing the solution parameters to the initial state. Due to this
specific properties, application of ELP in biotechnology, bioengineering and
biomedicine is the subject of extensive research. In this report we present
expression of the transgene coding for an ELP-GUSPlus fusion protein in
transgenic N. tabacum plant obtained by an A. rhizogenes mediated
transformation. Based on histochemical (GUS activity assay) and molecular
(Western blot analysis) analyses we report on targeting of ELP-functional
enzyme fusion to ER. This research was supported by the Ministry of Science
and Higher Education (grant No 2 P04B 029 29) and the University of Lodz (grant No 506/040996) 14. STUDY OF THE MOLECULAR MECHANISM AT THE
BASIS OF THE ANTI-INFLAMMATORY ACTION OF CYCLIC-AMP Karolina
Krzesaj1, Guy Hagemann2, Sarah Gerlo2 1Institute
of Automation, Technical University ul. Akademicka 16, 44–100 Gliwice,
Poland. 2Laboratory of Eukaryotic Gene Expression & Signal
Transduction (LEGEST), Department of Molecular Biology, Ghent University,
K.L. Ledeganckstraat 35, 9000 Gent, Belgium Cyclic-AMP is a second messenger that operates
through the activation of PKA or the more recently discovered guanine
exchange proteins directly activated by cAMP (epac). Alterations of
intracellular cAMP levels have been shown to have profound effects on
cytokine secretion. Intracellular cAMP concentrations are predominantly
increased via G-protein-mediated activation of adenylyl cyclase, which
converts ATP to cAMP. In addition, cAMP levels are affected via breakdown of
cAMP by phosphodiesterases. We and others have observed that pharmacological
or physiological (using serotonin or the β-adrenergic agonist
isoproterenol) elevation of intracellular cAMP levels represses the
expression of several pro-inflammatory cytokines by human monocytes. In this
project we wanted to investigate the molecular basis of this
anti-inflammatory effect. A major issue was the assessment of the effects of
cAMP and cAMP-inducing agonists on the activation of the NF-kB transcription
factor, which is pivotal in the expression of a multitude of inflammation-related
genes. As a model system we have used the human THP-1
leukemic monocytic cell line. Their inflamatory response was checked via
expression of interleukins six and eight. Interestingly, we observed that LPS, but not other pro-inflammatory
stimuli such as PMA or TNF, induced IL-6 secretion. PMA and TNF however did
induce the secretion of IL‑8, another NF-κB -dependent gene,
indicating gene-specific effects of these pro-inflammatory stimuli. In
addition, whereas cAMP also inhibited LPS-induces IL-8 expression, it did not
affect PMA-induced IL-8 and even synergized with TNF to induce IL-8. On the
other hand we could not detect any inhibitory effects of cAMP on IL-6
expression in Raw264.7 mouse macrophages and in human astrocytes, we explored
whether the inhibitory effect of cAMP was perhaps cell type specific. These
observations indicate the effects of cAMP are not only
stimulus-specific, but also cell-type specific. We also observed that whereas cAMP inhibits IL-6
expression in THP-1 cells, it also induced phosphorylation of p65 at the
serine 276 residue, a modification that has previously been demonstrated to
be associated with transcriptional activation of NF-κB-dependent genes
such as IL-6. We also show that both cytokines we investigated are
differently regulated and the regulation is changing with the developmental
stage of the cells. In summary, our findings indicate that cAMP can
inhibit pro-inflammatory gene expression via different gene- and
stimulus-specific mechanisms. Further experiments, perhaps using a
combination of classical gene expression tools in combination with
genome-wide bio-informatics approaches, will be required to elucidate the
multiple targets via which cAMP-dependent pathways can modulate pro‑inflammatory
gene expression. 15. HEAT SHOCK INDUCES
DOWN-REGULATION OF SEVERAL TRANSCRIPTION FACTORS EXPRESSION PRIOR TO THE
INDUCTION OF APOPTOSIS IN SPERMATOGENIC CELLS Małgorzata
Kus-Liśkiewicz1, Michał Jarząb2, Magdalena Olbryt2,
Wiesława Widłak2 1Branch Campus of the Faculty of Biotechnology,
Rzeszów University, Kolbuszowa 36-100, Sokołowska 26, Poland, 2Department of Tumor Biology, Maria Skłodowska-Curie
Memorial Cancer Centerand Institute of Oncology, Gliwice Branch, 44-101
Gliwice, Wybrzeże Armii Krajowej 15, Poland Elevated temperature and other stress conditions
cause denaturation of cellular proteins, which induces activation of the heat
shock transcription factor 1 (HSF1) and leads to elevated expression of heat
shock genes. Heat shock proteins (HSPs) are then rapidly synthesized and
their accumulation can result either in refolding of proteins to their native
state or in degradation of abnormal proteins, which is called the heat shock
response. However, not all cell types respond to cellular stressors in the
same manner. In the male germ cells (spermatocytes) activation of HSF1 does
not lead to HSP synthesis and cytoprotection. Instead, caspase-3 dependent
apoptosis is induced and spermatogenic cells are actively eliminated. To elucidate a mechanism of pro-apoptotic activity
of HSF1 in spermatogenic cells we carried out genome-wide transcriptional
analysis in control and heat-shocked cells, either male germ cells (where
HSF1 activation leads to apoptosis) or somatic cells (that survive in elevated
temperature). Spermatocytes and spermatids were isolated from mouse testes by
unit gravity sedimentation. Hepatocytes isolated by collagenase perfusion of
a mouse liver exemplified the somatic cells. RNA was isolated from control
and heat-shocked cells, either directly after treatment or after 1-3 hrs
recovery at physiological temperature. Approximately 36 000 transcripts,
representing the entire murine genome, were monitored using the Affymetrix
GeneChip system before and after the heat shock and 2 hrs recovery. In
addition, expression of selected genes was tested by RT-PCR. We identified
genes that are differentially expressed during hyperthermia in somatic and
male germ cells. In hepatocytes, the
heat shock stimulates expression of some genes involved in inflammatory and
immune response, like p38 (Mapk 14), CD14 antigen, interleukin 1 and
chemokine ligand 1. These genes, however, are not stimulated in
spermatocytes. More importantly, expression of many transcription factors,
for example subunits of AP-1 (Jun and Fos), Egr1 and 2, Zfp361l, Klf6, Atf3,
Nfkbiz, that is strongly induced by the heat shock in hepatocytes is
down-regulated in spermatogenic cells. Thus it seems possible that negative
regulation of transcription could be most essential for HSF1-dependent
induction of apoptosis in spermatogenic cells. This was supported by Ministry of
Science and Higher Education, grant 2 P04A 04030. 16. MOLECULAR SIGNATURE OF THE BRCA1
MUTATION IN BREAST CANCER, TRUE OR MYTH? 1Department
of Tumor Biology Maria Skłodowska-Curie Memorial Cancer Center and Institute
of Oncology, Gliwice Branch, Gliwice, Poland, 2International
Hereditary Cancer Center, Pomeranian Medical University, Szczecin, Poland 3Department
of Pathology Maria Skłodowska-Curie Memorial Cancer Center and Institute of
Oncology, Gliwice Branch, Gliwice, Poland, 4Department of Molecular Biology
Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology,
Gliwice Branch, Gliwice, Poland *currently: Department of Medical
Biology and Genetics, Grodno State Medical University, Grodno, Belarus Since wide implementation of mutation screening and
genetic counseling, breast cancer has been frequently regarded either as a
sporadic or a hereditary disease (hereditary breast cancer, HBC). There is an
ongoing debate whether pathology and clinical behavior of HBC is distinct
from those of sporadic breast cancer. Undoubtedly, patients with HBC develop
the disease at a younger age, but it is not clear whether they have worse
prognosis, as suggested in some studies. Pathologically and
immunophenotypically, BRCA1
mutation-linked breast cancer is regarded as the most distinct category.
