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POSTERS
EFFECT OF TNF ON
PROLIFERATIVE ACTIVITY OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVECs) IN NON-HODGKIN LYMPHOMA
Barilka V.A., Matlan
V.L., Piddubnyak V.A.*, Volodko N.A.*, Novak S.V.*, Chervinska O.D., Bilynsky B.T.*, Loginsky V.E.
Institute of Blood
Pathology and Transfusion Medicine,
Academy of Medical Sciences of Ukraine, Lviv, Ukraine
*Danilo Galytsky Lviv State
Medical University, Department of Oncology and Medradiology, Lviv, Ukraine
Angiogenesis plays a key role in tumor growth,
expansion and metastasis. The critical step in angiogenesis is the proliferation of endothelial cells, which is induced and
regulated by network of mitogens and cytokines such as tumor necrosis factor (TNF) a. o. TNF level is heightened in biological
liquids in many pathological conditions and plays an important role in pathogenesis of malignant lymphomas. However the role of
TNF in angiogenesis of non-Hodgkin lymphoma (NHL) is not clear. The aim of our study was to determine the proliferative activity
of HUVECs after influence of recombinant human TNF (rhTNF, Sigma) and supernatants from primary cultures of mononuclear cells
from lymph nodes (MNLN), peripheral blood (MNPB) and plasma of 10 patients with NHL.
Methods. HUVECs were obtained from
human umbilical vein by digestion with 0,2% collagenase and were cultivated as described (Jaffe E.A., 1973). TNF level was
detected by bioassay. TNF activity concerning HUVECs proliferation was measured by incorporation of 3H-thymidine and estimated
by index proliferation (IP). The NHL patients (Low-Inter Grade) were not treated before investigation. Results. Obtained data
indicated that in NHL patients, plasma as well as supernatants samples of MNLN, where TNF concentrations were 0,944plusmn;0,265
ng/ml and 0,142plusmn;0,067 ng/ml respectively, have comparatively strong proliferative activity on HUVECs (IP=1,507plusmn;0,360
and IP=1,460plusmn;0,213 respectively). MNPB more slowly induced proliferation of HUVECs (IP=1,072plusmn;0,215) and the TNF
level was lower in these supernatants (0,082plusmn;0,020 ng/ml); p<0,05. We found that low concentrations of rhTNF (from 0 -
1,550 ng/ml) inhibit the 3H-thymidine incorporation in HUVECs (IP<1). In a presence of intermediate rhTNF concentrations (1,550
ng/ml up to 13,630 ng/ml) the significant growth of endothelial cells was revealed (IP>1; r=0,966). However in higher
concentrations of rhTNF the cytolysis of HUVECs was observed. Conclusion. Both endogenic and rhTNF in vitro influence on HUVECs
proliferative activity in dose-dependent manner. These events may bear some relation to angiogenesis control in malignant
lymphomas.
SEMINIFEROUS EPITHELIUM DEGENERATION IN TRANSGENIC MICE
THAT CONSTITUTIVELY EXPRESS TRANSCRIPTIONALLY
ACTIVE
HEAT SHOCK FACTOR (HSF1) IN SPERMATOCYTES
Benedyk Konrad, Widłak Wiesława, Głowala Magdalena, Małusecka Ewa,
Krawczyk Zdzisław
Department of Tumor Biology, Centre of Oncology, M. Skłodowska-Curie Memorial Institute Gliwice,
Poland
Somatic cells exposed to elevated temperature and other non-physiological conditions respond to such a stress by a
rapid induction of the heat shock proteins (HSPs), which major function is to protect cells from the harmful effects of
denaturation of cellular proteins. The stress induced expression of hsps is mediated by the heat shock transcription factor 1
(HSF1). HSF1 exists in unstressed cells in a monomeric form that does not bind DNA. Stress conditions induce its conversion to
trimeric DNA-binding form. Mechanisms of the stress-response in mammalian germ cells are not clear at the moment. Mammalian
testes must descend from the abdominal cavity for normal development to occur and consequently the elevated testicular
temperature disrupts spermatogenesis and causes infertility. The role of heat shock proteins and HSF1 in this process is not
known. A previous studies suggested, that activation of HSF1 in testes would be a major trigger for the induction of apoptosis
in germ cells.
The aim of this work was to study the role of HSF1 in germ cells. We constructed transgenic mice that
constitutively express active trimeric form of HSF1 in spermatocytes. For this purpose, expression of the human HSF1 with
deletion of the regulatory domain (amino acids 221-315) was driven by the rat hst70 promoter. Hst70 gene is a unique member of
the multigene hsp70 family that is specifically activated during meiosis in pachytene spermatocytes. We obtained three
transgenic founders (only females) and established three lines. We confirmed the transgene expression in testes by RT-PCR.
Transgenic male mice mated with wild-type females did not sire any offspring despite normal copulatory behavior
(transgene-positive females were fertile). At autopsy, the testes of transgene-positive male mice were grossly smaller than
those of transgene-negative mice. Histopatological analysis of the testes of transgene-positive mice showed that the size of the
seminiferous tubules was reduced. Tubules of wild-type mouse contain mitotic spermatogonia at the periphery of seminiferous
tubules, spermatocytes, round spermatids and elongating spermatids being released into the tubule lumen. Seminiferous tubules
from transgenic mouse contain mitotic spermatogonia and disorganized mix of pachytene, leptotene/zygotene spermatocytes as well
as vacuoles and giant cells but no postmeiotic spermatids or spermatozoa. Apoptotic cells were detected in situ by TUNEL assay.
Such analysis revealed that seminiferous tubules in cross-sections of testes from transgene-positive mice contained a cluster of
apoptotic cells, whereas few apoptotic cells were detected in wild-type mice.
In somatic cells HSF1 induces expression of
HSPs and is an essential survival factor in cells exposed to thermal stress. We concluded that in germ cells HSF1 has an
opposite role. It inhibits spermatogenesis and induces death of germ cells by apoptosis. Apoptosis of germ cells depends on
death receptors and Bcl2-family proteins. We postulate that HSF1 acts as a transcriptional regulator of genes (yet unknown)
involved in this process.
INHIBITION OF 8-OXO-2'-DEOXYGUANOSINE 5'-TRIPHOSPHATE PYROPHOSPHOHYDROLASE (8-OXO-dGTPase)
ACTIVITY OF HUMAN MTH1 PROTEIN BY PHYSIOLOGICAL INHIBITORS, NUCLEOSIDE 5'-DIPHOSPHATES
Białkowski Karol
Department
of Clinical Biochemistry, The Ludwik Rydygier Medical University, Karłowicza 24, 85-092 Bydgoszcz; e-mail:
karolb@amb.bydgoszcz.pl
Human homologue of E. coli MutT protein (hMTH1), an enzyme decomposing
8-oxo-2c-deoxyguanosine 5c-triphosphate (8-oxo-dGTP) to 8-oxo-2c-deoxyguanosine 5c-monophosphate (8-oxo-dGMP) and inorganic
pyrophosphate, is thought to play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA. As
we found in our previous studies, 8-oxo-2c-deoxyguanosine 5c-diphosphate (8-oxo-dGDP) is a strong inhibitor of 8-oxo-dGTPase
activity (Białkowski, K. and Kasprzak, K.S., 1998, Nucleic Acids Res. 26, 3194-3201). In the present study we have tested the
inhibitory effects of canonical ribo- and deoxyribonucleoside 5'-diphosphates (NDP's and dNDP's) on the activity of human
8-oxo-dGTPase trying to answer the question, whether the enzyme can effectively decompose 8-oxo-dGTP in the presence of
physiological concentrations of NDP's and dNDP's. Among eight tested nucleoside diphosphates the strongest inhibitors of human
8-oxo-dGTPase were dGDP (Ki = 74 micro;M) > dADP (Ki = 147micro;M), and GDP (Ki = 502 micro;M). Other dNDP's and NDP's as dCDP,
dTDP, ADP, CDP, and UDP appeared to be equally very week inhibitors of 8-oxo-dGTPase activity. A potential of
diphosphonucleosides to inhibit 8-oxo-dGTPase activity in vivo is discussed. We also present a novel approach to investigating a
substrate specificity of MTH1 proteins that is based on 8-oxo-dGTPase activity competitive inhibition studies.
