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Gliwice Scientific Meetings 2009 ~~~ Lecture abstracts ~~~ Session I: Molecular Epidemiology of Cancer Genetic basis of familial aggregation of cancer Kari Hemminki and Asta Försti German Cancer Research Center (DKFZ), Im Neuenheimer
Feld 580, 69120 Heidelberg, Germany The recently published large genotyping studies,
mainly based on genome-wide scans, have identified a new repertoire of cancer
susceptibility genes and loci which are characterized by a high frequency of
the risk allele and a low relative risk, in line with the common disease-common
variant paradigm (1). A reason for these discrepancies is that the platforms
used for genome-wide studies have been built for relatively common variants
(minor allele frequency >0.1) constraining the results to variants with high
population attributable fraction (PAF) and low familial relative risk (FRR).
PAFs have been used extensively for environmental risk factors of cancer in
order to rank them and to assess the prospective gains in disease prevention.
Their use in cancer genetics is relatively new, probably because the mutant
variants of the ‘classical’ high penetrant cancer genes are so rare that their
contribution to the population burden is low compared to the high individual
risks. Some recent studies on low penetrant genes do cite PAFs and point to the
high conferred population burden in spite of the low relative risks.
Differences between high penetrant (relative risk some 5 or more) and low
penetrant (relative risks below 1.5 or 2.0) genes have recently been
illustrated by ‘molecular landscaping’. The PAF of a gene variant integrates
any unmeasured gene-gene and gene-environment interactions for the particular
study population. With the current volume of genetic data on susceptibility
genes, PAFs are useful in putting the findings into an etiologic perspective.
The calculation of joint PAFs for several genes gives a progress report into
the limits of understanding of the genetic basis of a disease (2). For breast cancer, the nine established loci gave a joint
PAF of > 60%, but explaining only some 8% of the empirical FRR. Recent
publications on colorectal cancer include the chromosome 8 locus, represented
by SNP rs6983267 and shared by prostate cancer, accounted for 0.4% of the
empirical FRR of 2.7. Another locus close to SMAD7 conferred a
marginally lower risk and it accounted for 0.3 % of the empirical excess FRR.
The joint PAF for these two loci was 27.8%; their FRR would be 1.01, accounting
for 0.7% of the empirical excess FRR of colorectal cancer. The available
data suggest that the majority of the familial aggregation of these cancers
cannot be explained by the known genes/loci. References: 1.
Hemminki K, Försti A, Lorenzo Bermejo J. Etiologic impact of known cancer
susceptibility genes. Mut Res Rev 2008;658:42-54. 2.
Hemminki K, Försti A, Lorenzo Bermejo J. New cancer susceptibility loci:
population and familial risks. Int J Cancer 2008; 123:1726-9. Search
for Modifiers of Hereditary Breast Cancer Risk Ute Hamann1, Stefan Wilkening1,
Michael Gilbert1, Chen Bowang1, Kari Hemminki1,
Anna Jakubowska2, Jan Lubinski2 1German Cancer Research Center (DKFZ), Heidelberg, Germany; 2International Hereditary Cancer
Center, Department of Genetics and Pathology, Pomeranian Medical University,
Szczecin, Poland. Germline mutations
in BRCA1 confer high risks of breast and ovarian cancer. The risk varies
by the age at diagnosis and the type of cancer in the index patient suggesting
that breast cancer risk in mutation carriers is modified by other genetic or
environmental factors that cluster in families. In this study we search for
genetic modifiers of hereditary breast cancer risk in Polish women carrying the
5382insC BRCA1 mutation using a genome-wide association study (GWAS) on
DNA pools. DNA samples were collected from 124 young breast cancer cases (age
of diagnosis < 45 years) and 119 older disease-free controls (age at
interview > 50 years) all harbouring the 5382insC BRCA1
founder mutation. Equimolar amounts of each DNA sample were added to either the
case or control pool. Pools were genotyped using Illumina HumaCNV370-Duo
arrays. Single nucleotide polymorphisms (SNPs) for individual genotyping were
selected using three methods including the cluster method, allele frequency