Among its characteristics are: high tumor grade, high mitotic index, pushing
margins, elevated lymphocyte infiltration and low estrogen receptor
expression. The question of putative molecular differences between hereditary
BRCA1 or BRCA2 mutation-linked and sporadic tumors was first analyzed by
Hedenfalk et al. who claimed that these three categories of breast cancer
could be easily distinguished on the basis of distinct gene expression
pattern (Hedenfalk et al., 2001). The aim of our study was to verify the magnitude of
difference in gene expression profile between BRCA1-associated and sporadic breast cancers. In our analyses we
took into account estrogen receptor status and molecular subtype of the
tumor, the two most significant features affecting global gene expression
pattern in breast cancer. We also considered that BRCA1 gene inactivation may be caused not only by its mutation
but also by the epigenetic silencing. Thus we checked for BRCA1 promoter methylation in the
tumor samples and analyzed the gene expression profile in all tumors with
inactive BRCA1 gene. Our results show that a marked difference between BRCA1-mutation linked and sporadic
breast cancer, previously reported by others, was probably due to uneven
stratification of ER(+)/ER(-) and basal-like/luminal tumor samples. Apparent
difference between BRCA1-linked and
other types of breast cancer observed in univariate analysis is diminished
when data are corrected for these features in multivariate analyses. In fact,
the difference in gene expression pattern of BRCA1-mutated and sporadic cancer is very discrete. These
conclusions were supported by the Q-PCR validation. We also found that BRCA1
gene inactivation due to promoter methylation had similar effect on general
gene expression profile as mutation-induced protein truncation. This suggests
that in the molecular studies of hereditary breast cancer, BRCA1 gene methylation should be
recognized and considered together with gene mutation. 17. ROLE
OF ELECTROSTATICS IN INACTIVATION OF KV 1.2 POTASSIUM ION CHANNEL. RANDOM
WALK SIMULATIONS Krzysztof Małysiak, Zbigniew J. Grzywna
Silesian University of Technology, Faculty of Chemistry, Department of
Physical Chemistry and Technology of Polymers, Section of Physics and Applied
Mathematics, Strzody 9, 44-100 Gliwice, Poland
Inactivation of the potassium ion channel is the
process in which the ball-like peptide blocks the pore of the ion channel. In
the intact (wild type) channel, inactivating peptides (four of them) are
bounded to the protein by the tethers. In the excised channel, inactivating
balls are removed, yet it is still possible to induce the inactivation by
adding peptides to the surrounding water-solution. In our model, inactivating ball behavior is
subjected to the overdamped Langevin dynamics. The influence of electrostatic
interactions of balls with channel protein and cellular membrane is included.
Comparison of predicted and measured (literature data) rates of inactivation
is presented. Common features and differences between different modes
of inactivation experiment are discussed. 18. PEAK
ALIGNMENT IN PROTEIN SPECTRA. A COMPARISON OF DIFFERENT ALIGNMENT TECHNIQUES Michał Marczyk, Joanna Polańska Institute
of Automatic Control, Silesian University of Technology, Akademicka 16,
44-100 Gliwice, Poland In protein
spectra features of scientific interest include peaks which represent relative levels of each protein within the mixture. However, as measurements are
affected by error, this causes peak
shifts between spectra. These shifts are nonlinear and persist even when we
use technical replicates. Concurrent analysis of many spectra is possible
only after their alignment. Alignment is a sequence of operations which settles localization of
common peaks in individual spectra. In
recent years mass spectrometry has been used to find disease related patterns
in complex protein mixtures. Detected markers need to be qualified,verified
and validated. This processes rejects almost all candidates. One of the
reason of this is that the true biochemical composition of the samples used
in the experiments is not known, so accuracy of spectra processing techniques
used can't be estimated. Creating three different data sets with 100
realistic spectra each, using a virtual mass spectrometer, developed by
Morris and Coombes [1], enabled
valuable comparison of efficiency, reliability, and accuracy of alignment
algorithms. From all the alignment algorithms, four
of them were chosen. First two are warping methods which received recognition
in chromatogram and NMR spectra alignment. Next two are methods used in two
different protein spectra processing software: SpecAlign [2] (method using Fast
Fourier Transform) and PrepMS [3] (method based on Matlab msalign function).
They were a good reference to old methods. Results of our
research is presented using both visual (heatmatps, mean spectrum, all
spectra view) and numerical (working time, precision) methods. For alignment
precision measurements peaks obtained after processing were compared with
expected values. Such operations describe advantages and disadvantages of the
tested algorithms. The important
thing that was observed concerns complexity and difficulty level with respect
of peak alignment of the investigated spectra and strictly linked to
concentration of the peaks in small areas. The fastest one and having very
good accuracy of alignment was the
algorithm used in SpecAlign. However, when the spectrum was too
complex (considerable peak shifts) it didn't align all spectra. One of the
warping methods, the slowest one, managed this problem. However, after using
this method accuracy of alignment was worse. Supported by
Ministry of Science and Higher Education grant no 2P05E06730(MM) and no
3T11F01029(JP). References: 1.
Coombes, K.R., Koomen, J.M.,
Baggerly, K.A., Morris, J.S., Kobayashi, R., Understanding the
characteristics of mass spectrometry data through the use of simulation, Cancer
Informatics, 2005;1: 2.
Wong, J.W.H., Cagney, G.,
Cartwright, H.M., SpecAlign - processing and alignment of mass spectra
datasets, Bioinformatics, 2005 May 1;21(9):2088-90. Epub 2005 Feb 2 3.
Karpievitch, Y.V., Hill, E.G.,
Smolka, A.J., Morris, J.S., Coombes, K.R., Baggerly, K.A., Almeida, J.S.,
PrepMS: TOF MS data graphical preprocessing tool, Bioinformatics, 2007 Jan
15;23(2):264-5. Epub 2006 Nov 22 19. ANALYSIS OF INDIVIDUAL RADIOSENSITIVITY ON THE BASIS OF EXPRESSION
PROFILING Agata
Meglicz1, Siamak Haghdoost2, Peter Svensson1,
Rebecca Esveldt-van Lange1, Harry Vrieling1, Mats
Harms-Ringdahl2, Micheline Giphart-Gassler1 1Leiden
University Medical Center, The Netherlands, 2 Stockholm
University, Sweden Current guidelines for radiation dose limits are
based on linear extrapolation of population-wide biological effects (like
cancer risk) obtained at relatively high doses of radiation (above 100 mGy).