THE
EXPRESSION OF A DNA REPAIR PROTEIN - XPA
IN NON-SMALL CELL LUNG CANCERS
Czarny Małgorzata, Chyczewski Lech*, Chorąży
Mieczysław, Rusin Marek
Center of Oncology - M.Skłodowska-Curie Memorial Institute, Department of Tumor Biology, Gliwice,
Poland
*Department of Clinical Molecular Biology Medical Academy, Białystok, Poland.
Lung cancer is the most common
cause of cancer death in industrialized countries. The procees of cancer development is connected with damage in DNA repair
pathways and accumulation of somatic mutation in genes coding for protein involved in regulation of cell proliferation,
migration and apoptosis. Deficiences in DNA repair correlate with an increased sensitivity to the cytotoxic effect of particular
DNA damaging agents, as well as the tendency for chromosomal abnormalities and mutations in specific oncogenes and tumor
suppresor genes. Studying the proceses preventing carcinogenesis e.g. DNA repair pathways may lead to better ways of cancer
prevention. There are several systems that repair damaged DNA in human cells. Nucleotide excision repair (NER) is considered to
be a major DNA repair mechanisms, involved in the removal of wide spectrum of bulky DNA adducts modyfying DNA double helix
conformation. The XPA protein is a component of NER and plays a central role in DNA lesion recognition. Regulation of XPA gene
expression has not been extensivelly studied. We analyzed expression of XPA protein in non-small cell lung cancer (NSCLC) tissue
using immunohistochemical metod. We detected high expression of XPA protein in cancer cells. This was surprising. We expected
low XPA expression in cancer cells because null mutation in XPA gene cause cancer-prone human disorder xeroderma pigmentosum. We
also compared the expression of XPA gene in primary human diploid fibroblasts (GMO7532) and lung cancer cell line A549. Using
western-blot and immunochemical metods we found overexpression of XPA in A549 line and low expression in GMO7532 cells.
Additionally, we found that expression of XPA is reduced in human diploid fibroblasts after contact inhibition of their growth.
It is possible that overexpression XPA in lung cancer contributes somehow to the cancerous phenotype of cells. The study on the
expression in an extensive set of lung cancer cell lines is currently under way.
FUNCTIONAL ANALYSIS OF SEQUENCES
UPSTREAM OF THE RAT HSP70.1 STRESS GENE
Fiszer-Kierzkowska A., Wysocka A., Vydra N., Lisowska K., Krawczyk
Z.
Department of Tumor Biology, Centre of Oncology Maria Skłodowska-Curie
Memorial Institute, Gliwice, Poland
The
HSP70 family constitutes the most conserved and best studied class of HSPs. There are two inducible histocompatibility complex
(MHC)-linked HSP70 genes in rat genome, which show highly differentiated expression pattern from being strictly inducible in
certain tissues to constitutively active in various tumours. Little specific knowledge is available about the regulation of
tissue- and cell-specific expression of hsp70i genes under normal and stress conditions in vivo. Some studies show that beside
HSE-HSF there are other cis-trans interactions regulating both basal and inducible transcription. The characterization of
noncoding sequences of the hsp70i genes has been suggested, in order to define the novel cis regulatory elements that could
influence the expression level of these genes.
Structure of the locus containing hsp70i genes was recently extensively
investigated in rat. A relatively extensive regions surrounding the hsp70.2 and hsp70.3 genes have been analyzed. Here we
present the promoter study of the hsp70.1 gene.
Functional studies of the promoter region were performed by transient
transfection assays with the use of series of constructs containing CAT reporter gene under control of various fragments of the
hsp70.1 gene promoter. We observed that out of the three HSE sequences present in the promoter the central one is crucial for
heat inducible transcription. This observation is in agreement with others' results (Konishi et al., 1995), which proved that of
two proximal HSEs only the HSE2 interacts with protein factors in heat shocked rat tissues. The level of heat inducible activity
can also be modulated by sequences other than heat shock element. Sequence located between -869 and -478 which does not contain
any evident regulatory elements enhances substantially heat inducible CAT activity. Sequences localized further upstream in the
region between position -1024 and -869 cause significant downregulation of transcription. This effect may be reversed when more
upstream DNA region containing the microsatellite sequence is incorporated into the construct. In our previous experiments we
observed forming of non-B-DNA structure within this microsatellite sequence in vitro. It remains to be established if any
non-B-DNA structure forms in vivo and if it is responsible for described regulation of the reporter gene.
HSP70
OVEREXPRESSION DOES NOT PROTECT MITOTIC SPINDLE
IN NORMAL (V79) AND TUMOR (B16) CELLS DURING AND AFTER EXPOSURE TO BENOMYL
AND GRISEOFULVIN
Głowala Magdalena, Mazurek Agnieszka, Piddubnyak Valeria, Fiszer-Kierzkowska Anna, Krawczyk
Zdzisław
Department of Tumor Biology, Centre of Oncology, M. Sklodowska-Curie Memorial Institute Gliwice,
Poland
Enhanced expression of hsp family genes results in increased resistance of cells to cytotoxic agents like eleveted
temperature or drugs. Previously we have shown that V79 cells transfected with rat hsp70 are more resistant to cytotoxicity
caused by heat shock or toxins from airborne pollutants. Interestingly, incubation with extract of airborne pollutants gave the
higher level of disturbed mitotoses for hsp70 transfected than for mock transfected cells.
Now we have shown that toxicity
of mitotic spindle poisons like benomyl and griseofulvin is more or less similar for hsp70 overexpressing and mock transfected
V79 and B16 cells as measured by survival assay. We also did not observe very relevant differences in hsp70 overexpressing
versus normal cells as far as mitotic spindle structure is concerned (the exception is benomyl in concentration of 5 ug/ml that
results in relatively less invalid mitoses in hsp70 clones of V79 and in B16 cell lines).
Experiments with 24h recovery
after basic 24h of incubation with benomyl or griseofulvin result in increased survival of V79 (hsp70 ) cells when compared to
mock transfected cells. Such a difference is also observed when cells are exposed to this toxins for more than 24h. There is no
difference in the percentage of disturbed mitoses between hsp70 and hsp70- cells after 24h of recovery. It means that although
mitotic spindle structure is not protected in hsp70 overexpressing cells after exposure to benomyl or griseofulvin their
survival is higher than cells with no hsp70 overexpression.
MEDIUM - MEDIATED BYSTANDER EFFECTS ON K562 CELLS
IRRADIATED
BY X-RAYS
Konopacka Maria1, Rogoliński Jacek1, Orlef Andrzej2, Rzeszowska-Wolny Joanna1
1Department of
Experimental and Clinical Radiobiology, 2Department of Medical Physics, Institute of Oncology, ul. Armii Krajowej 15, 44-100
Gliwice, Poland
Introduction
It has long been accepted that the radiation-induced DNA damage in cells is the results,
either by direct ionization or by the production of free radicals. Over the past decade it has been demonstrated that the
genetic damage occurred in cells that not direct irradiated but respond to signals transmited from cells irradiated. This
phenomen has been termed the 'bystander effect'. There are proposed at least two mechanisms for the transmission of signals from
irradiated cells to un-irradiated. One line of evidence indicates that the bystander effect is dependent on intracellular
communication through gap junctional. Another studies suggest a second mechanism in which irradiated cells secrete cytokines or
other soluble factors into the culture medium. The medium from irradiated cells (ICM-irradiation conditionen medium) can
initiate genetic changes in un-irradiated cells such as apoptosis, chromosomal aberrations, mutations and modulations of
specific proteins expression. It has been demonstrated that medium from irradiated human epithelial cells induced the bystander
response in un-irradiated cells but medium from irradiated human fibroblasts did not. Since this effect depends on the type of
cells it seems to be interest to test the medium-mediated bystander effect in human leucemic cells.