difference method and combined Z-test. Twenty-seven loci showing the largest
significant differences were selected for individual genotyping on 1,500
carriers. Genotyping analyses are currently performed. Preliminary results will
be presented. Evidence
for genetic basis in breast cancer survival Asta Försti Division of Molecular Genetic
Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany. Several earlier
studies have assessed survival in breast cancer based on familial risk of this
disease. The results have been conflicting and suggest that the risk and
prognostic factors of cancer are largely distinct. As a novel concept, we
searched for familial clustering of survival, i.e. concordance of survival
among family members. We used the nation-wide Swedish Family-Cancer Database to
estimate hazard ratios (HRs) for cause-specific and overall survival in breast
cancer. The study covered 1277 mother-daughter pairs with familial breast
cancer. The results were consistent in showing that both good and poor survival
in breast cancer aggregated in families, suggesting that the prognosis in
breast cancer is in part heritable. Accordingly, we searched for genes that
control heritability of prognosis among genes involved in metastatic cascades,
including angiogenesis-related genes, adhesion and extracellular matrix
degradation related genes, chromosomal instability genes and telomere length
related genes. For this study, we used a large Swedish study population with
detailed clinical data and up to 15 years of follow-up. The results showed that
variants in the angiogenesis-related genes, VEGF, KDR
and POSTN correlate with traditional prognostic factors in breast cancer. Variants of the gene for PAI-1, which is an
important factor for the invasive capacity of tumours, had prognostic bearing
in breast cancer. Integrins have similar
functions, also carrying genetic variants governing prognosis in breast cancer. Genes with implications for chromosomal instability
also had variants associated with prognosis in breast cancer. As a next step, we are carrying out a genome-wide
association study on survival, in which we compare the genotypes of women with
short survival (< 5 years) with women who have survived 11 years or longer,
in collaboration with the Nordic countries and Germany. Molecular
epidemiology of hereditary breast and ovary cancer in Silesia, clinical course
of breast and ovary cancer in respect to polymorphisms of PGR and MDR1 genes Karolina Tęcza1, Jolanta Pamuła-Piłat1, Sylwia Jędruś2, Ewa Telka3, Ewa Grzybowska1 1Molecular Biology
Department, MSC memorial Cancer Center and Institute of Oncology, Gliwice
Branch, Gliwice, Poland; 2III Oncologic Gynaecology Clinic/Teaching Hospital,
MSC memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice,
Poland; 3Radiotherapy
Department, MSC memorial Cancer Center and Institute of Oncology, Gliwice
Branch, Gliwice, Poland. Aim: We wanted to
analyze the influence of modifying genetic factors on the risk of BRCA(+) and
BRCA(-) breast and ovary cancers and on clinical parameters as the age of onset
and survival. Materials and Methods: 348 anonymous
healthy women (control group), 229 persons with ovary cancer, 45 persons with
breast and ovary cancer (case group). The patients under study developed
breast cancer prior to ovary cancer. RFLP and ASA PCR were used to analyze
mutations in BRCA genes and polymorphisms in PGR and MDR1 genes. Results: Alleles AA PGR
+331G/A and TT PGR20 slightly decreased the risk of ovary cancer. The presence
of allele T of 660 PGR significantly decreased the risk of second malignancy
(breast cancer) OR=0,44, p=0,039. Allele T of PGR 20 decreased also the risk of
second malignancy. Heterozygotes for PGR V660L and 20 were at statistically
significant decrease of second malignancy, OR=0,35, p=0,04. Heterozygotes
CT of MDR1 gene C3435T and genotypes AG and AT of G2677T/A were at lower risk
of developing ovary cancer. Genotype CT of C3435T polymorphism had protective
effect against developing the second malignancy while allele TT increased the
risk of breast cancer. Heterozygotes for both polymorphisms were at higher risk
of developing malignancy. Age of onset of ovary cancer
for GT heterozygotes of PGR V660L did not depend on the status of BRCA
mutation. For heterozygotes GA of PGR +331 polymorphism the age of onset for
breast and ovary cancer were lower in the group with negative BRCA status.