However, it is unclear whether such linear relationship models are valid at
low radiation doses. Other relationships between dose and effect are
imaginable: for example a threshold dose may exist below which little or no
effects occur. As a measure of biological consequences to low dose and dose
rate radiation we characterized genome-wide transcriptional responses. Human
lymphocytes from two different donors were isolated and subjected in
triplicate to various (low) doses of irradiation (0.4 mGy, 2 mGy, 50 mGy, 100
mGy and 200 mGy). All doses were administered either acutely (high dose rate
for short amount of time) or chronically (low dose rate for long time) after
which gene expression profiling was performed using Affymetrix GeneChips
HGU133plus2. From the outcome of these gene expression data we will determine
the lowest dose or dose rate for which a specific gene expression response
can be found and will determine whether this response follows a threshold or
linear dose relationship. Additionally we performed a time course experiment
on material from the second donor, in which we investigate gene expression
profiles 3h, 8h and 24h after irradiation with acute low doses (25mGy, 50mGy
and 200mGy). The goal of this experiment is to obtain information about
differences between genes acting at distinct time points post irradiation. In analogy to the observed inter-individual
variation in sensitivity to high dose radiotherapy, we expect a similar
variation in biological effects to low dose radiation. We will irradiate
lymphocytes from two groups of breast cancer patients displaying either late
normal tissue toxicity to radiation therapy or non-toxicity with the most
optimal low dose determined in the experiment described above. We hope to
find an intrinsic difference in gene expression between these two groups,
enabling to predict an individual’s radio-sensitivity to low-dose radiation. 20. NOVEL QUINAZOLINE DERIVATIVES, THEIR SYNTHESIS, STRUCTURE-ACTIVITY
RELATIONSHIP AND ANTIPROLIFERATIVE PROPERTIES A.
Mrozek1, R. Musioł1, A. Szurko2,3, J.
Rzeszowska-Wolny3, J. Finster1, P. Mazur1, 1University
of Silesia, Institute of Chemistry, Szkolna 9, 40-007 Katowice, Poland, 2University
of Silesia, Institute of Physics, Bankowa 12, 40-007 Katowice, Poland, 3Comprehensive
Cancer Centre, Maria Sklodowska-Curie Memorial Institute, Experimental and
Clinical Radiobiology Department, Poland Quinazolinone
moiety exists in number of bioefectors. This molecular scaffold has been
studied as antiamnestic and antioxidant compounds (1), and antimitotic anticancer agents (2). Series
of quinoline and quinazoline derivatives (Scheme 1) were synthesized and
evaluated for their anticancer activity.
Scheme
The
products were obtained in good to excellent yields, and structures were
confirmed by spectral data (NMR, IR). During the synthesis microwave
irradiation was successfully applied (3).
Antiproliferative activity was measured using HCT 116 cell line (human
colon carcinoma). We discuss the structure-activity relatioship between
chemical structure and biological activities of the evaluated compounds.
Several analogues have shown significant growth inhibitory activity against
HCT 116 cells. Anticancer activity was evaluated using MTS-reduction
colorimetric survival assay, and clonogenic cell survival method. Further
studies on docking quinazolinone based compounds to reverse transcriptase are
also discussed. This work was supported by grant no.
PB-W-03-036-00-08 References
1.
S. Kovalenko,
I. Belenichev, V. Nikitin, A. Karpenko: Search for substances with
antioxidant and antiamnestic activities among 2-substituted
4-(3H)-quinazolones. Acta Pol Pharm
Drug Design 2003, 60, 275-279. 2.
J.B. Jiang,
D.P. Hesson, B.A. Dusak, D.L. Dexter, G.J. Kang, E. Hamel: Sythesis and
biological evaluation of styrylquinazolin-4-(3H)-ones. A new class of
antimititic anticancer agents which inhibit tubulin polymerization. J Med
Chem 1990, 33, 1721-1728. 3.
R. Musiol, B. Podeszwa,
J. Finster, H. Niedbała, J. Polański, An efficient microwave-assisted
synthesis of structurally diverse styrylquinolines. Monatsh Chem 2006,
137, 1211-1217. 21. ACTIVITY OF NFkB TRANSCRIPTION FACTOR IS AFFECTED BY
HYPERTHERMIA AND ACTIVE HSF1 Małgorzata
Pakuła1, Magdalena Kalinowska-Herok1, Natalia Kashchak1,
Wiesława Widłak1, Marek Kimmel2, Piotr Widłak1 1Maria Skłodowska-Curie Memorial Center and Institute
of Oncology, Gliwice, 2Silesian
University of Technology, Gliwice, Poland NFkB
is a family of transcription factors, which in
resting cells are sequestered in the cytoplasm by association with IkB inhibitory
protein. Activation of NFkB requires degradation of IkB, which allows nuclear translocation of
NFkB
and their binding to cis-acting DNA regulatory elements. NFkB regulates
numerous genes important for pathogen- or cytokine-induced inflammation,
immune response and cell proliferation. NFκB also
activates several genes that promote cell survival, which contributes to
aggressive tumor growth and resistance to chemotherapy and radiation in
cancer treatment. Various reports have shown that experimental activation of
NFκB results in reduced apoptosis while its inhibition promotes
apoptosis and suppress tumor growth. HSF1 is the primary transcription factor
responsible for the transcriptional response to different forms of cellular
stress (e.g., a heat shock). The hyperthermia-activated HSF1 binds regulatory
DNA elements, termed heat shock elements (HSE), present in promoters of HSPs
genes, and activates their expression. In general, HSPs function as molecular
chaperones in regulation of cellular homeostasis and promoting survival. HSPs
overexpression is frequently found in many types of cancer, and is usually
associated with poor prognosis. Such up-regulation of HSPs putatively
increases resistance of cancer cells to therapy and apparently diminishes the
success of anti-cancer treatment. On the other hand, however, hyperthermia is
an adjuvant treatment used to sensitize cancer cells to radio- and
chemotherapy, possibly affecting pathways that promote cell survival. Here we aimed to address possible mechanisms by
which hyperthermia and HSF1-dependent signaling interfere with NFkB-dependent pathways. The U2OS
osteosarcoma human cell line has been used as an experimental model. The heat
shock response has been induced by means of hyperthermia. Alternatively,
cells have been transfected with mutated constitutively active HSF1 to
activate HSF1-dependent signaling in the absence of the heat shock. Cells were incubated with TNFa cytokine to activate NFkB, and then expression of NFkB-regulated genes
has been assessed by RT-PCR. We have observed that activity of NFkB was inhibited in cells subjected to hyperthermia, and four hours
recovery in physiological temperature was necessary to allow TNFa-induced activation of NFkB. On the other hand, NFkB remained to be
activatable by TNFa treatment in cells containing constitutively active
mutated HSF1 at normal temperature. Interestingly, however, several NFkB-activated genes were differently regulated in the presence of active
HSF1. Our findings clearly indicates functional interference among
hyperthermia, HSF1- and NFkB-dependent signaling pathways. This work was supported by the
Ministry of Science and Higher Education, grants 3T11A01929 and
PBZ-MNiI-2/1/2005. 22. CYTOTOXICITY OF ETOPOSIDE AND
CISPLATIN IS SYNERGISTICALLY INCREASED BY WORTMANNIN IN HUMAN GLIOMA CELLS Elzbieta
Pastwa1, and Urszula Lewandowska2 1Department
of Molecular Genetics, 2Department
of Medical Enzymology, Medical University of Lodz, 92-215 Lodz, Mazowiecka
6/8, Poland Etoposide and cisplatin treatment are used routinely
in glioma patients. However, resistance to these drugs is a major problem in
therapy and can be associated with accelerated repair of etoposide and cisplatin-induced
DNA damage. Etoposide induces DNA double-strand breaks (DSB), and cisplatin
induces DNA intra- and inter-strand cross-links (ICL). The latter agent may induce
DSB as an intermediate step during ICL repair. DSB are repaired mainly by
non-homologous DNA end joining (NHEJ). Important component in this repair
pathway is the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs).