Aim
In the present
study the medium-mediated bystander effects in human leucemic K562 cells was tested. The direct as well as bystander cells
responses to X-radiation were compared.
Material and Methods
The cultures of K562 cells were exposed to X-radiation at the
doses between 0 - 8 Gy. After one hour of incubation at 37oC the medium (ICM) was removed, filtered and transferred to
un-irradiated flasks containing the same line. The genetic changes in cells exposed to radiation or conditionen medium were
estimated as a frequency of apoptotic and micronucleated cells incubated at different time up 36h. The strand breaks were
measured using the single cell gel electrophoresis (comet) assay. The cytotoxicity of radiation or conditionen medium was
evaluated by trypan blue exclusion technique in cells cultured for 3 days.
Results
The increase of apoptotic as well as
micronucleated cells both, after X-ray or conditionen medium (ICM) exposure was observed. Only X-radiation but not ICM induced
the DNA strand breaks detected by the comet assay. The percent of viability was lower in cells incubated in ICM than in cells
exposed to radiation directly.
Conclusions
In the present study we observed the medium-mediated bystander response in
human leucemic K562 cells.
The DNA damage induced by the bystander effects is not the same as that induced by radiation
directly.
ASSESSING GENOTOXIC PROPERTIES OF cis-Pt(II) 3-AMINOFLAVONE COMPLEX IN COMPARISON WITH cis-DDP
Kośmider
Beata1, Osiecka Regina1, Zyner Elzbieta2, Ochocki Justyn2,
Ciesielska Ewa3
1 Cytogenetics and Plant Molecular Biology
Department, University of Lodz, ul. Banacha 12/16, 90-237 Lodz, Poland
2 Inorganic Chemistry Department, Medical University,
ul. Muszynskiego 1, 90-151 Lodz, Poland
3 General Chemistry Department, Medical University, ul. Lindleya 6, 90-131 Lodz,
Poland
Cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most commonly and widely used chemotherapeutic drug
today. Despite obvious benefits, it exhibits several deleterious side effects. This has warranted search for its novel analogs,
equally potent yet less toxic. Previous demonstration of anticancer properties of flavonoid compounds prompted synthesis of
cis-bis(3-aminoflavone)dichloroplatinum(II). The cis-DDP analog is less toxic and has proved its anticancer properties in in
vivo studies. To assess genotoxic properties of this compound a comet assay was performed using human peripheral blood
lymphocytes. Analysis of DNA damage was done following 1h incubation of cells with cis-Pt(II) 3-aminoflavone complex in
comparison with cis-DDP. Kinetics of DNA repair after 0.5h 1h and 1.5h incubation also were investigated. It is shown that
cis-Pt(II) 3-aminoflavone complex causes increase of tail moment value compared to cis-DDP. Besides cross-links, DNA breaks may
be induced by the investigated compound. Lower values seen, on the other hand, for cis-DDP seem due to the presence of DNA-DNA
and DNA-protein cross-links. A similar relationship was observed for DNA repair processes. A distinct decrease in tail moment
was observed for cis-Pt(II) 3-aminoflavone complexas soon as after 0.5h.
This work was supported by a grant from the
State Committee for Scientific Research (KBN) No:6PO4C08621.
DEGRADATION OF MODIFIED
DEOXYNUCLEOSIDE-5'-TRIPHOSPHATES
BY HUMAN TISSUE HOMOGENATES
Lichota Katarzyna D., Tudek Barbara, Kuśmierek Jarosław
T.
Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland
Bases in deoxynucleotide-5'-triphosphate (dNTP)
pool can be damaged by mutagens as bases in DNA. The level of bases damage in dNTP pool is much higher than in the DNA. Modified
dNTPs can be mutagenic, because they can be incorporated into DNA by DNA polymerases. The E. coli MutT protein is an enzyme
which sanitise dNTP pool of 8OH-dGTP by its hydrolysis to 8OH-dGMP and inorganic pyrophosphate. The human homologue of MutT,
hMTH1 protein, hydrolyses also oxidised forms of dATP, 2OH- and 8OH-dATP. An important role in dephosphorylation of damaged
dNTPs can also play other enzymatic activities, like phosphatases and nucleotidases, and final product of dephosphorylation, the
modified dN can be exerted from cells.
We studied degradation of dNTPs formed during oxidative stress, 8-OH-dGTP and etheno
derivatives - edCTP and edATP, by homogenates from human tissues (lung tumour and healthy surrounding). The HPLC profiles of
degradation of the examined dNTPs showed the sequential formation of dNDPs, dNMPs and dNs, what suggests that various enzymatic
activities are engaged in this process. We found that 8OH-dGTP and edCTP are dephosphorylated much more efficiently than their
unmodified equivalents, whereas edATP is dephosphorylated nearly as efficiently as dATP. This suggests that degradation of at
least some damaged dNTPs could involve enzymatic activities more specific for the damage than these which are involved in
degradation of unmodified dNTPs. In all tested patients, the rate of dephosphorylation 8OH-dGTP and edCTP by tumour tissue
homogenates was always higher than by healthy tissue ones, whereas the rate of dephosphorylation of unmodified dGTP by both
types of homogenates was similar. This indicates higher level of sanitising activities in tumor than in healthy
tissues.
Ackowledgments: This work was supported by EU grant No QLK4-CT2000-00286.
EXPRESSION OF HSP25 IN THE RAT
LIVER AFTER TREATMENT WITH HEPATOTOXICANTS INDUCING INFLAMMATORY REACTION
Małusecka E., Zborek A., Krzyżowska-Gruca S.,
Krawczyk K.
Department of Tumor Biology, Centre of Oncology, M. Skłodowska-Curie Memorial Institute, Gliwice,
Poland
There is an abundant literature concerning the role of high molecular weight HSPs in inflammation. However less is
known about involvement of small heat shock proteins in this process. Induced expression of HSP25 was described in inflammatory
cells as well as in epithelium accompanied by infiltrating inflammatory cells (in epithelial cells in pancreatitis, in
epithelium of periapical lesions of endodontic origin and in bronchial epithelium).
The present study was undertaken to
examine the induction of HSP25 in inflammatory processes in the rat liver. For this purpose animals were treated with single
intraperitoneal injections of one of the indicated hepatoxicants: tioacteamide (TAA), D-galactosamine or allyl alcohol, which
cause an inflammatory reaction around the central veins, diffusely distributed throughout the entire liver lobule or localized
in periportal areas, respectively. These toxicants were administered for different time lapse ranging from 6 - 96 hrs. In
control animals the immunoreaction for HSP25 was never observed. Expression of HSP25 appeared only after 36 hrs after TAA
administration when the centrilobular inflammatory reaction was highly pronounced. The immunostaining was localized in
hepatocytes directly adjoining the inflammatory cells. Appearance of the HSP25 protein initiated the gradual regression of
inflammatory reaction. After the D-galactosamine treatment the inflammatory cells were dispersed in the lobule and HSP25 was
present in many hepatocytes in centrilobular and intermediate regions. Intoxication with allyl alcohol induced expression of
HSP25 in hepatocytes surrounding the areas of necrosis and inflammation in the periportal zone. Our results suggest that HSP25
was induced by inflammatory cells, but the mechanism of this induction needs further elucidation.
THE INFLUENCE OF IDA IN
COMBINATION WITH GLUTARALDEHYDE ON THE HUMAN ERYTHROCYTES
Marczak Agnieszka, Łubgan Dorota, Jóźwiak
Zofia
Department of Thermobiology, University of Łódź, Banacha 12/16, 91-237 Łódź, Poland
Idarubicin (IDA,
4-demethoxydaunorubicin) is a cytostatic drug, highly effective in the treatment of various types of malignancies, especially in
leukemias. It is well known that cancer chemotherapy remains unfortunately still largely nonspecific, and drugs, including IDA,
are toxic for tumour cells as well as for normal cells.
We have been interested in using erythrocytes as a biocompatible and
biodegradable drug carriers, to diminish the toxic effect and improve therapeutic index of IDA. In our study glutaraldehyde was
used as the agent linking covalently the drug to the proteins of erythrocyte membrane.
Nowadays little is known about the
interactions of IDA and glutaraldehyde with the human erythrocytes.
Thus, in the present work we have compared the combined
effects of IDA and glutaraldehyde on the properties of erythrocytes (GSH GSSG and GSH concentration, conformational changes of
haemoglobin). It has been observed that human erythrocytes treated with glutaraldehyde were able to take up the increased amount
of IDA from the incubation medium.
The influence of IDA and glutaraldehyde on the structure of heamoglobin was examined by
Electron Spin Resonance Spectroscopy. The data imply that the glutaraldehyde (in concentrations 0,0005%, 0,0025%, 0,005%) alone
has stronger influence that the glutaraldehyde and IDA.
The concentration of GSH GSSG and GSH was determined by Akerboom and
Sies method (1981). A decrease of both GSSG GSH and GSH ratio was observed in the presence of all tested concentrations of
glutaraldehyde alone (0,0005%, 0,0025%, 0,005%) as well as in combination glutaraldehyde with drug.
UNDERSTANDING
THE IMAGES OF COMETS
Matulewicz Łukasz, Chwałek Agnieszka, Wideł Maria, Rzeszowska-Wolny Joanna
Department of
Experimental and Clinical Radiobiology, Center of Oncology Gliwice,
Armii Krajowej 15, 44-101 Gliwice, Poland, e-mail:
lucas@cometix.com
Single cell gel electrophoresis is a rapid and sensitive microscopic method for detection of DNA damage
on the individual cell level. This assay is relatively inexpensive. The images of comets we can get fast, but the interpretation
might be difficult. The comets can be evaluated quickly by visual scoring but for accurate measurement is necessary to use
computerised image analysis. To use naked eye for comparison of images of comets with different tail moments and/or tail
inertias is not possible because the human eye cannot add different intensity levels of comet image area.
Among various
parameters calculated by computerised image analysis the tail moment has been regarded as one of the best and most popular
indices of induced DNA damage. But the tail inertia provides more precise information about the distribution of individual DNA
fragments within a tail. The tail inertia was also found to be the most sensitive indicator of DNA damage in comparison with
other tail parameters. The advantage of tail inertia over tail moment for estimation of DNA damage induced by radiation and H2O2
will be presented.
Key words: comet assay, quantitative image analysis
PCC IN A PLANT CELL - THE WAY TO
APOPTOSIS
Maszewski Janusz, Rybaczek Dorota
Katedra Cytofizjologii, Uniwersytet Łódzki, ul. Pilarskiego 14, 90-231
Łódź
Cell-cycle checkpoints provide an efficient control mechanism to ensure that DNA synthesis and segregation of
chromosomes between two daughter cells at mitosis proceed with accuracy high enough to preserve genomic integrity. A number of
inhibitory pathways within G1, S, G2 and M phases give a cell both time and means needed for repair processes to occur before
genetic alterations are rendered irreversible and heritable. Experiments on root meristems of Vicia faba indicate that, when
exposed to caffeine (a putative inhibitor of Rad3-like protein kinase), 2-aminopurine (2-AP; CDK inhibitor), or sodium vanadate
(NaVO3; protein phosphatase inhibitor), the S phase-blocked cells may override the S-M control system and induce premature
condensation of incompletely replicated chromosomes (PCC). A variety of aberrant mitotic divisions includes chromosomal breaks
and gaps, lost and lagging chromatids and chromosomes, acentric fragments, chromosome bridges and micronuclei. Furthermore,
electron microscopic studies demonstrate that, depending on the chemical agent, ultrastructure of interphase and mitotic cells
reveals some specific features, comprising both chromatin condensation and the appearance of cytoplasmic structures. The
long-term experiments show that root cells displaying PCC become transferred into apoptotic cells.
CASPASE 3 INDUCTION
AND SOME APOPTOTIC GENES MODULATION
IN HUMAN ACUTE PROMYELOTIC LEUKEMIA CELL LINE HL-60
BY CARBOPLATIN IN COMBINATION
WITH AMIFOSTINE
Mirowski M., Różalski M., Krajewska U., Balcerczak E., 2Młynarski W., Wierzbicki R.
Department of
Pharmaceutical Biochemistry, Molecular Biology Laboratory, Muszyńskiego 1 Street, 90-151 Lodz,
2Clinic of Pediatrics,
Institute of Pediatrics, Medical University of Lodz, Poland
(e-mail: mirowski@ich.pharm.am.lodz.pl)
The
influence of carboplatin and carboplatin in combination with amifostine on the growth, caspase 3 activity and some apoptotic
genes expression was investigated in vitro in human acute promyelocytic leukemia cell line HL-60. The exposure of HL-60 cells to
amifostine (10-6 to 10-3 M) showed no or very little influence on number of HL-60 cells in culture. Proliferation of HL-60 cells
exposure to carboplatin dropped down with increasing dose of the drug. This effect was slightly higher when carboplatin in
combination with amifostine was used. The values of cytotoxic indexes (IC50) was estimated as 6.6x10-4 and 4.4x10-4 M (24 hours
incubation) and 3.3x10-5 and 2.5x10-5 M (48 hours incubation) for carboplatin and carboplatin with amifostine, respectively.
This effect was accompanied by caspase 3 activity induction, which seems to play a key role in the apoptotic process. HL-60
cells treated with carboplatin alone showed caspase 3 activity increase about 120 times. Combination of carboplatin with
amifostine intensify caspase 3 activity to 280 times. Furthermore, the expression of selected genes, which are involved in
apoptosis like bcl-2, c-myc, bax, and p65 gene, which function in this process is unknown, were determined. Semi quantitative
RT-PCR technique showed decrease of bcl-2 gene expression and increase expression of bax, c-myc and p65 genes in HL-60 cells
treated with carboplatin in combination with amifostine in comparison to the cells treated only with carboplatin.
On the
basis of our study we conclude that amifostine may increase corboplatin therapeutic efficiency regarding to human acute
promyelocytic leukemia cells.
ALTERATION OF MEMBRANE PHYSICAL STATE
ACTS AS "MOLECULAR THERMOMETER"
AND MODULATE
THE EXPRESSION OF HEAD SHOCK PROTEINS
Nagy Enikö, Balogh Gábor, Horvath Ibolya, Benkö Sándor, Gyöfry Zsuzsa, Hoyk Zsófia,
*Bensaude Olivier, Vigh László
Institute of Biochemistry, Biol.Res.Centre of Hung.Acad.Sci., Szeged, Hungary;
*Regulation de l'Expression Génétique CNRS, Paris, France
The classical model on the sensing of heat shock
proposes that accumulation of denatured proteins triggers the activation of the stress-response. Our alternative, but not
exclusive view is that the temperature-sensing mechanism is associated with the physical state of membranes (Horvath et al,
1998; Vigh et al. 1998;Vigh and Maresca, 2002;). In the present study we made the first attempt to extend the validity of this
concept to mammalian cells.
We have treated mammalian cells (K562) with benzyl alcohol and heptanol at concentrations that
at normal growth temperature induce the expression of heat shock proteins, including the synthesis of the major stress proteins
such as HSP70. The critical concentrations of each of the two membrane fluidizers were selected so that their addition to cells
caused an identical increase in the level of plasma membrane fluidity. Formation of isofluid states resulted in almost identical
downshifts in the temperature thresholds of the HSR of the treated cells. As in the case of heat stress, the initial fluidity
up-shifts induced by the membrane perturbants was accompained by rise of intracellular Ca and was followed by a fluidity
relaxation period. It is noted that the above treatments resulted in no measurable changes in the lipid class and/or fatty acid
composition. By monitoring the enzymatic activity of luciferase expressed in mammalian cells we have demonstrated that not like
the heat stress, benzyl alcohol and heptanol do not cause protein denaturation at concentrations that do induce the heat shock
response.
Our results suggest, that membranes seem to act as cellular thermometer where thermal stress is sensed and
transduced into a cellular signal leading to the activation of heat shock genes.
Horvath et al., Proc. Natl. Acad. Sci.