The aim of our work was to answer the question if the nonspecific DNA-PKcs
inhibitor wortmannin can sensitize human glioma cells to DNA damaging
agents: etoposide and cisplatin. Effect of wortmannin on drugs cytotoxicity
in T98G and MO59K human glioma cell lines was evaluated using XTT assay. The
potential mechanism of action of wortmannin on etoposide and cisplatin
cytotoxicity was examined by analysis of synergy. The results demonstrate that
wortmannin increases the cytotoxicity of etoposide and cisplatin in
combination in T98G and MO59K cells (reduction factor R>1). Pre-incubation
of T98G cells with 10 µM wortmannin potentiates about seven times the growth
inhibition of drugs (R=7,3), whereas pre-incubation of MO59K cells with 5 µM
wortmannin potentiates about four times the growth inhibition of drugs
(R=4,5). Moreover, there is a synergistic interaction between these two drugs
and wortmannin in both cell lines (combination index CI<1). Our data show that wortmannin
is able to modulate drugs toxicity to increase cell killing. This inhibitor,
in combination with etoposide and cisplatin, may have potential benefits in
cancer treatment. This work was supported by grant
401-117-32 from the Polish Ministry of Science and Higher Education and by
grants 502-19-677 and 503-00-78-3 from the Medical University of Lodz. 23. APPLICATION OF MALDI-TOF
ANALYSIS OF THE SERUM PROTEOME IN DETECTION OF BREAST CANCER PATIENTS; SAMPLE
PREPARATION METHOD-DEPENDENT CLASSIFICATION PERFORMANCE Monika
Pietrowska1, Łukasz Marczak3, Joanna Polanska2,
Katarzyna Behrendt1, Elżbieta Nowicka1, Anna Walaszczyk1,
Rafał Tarnawski1, Maciej Stobiecki3, Andrzej Polanski2,
Piotr Widłak1 1Maria Sklodowska-Curie Memorial Cancer Center and
Institute of Oncology, Gliwice, Poland, 2University of Technology, Gliwice, Poland, 3Institute of Bioorganic Chemistry, Poznan, Poland Proteomics is the study of the proteome – a complete
protein component of the cell. In contrast to the genome, the proteome
is dynamic and its fluctuations depend on combination of numerous internal
and external factors. Identifying and understanding changes in the proteome
related to a disease development and therapy progression is a subject of
clinical proteomics. Here we aimed to identify in the circulating blood a set
of polypeptide biomarkers that could be used in diagnostics and monitoring of
therapy of breast cancer patients. Analysis of the low-molecular-weight region of the
blood proteome (using either serum or plasma samples) by mass spectrometry
(MS) methods is one of the basic approaches of clinical proteomics. Although
no single peptide is expected to be a reliable bio-marker in such analyses,
multi-peptide sets of markers selected in numerical tests have been already
shown in a few studies to have prognostic and predictive value in cancer
diagnostics. In our study we have analyzed low-molecular-weight serum
polypeptides (<10 kD) using MALDI-ToF mass spectrometry. Blood samples were collected from the group of 100
breast cancer patients before the start of therapy, as well as in the group
of 400 healthy controls. Specific patterns of low-molecular-weight
polypeptides (1-10 kD) were identified thanks to mathematical analyses and
cross-correlated between experimental groups. A multi-component set of
polypeptides has been selected as a classifier that differentiate control and
cancer samples. Here we present a report from the project aimed to
identify a set of polypeptide biomarkers that could be used for diagnostics
and monitoring of a therapy of breast cancer patients. Preliminary data show
that cancer-specific multi-component polypeptide pattern could be identified
in serum of breast cancer patients. However, their importance for cancer
diagnostics remains to be verified. This work was supported by the Ministry
of Science and Higher Education, grant 2P05E06730. 24. Analysis of intracellular localization of
the human HspA2 protein in
cancer cells Wojciech Pigłowski, Piotr Filipczak, Zdzisław
Krawczyk, Dorota Ścieglińska Department
of Tumor Biology. Maria Skłodowska-Curie Memorial Cancer Center and Institute
of Oncology, Gliwice Branch. 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15,
Poland The human HSPA2 gene belongs to a HSP70
multigene family of heat shock genes. The HspA2 protein is necessary for
completing of meiosis and the progression of spermatogenesis. Reduced
expression of this protein was found to be related to male infertility. Expression
of the HspA2 is not heat inducible. The HSPA2 transcripts were
reported to be present in various human somatic tissues. However, the HspA2
expression in non-testicular cells, both at mRNA and protein level, is poorly
characterized. The HSPA2 gene activity was also observed in cell lines
derived from several human cancers and in cancer surgical samples. Depletion
of HSPA2 might be involved in diminished growth and survival of cancer
cells. The first aim of the study was to establish the
expression and intracellular localization of the HspA2 protein at
physiological temperature. We used qRT-PCR to determine transcription
level of HSPA2 in human cancer cell lines of various origin.
Intracellular localization of the HspA2 was analyzed using cell lines expressing
HspA2-GFP and mRFP-HspA2 fluorescent fusion proteins and confirmed by
immunofluorescence using cell lines naturally expressing HspA2 and the
specific anti-HspA2 antibody. In cells growing under normal culture
conditions the HspA2 protein was localized mainly in cytoplasm. The HspA2
protein was shifted into nucleus and nucleoli during heat shock. We also
observed that HspA2 is accumulated at centrosomes of interphase and mitotic
heat-shocked cells. Our results suggest, that the HspA2 protein can be involved
in protecting nucleoli and centrosomes integrity in cells subjected to heat
shock, and possibly to other cellular stressors. It seems that the HspA2 can
be considered as chaperone protecting cells against proteotoxic stresses. We
observed that overexpression of the HspA2 protein enhanced clonogenic
potential of cells treated with proteasome inhibitor. Funded by Polish Ministry of Science
and Higher Education; Grant Number: 3 PO5A 009 25. 25. CHLORITE
LEADS TO FORMATION OF CHLOROHYDRINS IN PHOSPHATIDYLCHOLINE VESICLES
CONTAINING UNSATURATED FATTY ACID RESIDUES
Robaszkiewicz
A.1, Spickett CM.2, Bartosz G.1, Soszyński
M.1 1Department
of Molecular Biophysics, University of Łodź, Banacha 12/16, 90-231 Łódź,
Poland, 2Strathclyde
Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27
Taylor Street, Glasgow, G4 0NR, Scotland, UK The activity of neutrophil-derived enzyme – myeloperoxidase is
associated with generation of HOCl/OCl-, which protects against
pathogen invasion but also brings about oxidative injuries in host tissues.