USA,95:3513, 1998
Vigh, L. et al., Trends. Biochem. Sci.(TIBS) 23:369, 1998
Vigh,L. and Maresca,B. in Cell and Molecular
Responses to Stress, eds. Storey, K.B. Storey DN (Elseviere, Amsterdam), pp.173-188.
MICROSATELLITE INSTABILITY AND DNA
REPAIR IN COLON CANCER
Obtułowicz T4, Skjelbred CF1a,2, Bowitz Lothe IM3, Hansteen I-L1a, Bock G1b, Nilsen B1b, Jorgensen
H3, Aase S1c, Tudek B4, Kure EH2,3
1 Telemark Central Hospital, Skien, Norway: 1a Department of Occupational and
Environmental Medicine, 1b Department of Surgery, 1c Department of Pathology;
2 Telemark University College, Bo,
Norway;
3 Department of Pathology, Ullevaal University Hospital, Oslo, Norway,
4 Institute of Biochemistry and Biohysics
PAS, Warsaw, Poland
The main causes of colorectal carcinoma (CRC) include: (i) inflammatory processes and high fat diet
resulting in the increase of DNA damage, among others - lipid peroxidation (LPO) derived etheno-DNA adducts formation; (ii)
deficiency in mismatch repair (MMR) resulting in the increased genomic instability.
We have compared microsatellite
instability (in 5 microsatellites: Bat26, Bat40, D2, D5 and D17), expression of MMR proteins (MLH1, MSH2, MSH6) as well as the
activity of DNA glycosylases specific for 1,N6-ethenoadenine (eA) and 3,N4-ethenocytosine (eC) in tumour and normal colon
epithelium obtained during surgical intervention on 50 sporadic CRC patients in Norway.
Out of 50 analyzed CRC cases, in 6
patients disturbances of genomic stability and/or MMR deficiency was observed. Four of them revealed MSI in tumour tissue: two
in all five microsatellites, one in BAT26, BAT 40 and D5, and one in BAT26, BAT40 and D2. Disturbances in MMR proteins
expression were noted in tumour tissue of only 3 out of 50 patients, and they involved exclusively deficiency of MLH1 protein
expression as measured by antibody staining. Interestingly, only in one case (patient no 4) microsatellite instability coincided
with the dysfunction of MLH1 protein. In one case with MSI, MMR deficiency has not been measured yet. In the other four cases
there was no correlation between microsatellite instability and expression of MMR proteins, suggesting that other factors than
MMR deficiency can also contribute to genomic instability in colon cancer.
DNA glycosylases were measured only in 2 out of 6
patients, who revealed either MSI or MMR disturbances. For one of them (patients No 4), neither eA- nor eC-glycosylase activity
was detectable in normal and tumour colon epithelium. This patient had also defective MMR, thus high level of DNA damage could
be expected. For the second one, both eA- and eC-glycosylases activities had significantly higher values in normal colon and in
tumour than in other patients, and moreover the eA-glycosylase activity increased tremendously in the tumour to the value 41.8
eA pmoles/h/mg protein, which was at least 3-fold higher value than in any other tissue measured in this study. Further
investigations are now in progress to evaluate the role of etheno-DNA adducts and DNA-glycosylases activities for the genomic
stability in colon cancer.
Ackowledgments: This work was supported in part by EU grant No
QLK4-CT2000-00286.
FASTER DNA REPAIR IN LYMPHOCYTES IRRADIATED IN VITRO CORRELATES WITH A LATER MAXIMAL ACUTE REACTION
DURING RADIOTHERAPY
Palyvoda O.1,2, Wygoda A.1, Drobot L.2 , Rzeszowska-Wolny J.1
1Department of Experimental
and Clinical Radiobiology, Oncology Center, M.Skłodowska-Curie Memorial Institute, 44-100 Gliwice, Poland;
2Institute of
Cell Biology, Ukrainian National Academy of Sciences, 49005 Lviv, Ukraine
It is commonly known that individuals show
marked differences in radiation sensitivity. To study the relation between the sensitivity of healthy tissues and the side
effects occuring during radiotherapy, we conducted a population study of DNA damage and repair in lymphocytes from 82 healthy
donors and patients with head and neck cancers before radiotherapy. DNA damage and the kinetics of repair were determined by
comet assays in isolated lymphocytes irradiated in vitro with 2 Gy. The DNA repair curves for every donor were computer fitted
to an exponential equation and the para-meters reflecting induced DNA damage (a), rate of DNA repair (t) and residual DNA damage
(c) were calculated. The patient group showed a significantly higher level of background DNA damage in lymphocytes before
irradiation than the healthy donors, while the parameters of repair were greatly scattered in both groups. In the patient group
we compared the response of lymphocytes irradiated in vitro with the patient's acute reaction to radiotherapy estimated with the
method described by Dische. We did not find a correlation of the level of the maximal acute reaction appearing during
radiotherapy with any of the parameters describing the kinetics of DNA repair in lymphocytes. However, a significant difference
(p=0,029) was observed in the time (or cumulative dose) after which the maximal acute reaction was seen between patients
characterized by high and low value of the parameter t describing the rate of DNA repair.
Supported by KBN Grant
4P05A01519
BRCA1 AND BRCA2 GERMLINE MUTATIONS IN SPORADIC BREAST AND OVARIAN CANCER CASES FROM UPPER
SILESIA
Pamuła Jolanta1, Zientek Helena1, Siemińska Marzena1, Rogozińska-Szczepka2,
Chmielik Ewa3, Michalska Jadwiga1,
Kalinowska Ewa1, Utracka-Hutka Beata2,
Kaźmierczak-Maciejewska Maria5, Budryk Magdalena1, Mańka Grzegorz4, Olejek Anita4,
Grzybowska Ewa1
1Department of Tumor Biology, 2Chemotherapy Clinics, 3Department of Pathology, 5Outpatient Clinic, Centre
of Oncology, Maria Skłodowska-Curie Memorial Institute, Wybrzeże Armii Krajowej 15, 44 - 101 Gliwice, Poland
4Department of
Obstetrics and Gynecology, Silesian Medical Academy, ul. Batorego 15, 41 - 902 Bytom, Poland
Mutations in BRCA1 and
BRCA2 genes are responsible for familial breast and ovarian cancer syndrome and they were found in the families with strong
history of cancer cases. The aim of our study was to estimate the frequency of germ-line mutation in sporadic cases of breast
and ovarian cancer. Previous studies demonstrated that screening for local founder mutations might identify the majority of
BRCA1 and BRCA2 carriers in Polish population. We used allele specific oligonucleotide PCR-based assays (ASA-PCR) for
pre-screening of 49 sporadic breast and ovarian cancer cases. Four different disease predisposing mutations in BRCA1 gene
(185delAG, 300T/G, 4153delA, 5382insC) and two mutations in BRCA2 gene (6174delT, 9631delC) were chosen for analysis.
In
total, germ-line mutations were found in five (10,2%) of 49 patients without family histories of breast and/or ovarian cancers
and 5 mutation carriers of mutation were identified among their family members. Four probands with detected mutation had ovarian
cancer and one breast cancer. Germline mutations were found only in BRCA1 gene (185delAG, 300T/G, 4153delA, 5382insC). There
were cancer cases in these families (gastric cancer, leukemia, head and neck cancer, prostate cancer, lung cancer, colorectal
cancer and breast cancer) but the families did not meet the criteria for HB(O)C. Three probands inherited mutation via male line
so the observed phenotype were not typical and in one of the families only men inherited the mutation.
This percentage of
mutations in sporadic cases is significant in comparison with the total of 10% among probands with the strong family history of
breast or/and ovarian cancer, 27% in bilateral breast or/and ovarian cancer cases and 18% in early onset breast and ovarian
cancer cases which are observed in Silesian population.
We conclude that a significant fraction of sporadic breast/ovarian
cancer patients carry inherited founder BRCA1 and BRCA2 mutations. These findings indicate that not only patients with the
strong family history, bilaterality, multifocality or early onset breast and/or ovarian cancer can benefit from genetic
susceptibility testing.
POLYMORPHISMS AND EXPRESSION OF HUMAN MGMT GENE
Pawlas Małgorzata, Butkiewicz Dorota,
Samojedny Arkadiusz, Chorąży Mieczysław,
Rusin Marek
Department of Tumor Biology, Center of Oncology - M.
Skłodowska-Curie Memorial Institute, Gliwice, Poland
Genetic polymorphism of DNA repair genes may be associated with
modulation of cancer risk. Our investigation focused on MGMT gene encoding an alkyltransferase involved in direct DNA repair.
The aim of our study was to determine the frequency of four polymorphic alleles of MGMT gene: 84Leu>Phe, associated
substitutions 143Ile>Val-178Lys>Arg, enhancer polymorphism 1099C>T (Acc. X61657) and 79G>T (Acc.U95038) in 96 non-small cell
lung cancer (NSCLC) cases and 96 cancer-free individuals from Upper Silesia. We also performed genotyping of three frequent MGMT
alleles (84Phe, 143Val-178Arg, 1099C>T) in 164 NSCLC cases to examine the association of MGMT alleles with basic clinical and
epidemiological characteristics of the patients. We studied "differential expression" of MGMT alleles and its association with
polymorphisms in regulatory region of MGMT. Material for the expression study was RNA and DNA from white blood cells of 45
healthy inhabitants of Upper Silesia. We found that no MGMT allele showed statistically significant frequency difference between
cancer cases and controls. The enhancer polymorphism was less frequent in never smokers (4%) than in smokers (18%), in females
(5%) than in males (17%) and in never smoking cancer patients (6%) compared with never smoking controls (24%). We detected
strong association between 84Phe allele and the clinical stage of cancer. MGMT alleles in heterozygotes revealed "differential
expression" in white blood cells (14% of informative samples) and this phenomenon was associated with the enhancer polymorphism
of MGMT. To further investigate the functional significance of the enhancer polymorphism, we cloned 275 bp wild-type and
polymorphic promoter-enhancer element into pGL-3 vector in a way that the element controlled the expression of firefly
luciferase gene. After transfection into three different cell lines, we found that the polymorphic variant was invariably
associated with significantly increased activity of the luciferase enzyme. These observations are consistent with the hypothesis
that the enhancer polymorphism is of functional significance since it is associated with increased expression of the MGMT gene
and may decrease lung cancer risk at low carcinogen exposure.
CYTOTOXIC EFFECT OF DOXORUBICIN CONJUGATES WITH
POLY-3-HYDROXYBUTYRATE. PRELIMINARY STUDY
Piddubnyak Valeria, Jedliński Zbigniew*, Matuszowicz Andrzej*, Głowala
Magdalena, Kurcok Piotr*, Juzwa Maria*, Śnietura Mirosław#, Lange Dariusz#, Krawczyk Zdzisław
Department of Tumor
Biology, Centre of Oncology - Maria Skłodowska-Curie Memorial Institute, Gliwice;
*Centre of Polymer Chemistry, Polish
Academy of Science, Zabrze;
#Department of Tumor Pathology, Centre of Oncology - Maria Skłodowska-Curie Memorial Institute,
Gliwice
Chemotherapy is one of the most important anticancer treatment modes. Several approaches have been undertaken to
elaborate a strategy of a cancer-targeting drug delivery system that could increase the concentration of pharmaceutical drugs in
tumor tissue, to reduce drug exposure and toxicity to other tissues, and to overcome drug resistance. There is an increasing
interest in designing vectorized drugs in which a given chemotherapeutic is linked by a covalent hydrolysable bond to either a
peptide or a polymeric substance. One of such polymeric substances, a potential vector for drug delivery, is
poly-(3-hydroxybutyrate) (PHB in short). PHBs are a ubiquitous constituent of cells and have been found in bacterial cell
membranes, in a variety of plant tissues, as well as in plasma membranes, mitochondria, and microsomes of animal cells. Novel
methods developed at the Centre of Polymer Chemistry, Zabrze enable synthesis of tailor-made oligo- and poly-(3-hydroxybutyrate)
with well-defined structure and properties. Recently, the conjugates of doxorubicin with poly-(3-hydroxybutyrate) were also
synthesized.
The aim of the present study was to compare the cytotoxicity of free and conjugated doxorubicin as well as to
compare the kinetics of uptake and subcellular localization of these two forms of this cytostatic drug. We first determined
whether short-chain PHBs could exert a cytotoxic effect on cells grown in culture. Cytotoxicity MTT assays were performed on
hamster V79 cells, murine melanoma Bl6 cells and human breast cancer cell line MCF7. We found that the majority of several PHB
oligomers tested did not significantly affect cell growth; this was also confirmed in clonogenic assay. We also found that the
PHBs tested do not induce cytoprotective mechanisms manifested by induction of hsp70i genes. In order to compare a cytotoxic
effect of free and PHB-bound doxorubicin we performed MTT assays using cell lines mentioned above, and found that both forms of
the cytostatic drug kill cells at a similar rate.
To study the intracellular distribution of the free and PHB-conjugated
doxorubicin we used confocal laser scanning microscopy (CLSM). Owing to doxorubicin autofluorescence the CLSM was found to be a
highly useful method for detecting intracellular localization of this compound. The study was performed on V79, B16 and MCF7
cells, as well as on the doxorubicin-resistant variant of MCF7 cells. We found that, in contrast to doxorubicin, which is
confined to the nucleus, PHB-doxorubicin conjugates are localized predominantly in cytoplasm. A very distinct feature of the
PHB-bound doxorubicin is its significantly faster cellular uptake, compared to the free drug. Of special importance is the
observation that while free doxorubicin is effectively removed from doxorubicin-resistant MCF7 cell variant, PHB-bound drug is
not.
Our data show that the PHB-conjugated doxorubicin is a very effective drug-carrier molecule. It can be considered a
novel form of this cytostatic drug, potentially able to overcome drug resistance phenomenon.
CHARACTERISTICS OF
REPETITIVE DNA SEQUENCES
IN HUMAN GENOME
Piwowar Monika, Grzybowski Piotr, Meus Jan, Roterman Irena
Collegium
Medicum - Jagiellonian University Kraków, Poland
Metaanalysis of simple repeated repetitive sequences in human
genome requires general characteristic. Simple repeats were selected from GenBank. These repeats comprised tandemly repeated
units 1-6 nucleotides (nt) long and have been extracted from genomic DNA sequences. Of the 501 theoretically possible, different
types of repeat only 67 were present in the analysed database in at least two different size ranges over 12 nt. (they present ~
1% of the total DNA taken to the analysis) [1].
The chi-square test is usually used to analyse the relation between
qualitative variables (type of amino acid sequence, its localisation etc). The new Z- coefficient method for quantitative
analysis of contingency tables is introduced. This method allows to put all combinations from all forms of analysed two
qualitative variables in the ranking order. The information about such order allows to reveal the combinations, which are more
and those which are less responsible for the presence of significant dependence between two analysed variables. It was found
that in the dependence between length of repeat units and degree of dispersion the tri-nuleotides fragments represent the
highest localisation stability. 1-, 2-, 4-, 5- and 6-nucleotides fragments exhibit the higher ability to be dispersed in genome.
The expandability of repetitive sequences in human genome is assumed to cause the neurodegenrative diseases so it can be
important in clinical image.
THE URINARY EXCRETION OF 8-OXOGUANINE AND 8-OXO-2'-DEOXYGUANOSINE IN NON SMALL CELL LUNG
CANCER PATIENTS
Rozalski Rafal, Gackowski Daniel, Roszkowski Krzysztof, Foksinski Marek, Siomek Agnieszka, Kowalewski
Janusz, Jurgowiak Marek, Olinski Ryszard
The Ludwik Rydygier Medical University in Bydgoszcz, Karlowicza 24, 85-092
Bydgoszcz
Using HPLC prepurification/isotope dilution GC/MS technique, we examined whether the amount of 8-oxoguanine and
8-oxo-2'-deoxyguanosine excreted into urine is higher in cancer patients when compared with the control group. The control group
consisted of 38 healthy subjects and the patient group comprised 57 non small cell lung cancer patients.