It is known, that HOCl/OCl- is able to induce lipid peroxidation;
however the analysis of the mechanism of this reaction and the products
pattern are still to be elucidated. Therefore, we selected three
phosphatidylcholines with known fatty acid content: 1-Steraroyl-2-Oleoyl-sn-Glycero-3-Phosphocholine
(18:0-18:1 PC), 1-Steraroyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine
(18:0-18:2 PC) and 1-Steraroyl-2-Arachidonyl-sn-Glycero-3-Phosphocholine
(18:0-18:4 PC). The lipid vesicles were prepared in sodium phosphate buffer
(pH = 6.0), sonicated and treated with HOCl/OCl- (5 molar excess
per one double bond). The excess of HOCl/OCl- was washed on
reverse phase Sep-Pak columns. Lipids were washed with organic solvent system
and after evaporation of solvents lipids were reconstituted in This study was performed within the
framework of COST B35 action and supported by Grant-in-aid No.
83/N-Cost/2007/0. 26. INSIGHTS INTO DNA REPAIR SYSTEM IN
COLON CANCER CELL LINES EXPOSED TO IONIZING RADIATION M.
Skonieczna1, A. Lalik1, A. Cieślar-Pobuda1,
S. Student1, J. Rzeszowska-Wolny1,2 1Silesian
University of Technology, 44-100 Gliwice, Poland, 2Maria
Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Branch in
Gliwice, 44-100 Gliwice, Poland The oxidative stress generated by ionizing radiation
during radiotherapy induces different type of DNA damage. The formation of
DNA lesions leads to activation of DNA repair enzymes or to genomic
instability and apoptosis. In this process, the tumor suppressor gene p53 plays
a major role in allowing DNA repair or triggering apoptosis when DNA
alteration has reached an unacceptable level. Here we compared the level of DNA strand breaks and
their repair in two cell lines differing in p53 status (HCT116 p53+/+ and
HCT116 p53-/-) exposed to ionizing radiation. The level of DNA strand breaks
was measured by the comet and micronuclei assay. The changes in expression of
genes coding for DNA repair proteins were analyzed by quantitative RT-PCR.
The level of poly(ADP-ribose) in the first minutes after exposure to IR was
also assessed. Our results suggest that wild type and p53 mutated HCT 116
cells differ in mechanisms of DNA repair. The differences in DNA repair did
not concern the first steps of DNA repair since the level of DNA damage and
poly(ADP-ribose) production in the first minutes after exposure to IR were
almost the same in both cell lines. However, transcripts of genes from DNA
repair pathways analyzed by quantitative RT-PCR showed different trends in
expression after exposure to irradiation in HCT p53+/+ and HCT p53-/- cells. Also DNA breaks level at
30-60 min. after irradiation was statistically higher in HCT p53-/- than in HCT p53+/+. 27. NFkB SUPPRESSES P53-DEPENDENT UV-INDUCED APOPTOSIS Katarzyna
Szołtysek1, Katarzyna Pietranek1, Nataliya Kashchak1,2,
Magdalena Kalinowska-Herok1, Jakub Hanus1, Monika
Pietrowska1, Marek Kimmel3, Piotr Widłak1 1Department of Experimental and Clinical
Radiobiology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology,
Gliwice Branch, Poland, 2Institute of Cell Biology, NAS of Ukraine, 3Institute of Automation, Memorial
Silesian University of Technology, Gliwice, Poland Signaling pathways that depend on p53 or NFκB transcription
factors are essential components of cellular responses to stress. In general,
p53 is involved in either activation of cell cycle arrest or induction of
apoptosis, while NFκB exerts mostly anti-apoptotic functions; both
regulatory pathways apparently interfere with each other. Here we aimed to analyze effects of NFκB
activation on DNA damage-induced apoptosis, either p53-dependent or
p53-independent, in a set of human cell lines. Four cell lines, HCT116 and
RKO colon carcinoma, NCI-H1299 lung carcinoma and HL60 myeloblastoma, each of
them in two congenic variants either containing or lacking transcriptionally
competent p53, were used. Cells were incubated with TNFα cytokine to
activate NFκB and then treated with ultraviolet or ionizing radiation to
induce apoptosis, which was assessed by measurement of the sub-G1 cell
fraction. We observed that treatment with TNFα resulted in an
approximately 2-fold reduction in the frequency of apoptotic cells in
UV-irradiated p53-proficient lines (with exception of UV-resistant NCI-H1299
cells). This anti-apoptotic effect was lost when cells were
pretreated with parthenolide, an inhibitor of NFκB activation. In marked
contrast, TNFα-pretreatment of p53-deficient lines resulted in an
increased frequency of apoptotic cells after UV irradiation (with exception
of HL60 cells). Such anti- and pro-apoptotic influence of TNFα was less
obvious in cells treated with ionizing radiation. The data clearly indicates
functional interference of both signaling pathways upon the damage-induced
apoptotic response, yet the observed effects are both cell type- and
stimulus-specific. 28. PHOTOPHYSICAL AND
BIOLOGICAL STUDIES OF SOME NOVEL PHOTOSENSITIZERS WITH PROSPECTS FOR USE IN
PDT Agnieszka
Szurko*,1,2,
Marzena Rams1, Aleksander Sochanik2, Franz-Peter
Montforts3, Piotr Kuś4, Violetta Kozik4,
Marta Stefaniak4, Marcin Rojkiewicz4, Grzegorz Zięba4,
Anna Pasewicz1, Alicja Ratuszna1 1A. Chelkowski
Institute of Physics, University of Silesia, Katowice, Poland, 2Maria Sklodowska-Curie
Memorial Cancer Center and Institute of Oncology, Gliwice, Poland, 3Institute of
Organic Chemistry, University of Bremen, Bremen, Germany, contact: mont@chemie.uni-bremen.de) 4Institute of Chemistry; University of Silesia,
Katowice, Poland Photodynamic therapy (PDT) is a promising approach
to cancer treatment which combines light, a photosensitizer and oxygen
action. However, search for novel, better photosensitizers is continuing as
majority of photosensitizers that have been synthesized do not exhibit desirable combinations of chemical, photophysical and
biological properties which is a perequisite for improved efficacy of PDT
against varying types of cancer. The aim of the presented study was to investigate
selected properties of various photosensitizing compounds with special
interest focusing on porphyrin-type and chlorin-type photosensitizers.
Preliminary in vitro cytotoxicity
studies were performed using colon adenocarcinoma cells (Hct116) and mouse
Lewis lung carcinoma cells (LLC). Cell proliferation was evaluated by MTS-tetrazolium
reduction assay for both types of photosensitizers. More detailed studies
were carried out for a chlorin-type photosensitizer, for which dark toxicity,
PDT efficiency and some chemical as well as photophysical properties were
evaluated. To verify chemical structure of the examined chlorin photoelectron
spectroscopy (XPS), Raman and infrared (IR) spectroscopy were used.
Photophysical properties such as absorption in the therapeutic window (600 –
800nm) were determined by UV-VIS spectroscopy (emission and excitation
spectra). The obtained Raman, IR and XPS spectra confirmed the
chemical structure of the examined chlorin derivative. Chemical purity,
photophysical and biological properties of this photosensitizing compound
fulfill suitability requirements for application in PDT. The investigated
porphyrin-type photosensitizers, on the other hand, require further study as
primary results indicate that high dark toxicity of some of them makes them
rather useless in PDT whereas others should be investigated thoroughly. The study has been financed by a grant
from the Ministry of Science and Higher Education, No. 0538/R/T02/2007/03 29. THE INTERACTION OF TRANSFRRIN
- DOXORUBICIN CONJUGATE WITH CCRF-CEM LEUKEMIA CELLS DOX
was coupled to human TRF by using glutharaldehyde crosslinking method. Apoptosis was detected in cells using Hoechst
33258/Propidium Iodide double staining after 48 hours incubation with drugs.
The results demonstrate that TRF-DOX induced a higher level of apoptosis and
necrosis in cultured cells than DOX. However, more studies are needed to show
if that conjugate of DOX could replace the free anthracycline in
chemotherapy. 30. The effect of Pirolin on the
level of oxidative stress induced in heart tissue OF Wistar rats treated with
anticancer drugs (Doxorubicin and taxanes) Sabina
Tabaczar1, Anna Pieniążek2, Jan Czepas1,
Joanna Zelga3, Krzysztof Gwoździński1 1Department
of Molecular Biophysics, 2Department
of Thermobiology, University of Łódź, 90-237 Łódź, Banacha 12/16, Poland, 3Nofer Institute of
Occupational Medicine, 91-348 Łódź, św. Teresy 8, Poland Doxorubicin (DOX) is an anticancer drug commonly
used in chemotherapy of various tumours.
However, antitumour therapy with this anthracycline is limited by the risk
of developing heart failure. Generation of reactive oxygen species (ROS)
contributes to the cardiotoxicity of doxorubicin, which is involved in the
treatment. It has been shown that risk of heart disorders is higher upon
taxanes therapies (especially in combined therapies with paclitaxel (PTX and
DOX). It seems that addition of antioxidants may decrease side effects of
anticancer drugs. Nitroxides are low molecular weight, stable free radicals
reacting with free oxygen radicals and present antioxidant properties. The aim of this study was to
analyze the effect of nitroxide Pirolin (PL, 3-carbamoyl-2,2,5,5-tetramethylpirroline-1-oxyl) on the oxidative stress level induced by DOX and
taxanes in the rats hearts. Rats were injected intraperitoneally with tested
compounds and sacrificed four days later. The investigated compounds were
injected alone or in combinations. Assessments of the amount of lipid
peroxidation products (TBARS) and peroxides level were performed. The
increase of the level of peroxides was found in the hearts treated with the
combination of doxorubicin and Pirolin in comparison with single treatment
with PL. In the case of docetaxel significant changes in the oxidative stress
markers were not detected. Neither protective nor prooxidative properties of
Pirolin were observed when used in combination with docetaxel. However,
results obtained after paclitaxel injection have shown increase of the level
of peroxides in heart tissue in comparison with control group. Pirolin in
combination with PTX significantly decreased the effect induced by
paclitaxel. DOX caused elevation of the level of TBARS in comparison with
control group. Induction of TBARS by Pirolin and taxanes in comparison with
control group was lower than that for DOX. Pirolin used simultaneously with
DOX or PTX decreased production of TBARS in comparison to drugs injected
alone. The
obtained results indicate that Pirolin influences prooxidative effect induced
by paclitaxel. Presented data suggest that Pirolin interacts with peroxides producing
thiobarbituric acid reactive substances in heart tissue. On the other hand,
in combination with DOX, Pirolin lowers the level of TBARS, thus acts as an
antioxidant. This work was supported by Ministry of
Science and Higher Education grant 31. P53 RELATED
RESPONSE OF HUMAN COLON CARCINOMA CELL LINES TO RADIATION AND BYSTANDER
SIGNALS Maria
Widel1,2, Waldemar Przybyszewski1, Agnieszka Szurko1,3,
Anna Lalik2, 1Department
of Experimental and Clinical Radiobiology, Center of Oncology Gliwice, 2Institute
of Automation, Silesian Technical University Gliwice, 3Institute
of Physics, University of Silesia, Katowice Radiation-induced bystander effects are biologic
responses of nonirradiated cells that were not traversed by an ionizing
radiation track. These bystander effects take place in the
neighbors of irradiated cells, bystander cells, via cell-to-cell gap junctions or in other
nonirradiated cells that have received secreted signals (soluble
factors) from irradiated cells. It
is likely that multiple signaling cascades involving both an initiating event
and downstream signaling steps are necessary to mediate the bystander
process. P53 gene seems to play an important role in the bystander effect
since it coordinates cellular response to oxidative stress created by
ionizing radiation through cell cycle control and apoptosis. The aim of our studies was focused on comparing the
response of two human colorectal carcinoma HCT116 cell lines differing in p53
status when they were directly exposed to radiation or exposed to soluble factors during
co-culturing with irradiated ones. As
the first step of experimental cycle the radiation (0-6Gy) sensitivity of
HCT116 p53+/+ and HCT116 p53-/- measured as clonogenic cell survival,
induction of micronuclei and apoptosis
was assessed. The dose dependent clonogenic cell survival was similar
for p53 wild type and p53 knocked-out cells; however, both cell lines
differed considerably in induction of micronuclei and apoptosis. Both cell
lines differed also in the expression of some genes (CASP6, IRAK1,CHUK)
engaged in apoptosis as measured by qRT-PCR. The
bystander experiments were then performed and micronuclei and apoptosis
frequencies were measured in radiation-exposed and bystander cells. Signal
molecules released from irradiated cells induced apoptosis in p53 knocked-out
cells with higher efficiency than in wild type cells. The molecular nature of
possible p53-independent apoptosis pathway needs further exploration. Supported
by the Polish Ministry of Science and High Education, granst: no N406
101/31/3870 and PBZ-MNiL-2/1/2005 32. D-METHIONINE IN NOISE INDUCED HEARING LOSS Wiktorek-Smagur A.1,
Jamesdaniel S.1, Politański
P.1, Rajkowska E.1, Sha S.2, Schacht J.2,
Śliwińska-Kowalska M.1 1Nofer Institute of Occupational
Medicine, 8 Teresy street, 91-348 Lodz, Poland, 2Kresge Hearing Research Institute,
University of Michigan, Ann Arbor, MI, USA We tested D-methionine (D-Met), a sulfur
containing compound, as an otoprotectant in male C57BL/6J/Han/IMP mouse
strain. D-Met was administrated 1 h
before and 1 h after noise exposure. One additional dose each was given on
days 1 and 2 after noise exposure. The changes in superoxide dismutase (SOD),
catalase, lipid peroxidation (LPO) were measured in the cochlea 3, 7, 14
after noise exposure (4 kHz octave band at the intensity of 110 dB SPL for 4
hours) in C57BL/6 mice. ABR were measured for each animal before and after
noise exposure and after 14 days. The ABR indicates significant functional
deficits due to noise exposure which stabilize in two weeks with a permanent
threshold shift (PTS) of 15 dB for both 4 kHz and 8 kHz. In addition we also
studied the action of an antioxidant D-methionine (D-met) to investigate its
role in preventing this noise induced oxidative stress and hearing loss.