The mean level of
8-oxoguanine in urine samples of 57 cancer patients was 191 ( 102 nmol/24h. It was significantly higher (p=0.0086) than in the
urine of the control group, where the level reached the value of 138 ( 82 nmol/24h. The mean levels of 8-oxo-2'-deoxyguanosine
in the control group and in cancer patients were very similar and reached the mean values of 35 ( 21 nmol/24h and 32 ( 18
nmol/24h respectively. This difference was not statistically significant.
We have found that the amount of the modified base
(but not the nucleoside) excreted into urine is about 40% higher in cancer patients than in the control group. Since the
presence of the modified base in urine may represent the primary repair product of the oxidative DNA damage in vivo our results
suggest an important role of DNA glycosylases (most likely OGG1) in removal of the damage induced as a result of cancer
development.
HEAT SHOCK COGNATE 70 (HSC70) GENE SEQUENCE ALTERATIONS
IN NON-SMALL-CELL LUNG CANCER PATIENTS -
ASSOCIATION WITH P53 GENE MUTATIONS AND IMMUNOHISTOCHEMICAL STAINING
OF HSC70 AND P53 PROTEINS
Rusin Marek*, Zientek
Helena*, Krześniak Małgorzata, Małusecka Ewa, Zborek Anna, Krzyżowska-Gruca Stefania, Butkiewicz Dorota, Lisowska Katarzyna,
Grzybowska Ewa, Krawczyk Zdzisław
Department of Tumor Biology, Centre of Oncology - Maria Skłodowska -Curie Memorial
Institute, 44-101 Gliwice, Poland
The loss of heterozygosity at the locus 11q23.3 is a frequent event in various human
cancers. Recently, somatic mutations of 11q23.3-linked heat shock cognate HSC70 gene, in coincidence with allelic imbalance,
were found by others in sporadic breast carcinomas. To find out whether intragenic somatic mutations of HSC70 occur also in lung
carcinomas, we sequenced exons 2 - 8 of the gene as well as adjacent intronic sequences in a series of DNA samples isolated from
non-small-cell lung cancer (NSCLC) tissues. No somatic mutations were detected, however we identified 22 polymorphic sequence
changes, 19 of which were not reported previously. We found a significant association between the most frequent HSC70
polymorphism (1541-1542delGT) and the immunohistochemical staining pattern of the HSC70 protein and p53 tumor suppressor
protein, determined by us previously. Generally, carriers of that polymorphism showed weaker staining of HSC70 and p53 in tumor
tissues, as compared to wild-type homozygotes. We also found that 1541-1542delGT polymorphism is associated with frequency of
the deletion/insertion mutations of p53 gene. For the latter comparison we re-examined the mutation frequency of the p53 gene in
our series of lung cancer patients. Here, we report 17 newly detected p53 mutations. The overall p53 mutation frequency
(including the alterations that were previously found by us) reached 65%, a value that is among the highest found in NSCLC so
far. Our data indicate that the 1541-1542delGT polymorphism of HSC70 gene may be functional and associated with modulation of
cellular levels of HSC70 and mutant forms of p53 protein.
*These authors contributed equally to this work.
PREMATURE
MITOSIS - CHROMOSOMES REPLICATE THE DNA
Rybaczek Dorota, Maszewski Janusz
Katedra Cytofizjologii, Uniwersytet
Łódzki, ul. Pilarskiego 14, 90-231 Łódź
The prolonged arrest of DNA replication invokes mechanisms that relay a signal
(or signals) to effector molecules which implement a number of checkpoint-dependent responses, including a decrease in the
activity of CDK-cyclin B complexes. In consequence, the commitment of cells to enter mitosis become blocked. Experiments on root
meristem cells of Vicia faba indicate that caffeine allows a number of cells to override the intra-S-phase checkpoint control
and to induce mitotic condensation of incompletely replicated chromosomes (PCC). Using Feulgen cytophotometry and 3H-thymidine
autoradiography, we have evidenced that their appearance varies significantly depending on the stage at which
hydroxyurea-blocked S-phase cells were stimulated to enter mitosis. Furthermore, prematurely condensed chromosomes maintain the
ability to continue DNA replication. Since 3H-thymidine incorporation is restricted merely to cells at anaphase and telophase,
it seems reasonable to suppose that the commitment of chromatin to the resumption of DNA synthesis depends on the transition
throughout the APC/C-mediated checkpoint, which gates the cells into the exit from mitosis.
THE COMBINED TREATMENT
OF TRANSPLANTABLE SOLID MAMMARY CARCINOMA IN WISTAR RATS BY USE OF PHOTODYNAMIC THERAPY
AND CYSTEINE PROTEINASE
INHIBITOR
Siewiński Maciej1, Saleh Yousif, Gryboś Marian
1Department of Obstetrics and Gynecology, Medical
University of Wrocław, Chałubińskiego 3, PL-50-368 Wrocław, tel. 71-7842413
Numerous studies showed that lysosomal
proteinases play important role in carcinogenesis. Enzymatic activity of tumor-associated proteases is counter-balanced by
specific inhibitors. PDT is technique which involves photoexcitation of sensitizing drug retained in neoplastic tissue that is
subsequently destroyed. Intraperitoneal injections of hematoporphyrin derivative (HpD) were made in dose 20 mg/kg in rats
transplanted with mammary carcinoma. Halogen lamp was used 24 hrs later at 630 /-20 nm and total dose - 200 J/sq. cm. Cysteine
proteinase inhibitor (CPI) was dissolved in saline and injected subcutaneously in doses 50 mg and 200 mg per animal.
Effectiveness of treatment was evaluated with regard to survival time and tumor response and to depth of necrosis. In several
cases tumors completely disappeared following HpD-PDT CPI. Number of complete tumor responses was higher when PDT 200 mg of CPI
was used, i.e. 6/10 rats. Promising results have also been obtained with regard to survival time of treated animals and to
induction of tumor necrosis.
DNA ADDUCTS AND REPAIR CAPACITY IN LUNG CANCER
Speina Elżbieta1, Zielińska Maja1
Barbin Alain2, Gackowski Daniel3, Kowalewski Janusz4, Oliński Ryszard3, Tudek Barbara1
1Institute of Biochemistry and
Biophysics PAS, Warsaw, Poland;
2International Agency for Research on Cancer, Lyon, France;
3Department of Clinical
Biochemistry, 4Department and Clinic of Thoracic Surgery and Tumours, Ludwik Rydygier Medical University in Bydgoszcz,
Poland
Oxidative stress and lipid peroxidation (LPO) generate promutagenic DNA lesions such as 1,N6-ethenoadenine (eA)
and 3,N4-ethenocytosine (eC) that are likely to contribute to lung cancer development and progression.
We measured the levels
of eA and eC by immunoaffinity/32P postlabeling in the DNA of tumour and normal lung tissue of lung cancer patients (33 cases)
as well as in leukocytes of the same group. Activities of DNA-glycosylases repairing eA and eC were also measured in the same
tissues (57 cases) and in leukocytes of healthy volunteers (26 individuals).
High individual differences (up to 10-fold) were
observed both in adduct level and glycosylase activities. No difference in eA and eC level between tumour and non-affected lung
tissue was recorded. However, leukocytes accumulated statistically significant higher number of DNA adducts than lung tissues.
Repair activities for both eA and eC were significantly higher in tumour than in normal lung tissue. There was inverse
correlation between the level of eC and the activity of eC-glycosylase in normal and tumour lung tissue, however for eA such
correlation was found only in tumours. This suggests that eC-DNA-glycosylase, is the "first choice" enzyme for removing this
lesion from DNA in humans.
There was no difference in eA and eC-glycosylases activities between men and women as well as
smokers and ex-smokers.
eC-glycosylase activity was decreasing gradually with age in normal lung (3.22-fold difference
between groups of 40-50-years old and over 70-years old patients).