D-met was able to scavenge the free radicals resulting in a significant
decrease in LPO levels from noise exposure controls on the 7th
day, apart from attenuating the changes in enzyme activity to control limits
on the 3rd and 7th day. It also significantly reduced
the PTS observed on the 14th day from 15 dB to 5 dB for 4 kHz and
from 15dB it to 7 dB for 8 kHz . In summary, we established that D-met
significantly protected against permanent noise-induced hearing loss. The study was supported by the NoiseHear project
(contract No. MTDK-CT-2004-003137) under the 6th European
Community Framework Programme, the Marie Curie Host Fellowship for the
Transfer of Knowledge. 33. COMBINED
ANTICANCER THERAPY WITH ANTIVASCULAR PEPTIDE AND CHEMOTHERAPEUTIC-LOADED
LIPOSOMES Aleksander Sochanik, Iwona
Mitrus, Ryszard Smolarczyk, Tomasz Cichoń, Stanisław Szala Department of Molecular Biology, Maria
Skłodowska-Curie Memorial Cancer Center, Wybrzeże Armii Krajowej 15, 44-101
Gliwice, Poland The study has sought to validate the combination of
a vascular-targeting RGD-4C-GG-D(KLAKLAK)2 oligopeptide and a
chemotherapeutic (doxorubicin) entrapped in long-circulating liposomes
(targeted by folate ligand and non-targeted) in destroying malignant melanoma
tumors in mice. The RGD-motif allows selective binding of the oligopeptide to
αVβ3 integrin receptors The KLAKLAK2-motif domain mediates
apoptosis of endothelial cells with subsequent necrosis of neighboring
neoplastic cells. The liposomes allow the chemotherapeutic to penetrate and
destroy neoplastic cells. Mice bearing B16(F10) tumors were subjected to the
combined treatment to seek improvement of the therapeutic outcome. Liposomes
(DSPC/cholesterol/DSPE-PEG or DSPE-PEG-folate) were prepared using ammonium
sulphate solvation procedure followed by extrusion and dialysis into sucrose
solution. Doxorubicin was entrapped inside liposomes using transmembrane pH
gradient. Animals were administered the peptide intratumorally and liposomes
were injected intravenously. Tumor growth rate and animal survival were
monitored. Monotherapy of B16(F10) melanoma tumor-bearing
C57BL6 mice with therapeutic peptide alone (4× 250μg) resulted in inhibited tumor growth but did not extend
animals’ survival. A single
round of alternating repeated administrations of KLAK (4×250μg of
peptide) and/or doxorubicin-carrying liposomes (4×40ug of drug) to
tumor-bearing mice showed no sizeable differences in tumor growth inhibition
or survival between groups treated with non-targeted or targeted liposomes, although both combinations were
better than controls or free doxorubicin. On the other hand, when growth of
tumors was rechallenged with a second round of combination therapy, tumor
growth was inhibited several-fold and survival of animals was significantly
extended compared to controls. Repeated combination therapy revealed
differences between antivascular peptide combination with FTL and that with
NTL liposomes in favor of pegylated targeted liposomes. Peptide-mediated monotherapy resulted in multifocal
cluster-type necrosis involving ca.
20% of tumors whereas liposomal doxorubicin produced multiple necrotic foci
spread throughout tumors (ca. 15%). Combination therapy caused diffuse type
of necrosis involving ca. 60% of
tumors. 34. EFFECTS OF
POLY(ADP-RIBOSE) POLYMERASE INHIBITION IN MYELOGENOUS LEUKAEMIA CELLS K562 Katarzyna
Badura, Anna Byczek,
Magdalena Gwarda, Artur Cieślar- Pobuda Silesian University of Technology,
44-100 Gliwice, Poland Poly(ADP-ribose) polymerase 1 (PARP-1 or PARP), is a
nuclear enzyme catalyzing the synthesis of long and branched homopolymers of
ADP-ribose from NAD+ molecules. The enzyme is well known for its
multiple regulatory functions: from DNA repair and transcription to cell
survival and death. Modulating its activity, from stimulation to inhibition,
is claimed to be applicable in treatment or prevention of many disease states
including cardiac infarct, diabetes, inflammation,
retroviral infection and cancer. So far, several
PARP inhibitors have been developed, mostly to sensitize tumor cells
to chemo- and radiotherapy through inhibition of DNA repair. We used an
immunocytochemical approach in our study of myelogenous leukaemia cells
(K562) and mouse Lewis lung carcinoma cells
(LLC)
treated with 100 μM H2O2 to examine in situ the
PARP inhibitory efficiency of 8-hydroxy-2-methyl-4(3H)-quinazolinone (NU1025). We also used comet assay to
compare kinetics of DNA repair with the level of poly(ADP-ribose), PARP and
its spatial distribution in nuclei. According to the obtained data, NU1025 indeed
inhibits PARP which is demonstrated by significant decrease in the level of
poly(ADP-ribose) during DNA strand break rejoining. The results of microscopic observations suggest
hat there are also differences in the distribution of PARP in cells with
inhibitor as compared to untreated cells. Cells treated with NU1025 showed
tendency to cumulate PARP in larger aggregates, whereas poly(ADP-ribose)
polymerase in untreated cells exhibited more uniform distribution. 35.