We observed differences between two histological types of
lung cancer: squamous cell carcinoma (SQ) and adenocarcinoma (AD). In patients with SQ the ratio of eA/eC was higher in
non-affected lung tissue than in AD patients. In AD individuals eA and eC DNA-glycosylase activities were significantly lower
than in SQ, however normal lung of AD patients revealed higher deficiency in eA-glycosylase (2.72-fold decrease) than in
eC-glycosylase (1.8-fold) in comparison with SQ type of tumour. This might suggest that people developing inflammation-related
adenocarcinoma, might have defective "first choice" defense mechanisms against LPO-induced DNA damage.
Plasma of healthy
volunteers contained higher level of vitamin E and A in comparison with that of cancer patients.
THE 'cis' REGULATORY
SEQUENCES ESSENTIAL FOR TRANSCRIPTION
OF THE hst70 GENE ARE LOCALIZED WITHIN THE 5'UTR REGION
Ścieglińska Dorota,
Widłak Wiesława, Krawczyk Zdzisław
Department of Tumor Biology, Centre of Oncology, M.Skłodowska-Curie Memorial
Institute, Gliwice, Poland
The rat hst70 gene belongs to the hsp70 family of heat shock genes and codes for a molecular
chaperone protein necessary for synaptonemal complex disassembly, progression of spermatogenesis beyond meiotic phase and
probably development of spermatids.
Previously, we identified a short intron in 5'UTR of the hst70 gene and demonstrated that
the transcription of hst70 gene could be initiated from two main start sites, localized 353 bp (T1 start site) and 116 bp (T2
start site) upstream of the ATG codon. T2 start site is localized within the intron sequences, T1 precedes the untranslated
exon1. Functional studies of the hst70 gene promoter activity revealed, that a 367 bp region upstream of ATG is sufficient to
direct expression of CAT reporter gene specifically to testes of transgenic mice.
In the present study we demonstrate the
expression pattern of series of pHST-CAT constructs in which the hst70 gene promoter was truncated either from 5' or 3'
directions. We showed, using transfection in vitro, that sequences localized in exon1 and a 5' part of intron are essential for
expression of the hst70 gene. Searching for "cis" regulatory elements localized within this hst70 promoter fragment we found two
short stretches of almost perfect homology (described as boxA and boxB) in homologues genes of rat, mice and human. We
determined that sequences containing only boxes A and B are capable to direct expression of the CAT reporter gene specifically
to testis of transgenic mice. Next, we analyzed the binding of nuclear proteins from testes of immature (hst70 gene inactive)
and sexually mature rats (hst70 gene highly active) to boxA and boxB sequences using the "band shift assay". We obtained a
specific complex only between nuclear proteins from testes of immature rats and the octamer sequence within boxA. We speculate
that this protein/proteins could repress the activity of the hst70 gene in immature rats.
EFFECT OF DIFFERENT
TRANSFECTION AGENTS
ON THE ACTIVITY OF THE RAT hsp70i GENE PROMOTER
Vydra Natallia, Wysocka Aleksandra,
Fiszer-Kierzkowska Anna, Lisowska Katarzyna
Department of Tumor Biology, Centre of Oncology - Maria
Skłodowska-Curie
Memorial Institute, Gliwice, Poland
In normal mammalian cells, under physiological conditions the
hsp70i (i-inducible) genes are either inactive or expressed at a very low level. Stress response triggers heat shock factor
(HSF) activation, its interaction with regulatory sequence HSE and finally the hsp70i genes expression. The hsp70i genes are
also constitutively expressed in some primary tumors and tumor cell lines (Morimoto, 1999). However, the mechanism of that
phenomenon is poorly understood. It seems that except the well-recognized HSE-HSF interaction other cis-trans interactions may
be involved in the regulation of inducible hsp70 genes in some circumstances.
To search for possible other cis-trans
interactions that could regulate hsp70 gene expression we have constructed series of vectors in which GFP or CAT reporter genes
were fused with different fragments, (up to -2.0 kbp, relative to the transcription start site) of the promoter region of the
rat hsp70.1 gene. Surprisingly, when we used Lipofectin Reagent (Gibco BRL) for transient transfection of the cells with these
constructs, a strong activation of the reporter gene was observed regardless of incubation temperature. High hsp70.1 promoter
activity was observed between 16 and 48 hours after lipofection of B16 and FTO 2B cells. Heat shock did not significantly
increase CAT activity in lipofected cells. This was in contrast to cells transfected by DEAE-dextran method that did not exhibit
CAT activity at 37°C while they did at 42°. This suggested that Lipofectin itself induced hsp70 gene promoter
activity. The electrophoretic mobility shift assay (EMSA) with the HSE oligonucleotide and the extracts from Lipofectin treated
cells showed that Lipofectin activation is independent of the HSF-HSE interaction. Northern blotting experiment revealed that
only transiently transfected hsp70 promoter sequences were susceptible to the Lipofectin treatment while the endogenous hsp70
was not induced. Preliminary search for the sequences responsible for Lipofectin induced expression showed that most important
regulatory sequences are localized within 350 bp region encompassing 85 bp of the 5'UTR and 260 bp of the promoter. We also
analyzed the effect of other liposomal transfection agents on the hsp70.1 promoter activity and we found that Lipofectamine PLUS
(Gibco BRL) had similar properties to Lipofectin while two home-made liposomal formulations (Arg-Chol/DOPE and DDAB/DOPE) did
not induce reporter gene transcription by themselves. Our results indicate that caution must be taken when performing promoter
studies of hsp genes, as the type of compounds used for transfection may seriously affect the results.
GFP FLUORESCENCE
IN SOMATIC CELLS OF TRANSGENIC MICE CONTAINING EGFP REPORTER GENE UNDER THE CONTROL
OF THE RAT TESTIS-SPECIFIC hst70 GENE
PROMOTER
Widłak Wiesława, Konopka Witold, Zborek Anna, Ścieglińska Dorota, Krawczyk Zdzisław
Department of
Tumor Biology, Centre of Oncology, M. Skłodowska-Curie Memorial Institute Gliwice, Poland;
Department of Molecular and
Cellular Neurobiology, Nencki Institute, Warsaw, Poland.
Rat hst70 gene (in mouse called hsp70.2) is a unique member of
the multigene hsp70 family. The gene is activated during meiosis in spermatogenic cells.and codes for molecular chaperone
protein involved in desynapsis of synaptonemal complexes as well as chaperoning CDC2 kinase required for completion of the
meiotic division. Homozygotic removal of the hsp70.2 gene leads to male infertility (due to spermatocytes apoptosis), whereas
females remain fertile. We have previously shown using RT-PCR and transgenic mice with CAT reporter gene, that an expression
level of hst70 in tissues other than testis is 10-100 times lower. The highest hst70 expression can be detected in the brain
while in the liver the gene is fully repressed.
To study the expression of the hst70 gene at a single cell level we have
constructed transgenic mice bearing enhanced green fluorescent protein gene (EGFP) under the control of about 800bp of hst70
promoter. We have obtained 17 founders of transgenic mice, and established 6 transgenic lines. All transgenic males exhibit EGFP
fluorescence in testes, epididymides and vasa deferentia. EGFP is expressed in testes in cell- and stage-specific manner
resembling the pattern observed for the endogenous hst70 gene. The hst70 gene is strongly activated in pachytene spermatocytes
that appear during maturation of testes in two weeks-old mice. EGFP fluorescence is present in cytoplasm and nucleus of
spermatocytes, spermatids, residual bodies and spermatozoa. EGFP fluorescence is detected (in some transgenic lines) in female
reproductive organs, especially in uterus, and in one-cell and two-cell stage embryos. In all transgenic mice EGFP fluorescence
is present in the brain. Depending on the line, transgene may be active in other somatic organs: pancreas, pituitary, salivary
gland, kidney, adrenal glands, bladder, heart, lung, and spleen. Differences in pattern of the transgene expression observed
between the founders may result from site of its chromosomal integration.
Transcription of hst70 in restricted areas of
somatic tissues may suggest novel function for its protein. However, it is unclear whether hst70 transcripts are efficiently
translated in somatic cells. In order to elucidate this problem we are going to construct transgenic mice expressing HST70-EGFP
fusion protein.
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