RADIATION INDUCED BIOLOGICAL BYSTANDER EFFECT ELICITED IN VITRO BY TARGETED RADIOPHARMACEUTICALS LABELED WITH a-, b- AND AUGER ELECTRON EMITTING RADIOHALOGENS - lecture Marie
Boyd Radiation Biology Group, Division of Cancer
Science and Molecular Pathology, Glasgow University, Cancer Research UK
Beatson Laboratories, Glasgow, G61 1BD Scotland; tel: +44 (0)141 330 4126;
FAX: +44 (0)141 330 4127; e-mail: r.mairs@beatson.gla.ac.uk Recent studies have shown that indirect effects of
ionizing radiation may contribute significantly to the effectiveness of
radiotherapy by sterilizing malignant cells that are not directly hit by the
radiation. However, there have been few
investigations of the importance of indirect effects in targeted radionuclide
treatment. Our purpose was to compare
the induction of bystander effects by external beam γ-radiation with
those resultant from exposure to three radiohaloanalogues of meta-iodobenzylguanidine (MIBG): [131I]MIBG
(low linear energy transfer (LET) β-emitter), [123I]MIBG
(potentially high LET Auger electron emitter), and meta-[211At]astatobenzylguanidine
([211At]MABG) (high LET α-emitter). Methods: Two human tumor cell lines - UVW (glioma) and EJ138
(transitional cell carcinoma of bladder) – were transfected with the
noradrenaline transporter (NAT) gene to enable active uptake of MIBG. Medium from cells that accumulated the
radiopharmaceuticals or were treated with external beam radiation was
transferred to cells which had not been exposed to radioactivity and
clonogenic survival was determined in donor and recipient cultures. Other
endpoints were also examined to determine the mechanisms involved in the
observed effects. Results: Over the dose
range 0 to 9 Gy of external beam radiation of donor cells, 2 Gy caused 30 to
40% clonogenic cell kill in recipient cultures. This potency was maintained but not increased
by higher dosage, thus indicating lack of a dose response relationship with
respect to the generation of bystander signals after a particular dose
administered to the donor cells. In
contrast, no corresponding saturation of bystander cell kill was observed
after treatment with a range of activity concentrations of [131I]MIBG,
which resulted in up to 97% death of donor cells. Cellular uptake of [123I]MIBG
and [211At]MABG induced increasing recipient cell kill up to
levels that resulted in direct kill of 35 to 70% of clonogens. Thereafter, the administration of higher activity
concentrations of these high-LET emitters was inversely related to the kill
of recipient cells. Over the range of
activity concentrations examined, neither direct nor indirect kill was
observed in cultures of cells not expressing the noradrenaline transporter
and thus incapable of active uptake of MIBG. Conclusion:
Potent toxins are generated
specifically by cells which concentrate radiohalogenated MIBG. These may be LET-dependent and distinct
from those elicited by conventional radiotherapy. 36. BINDING OF GENISTEINE DERIVATIVES TO ABL AND LCK PROTEIN
KINASES, AND TO MICROTUBULES - MODELLING STUDIES - lecture Krystiana Krzysko, Bogdan Lesyng Centre of Excellence ”BioExploratorium” and Department of Biophysics,
University Genistein
derivatives inhibit activity of Abl and Lck tyrosine kinases. These
derivatives also cause disintegration of microtubules of the central spindle when
applied at micromolecular concentration to the studied cell lines, as well as
inhibit polymerization of tubulin. In
our modeling studies we optimized structures of selected genistein
derivatives. Their most stable conformers were identified. Structures of the
Abl and Lck kinases were also refined. Possible binding sites were found, and
the genistein derivatives were docked to the mentioned targets. Also, 3D
structures of microtubules were build up and optimized. Their end-parts
contain GTP and GDP nucleotides. Experimental results reported by A. Rusin
during this conference were accounted by our modelling studies. During this conference novel results of our modeling studies will be
reported, in particular:
Conclusions will be formulated in
context of the experimental results obtained by the collaborating
experimental groups. Acknowledgements: These studies are supported by the Ministry of Science
and Higher Education (grant 7C-COI/PBZ/2008) 37. IMPACT OF ACIDOSIS ON MODULATION OF GABAERGIC IPSCS BY
BENZODIAZEPINE RECEPTOR AGONISTS - lecture Jerzy Mozrzymas, Tomasz Wójtowicz, Paulina
Wyrembek, Katarzyna Lebida, Michał Piast, Katarzyna Mercik Lab.
Neurosci. Dept. Biophysics, Wrocław Medical University, Wrocław, Poland Benzodiazepine (BDZ)
receptor agonists are known to increase the amplitude and duration of IPSCs.
Moreover, at low [GABA], BDZs strongly enhance the current responses
suggesting the up-regulation of agonist binding while their action on gating
remains a matter of debate. Importantly, BDZs are widely used in clinical
practice while several brain pathologies are associated with local
alterations of extracellular pH (most typically acidosis) in the brain.
Taking this into account, we have examined the impact of combined action of
BDZ receptor agonists and acidification of extracellular medium on GABAergic
currents. For this purpose, the effects of these factors on GABAAR
binding and gating were investigated by a parallel analysis of mIPSCs and the
current responses to rapid GABA applications. At control pH (7.2), flurazepam
and zolpidem enhanced the amplitude and prolonged decay of mIPSCs. Both BDZ
receptor agonists strongly enhanced responses to low [GABA] but,
surprisingly, decreased the currents evoked by saturating or half-saturating
[GABA]. Analysis of current responses to ultrafast GABA applications
indicated that flurazepam and zolpidem enhanced binding and desensitization
of GABAA receptors. These BDZ receptor agonists markedly prolonged
deactivation of responses to low [GABA] but had almost no effect on
deactivation at saturating or half-saturating [GABA]. Recordings of responses
to half-saturating [GABA] applications revealed that appropriate timing of
agonist exposure was sufficient to reproduce either a decrease or enhancement
of currents by flurazepam or zolpidem. Recordings of currents mediated by
recombinant (“synaptic”) a1b2g2 receptors reproduced
all major findings observed for neuronal GABAARs. Thus the results
obtained at pH = 7.2 indicated that extremely brief agonist transient renders
IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.
Our previous studies (Mozrzymas et al. 2003, J.Neurosci.) have shown that
binding and desensitization are strongly affected also by extracellular pH,
the factor that may be severely altered in brain pathology. We have found
that at acidic pH (6.0), flurazepam produced a stronger enhancement of mIPSC amplitudes
than at physiological pH. At low [GABA], flurazepam markedly enhanced current
amplitudes both at normal and at acidic pH but at the latter, the relative
effect was larger. On the contrary, at saturating [GABA], flurazepam reduced
current amplitudes both at pH = 7.2 and 6.0. Slowing down of deactivation kinetics by flurazepam decreased
with GABA concentration but at pH = 6.0, this trend was shifted towards
higher [GABA]. Pharmacokinetic analysis of current responses to ultrafast
GABA applications indicated that the effects of flurazepam and protons are
additive. We conclude that changes in extracellular pH not only affect the
amplitude and time course of mIPSCs but also alter their susceptibility to
modulation by benzodiazepine receptor agonists. Support:
Wellcome Trust International Senior Research Fellowship in Biomedical Science
(grant No. 070231/Z/03/Z) 38. STRUCTURE OPTIMIZATION OF THE LCK KINASE
CATALYTIC DOMAIN AND DESIGN OF ITS GENISTEIN DERIVATIVE INHIBITORS Joanna Panecka, Krystiana
Krzysko, Bogdan Lesyng Centre of Excellence ”BioExploratorium” and
Department of Biophysics, University of Warsaw, Faculty of Physics, Zwirki i
Wigury 93, 02089 Warsaw (panecka@bioexploratorium.pl) Aberrant
activity of many protein tyrosine kinases has been identified as one of the
reasons for cancer development. There is some evidence suggesting, that
genistein and its derivatives are promising candidates for inhibitors of the
Lck kinase, upregulation of which is connected to various cancers, including
human leukaemia. In the present work we used computational modelling
techniques to study interactions of the selected genistein derivatives with
the Lck kinase. We modelled and optimized structure of the protein. Then, we
performed docking simulations of the ligands to the active site. Stability of
the most energetically favorable binding modes has been verified by means of
molecular dynamics method. Based on the results, we selected the most stable
binding modes and suggested certain modifications of the molecules, that
could lead to stronger and more specific binding.
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