Carriers of TT and AT alleles of MDR1 G2677A/T and carriers of TT allele
together with negative status of BRCA had earlier onset of ovary cancer than
carriers of the same alleles, with mutation in BRCA genes. Carriers of T allele
of MDR1 G2677A/T had later onset of ovary cancer regardless the status of BRCA
genes. Alleles GA of PGR +331G/A and
V660L shortened the survival of ovary cancer patients with negative BRCA
status. Alleles GG of PGR +331G/A and GT for V660L polymorphisms increased the
survival of ovary cancer patients with mutation in BRCA genes. Patients with
mutation in BRCA genes, two primary malignancies, carrying allele GA of
PGR+331G/A polymorphism had the longest survival. Conclusions: Polymorphic
variants of PGR and MDR1 genes modified more the clinical course and the risk
of second malignancy than the risk of developing ovary cancer alone. Pregnancy
hormones and maternal cancer Annekatrin Lukanova, Rudolf Kaaks Division of Cancer
Epidemiology, DKFZ, Heidelberg, Germany. Pregnancy is the
strongest natural protection against breast and ovarian cancers. Nevertheless,
particularly for breast cancer, the effect appears to be complex. Before the
long-term protective effect is established, there is a transient increase in
risk, which is especially pronounced in women who delay childbearing until
after age 30-35 or in women with a strong family history of the disease. The
mechanisms underlying the association of pregnancy with cancer risk are poorly
understood, but animal experiments strongly suggest that the hormonal changes
during gestation are involved. Using the resources of large
Maternity Cohorts from Finland and Northern Sweden the association of first
trimester pregnancy human chorionic gonadotropin, estrogens, progesterone,
testosterone, SHBG and IGF-I with maternal breast cancer were explored. Initial
preliminary results strongly suggest that hormone concentrations during the
first trimester of a first full-term pregnancy play an important role for both
the beneficial and adverse effect of pregnancy on maternal risk of breast
cancer. In the past century dramatic
changes in the reproductive pattern has taken place world-wide, with a delay of
the onset of childbearing and reduction of the average number of children per
woman. While the societal and economic forces that are at the roots of such
profound, rapid changes are not likely to be modified, a better understanding
of the factors mediating the protective association of childbearing could
ultimately lead us to the design and launching of preventive interventions
seeking to mimic nature’s plans. Genetic
influences on survival in lung cancer Dorota Butkiewicz Maria Sklodowska-Curie
Memorial Cancer Center and Institute of Oncology, Gliwice, Poland. Lung cancer
remains the leading cause of cancer-related mortality among men and women
worldwide. Despite recent advances in therapy, the overall 5-year survival rate
for the patients is still low. Molecular genetic studies have shown that
polymorphic variations (SNPs) in genes participating in many pathways may
modulate susceptibility to lung cancer. Also, they have been suggested to
affect response to therapy, its toxicity and prognosis. While it is unlikely
that a single SNP can adequately assign cancer treatment options, predict
survival or allow early detection, a better understanding of the mechanisms and
pathways involved in lung carcinogenesis may lead to improved clinical outcomes
in the future. In the present report several biomarkers, mainly associated with
DNA repair, and their role in lung cancer prognosis will be evaluated. Genome wide studies in cancer Federico Canzian Genomic Epidemiology Group,
German Cancer Research Center, Heidelberg, Germany. In the last few
years, genome-wide association studies (GWAS) have proven to be a powerful tool
to explore the role of common polymorphisms on risk of common diseases,
including cancer. Existing GWAS results are
still far from showing a complete picture of genetic susceptibility to cancer.
New GWAS on specific types of common cancers, as well as on rare cancers, are
needed. The NCI-sponsored Cohort
Consortium provides excellent opportunities to perform these studies. In
particular, the Breast and Prostate Cancer Cohort Consortium (BPC3), which
includes large prospective cohorts from Europe and the USA, is performing two
new GWAS on estrogen receptor negative (ER-) breast cancer and aggressive
prostate cancer, respectively. Estrogen receptor negative
(ER-) breast cancers have specific biological and epidemiologic characteristics
and greater lethality. Aggressive forms of prostate cancer differ
epidemiologically from the more common indolent forms of prostate cancer and
are of greater clinical importance. The current generation of GWAS is
underpowered to discover gene variants associated with these tumors. Over 2,000
cases of ER- breast cancer and of aggressive prostate cancer, and matched
controls, are being currently scanned within BPC3. In parallel, by pooling all
available cases from the cohorts, we can study over 10,000 cases of both breast
and prostate cancer. This enables us to further study polymorphisms shown by
previous GWAS to be associated with risk of these cancers. We have so far
genotyped 16 SNPs for breast cancer and 29 for prostate cancer. We use these
data to 1) replicate previously reported associations, 2) examine interactions
between SNPs and established non-genetic risk factors and 3) set up
multifactorial scores to predict cancer risk. Finally, a GWAS on pancreatic
cancer is ongoing within the Cohort Consortium. By using nearly 4,000 cases
from cohorts and case-control studies, we have identified for the first time 4
genomic regions harboring polymorphisms that influence the risk of pancreatic
cancer. We are performing studies on pancreatic tumor samples to elucidate the
functional relevance of these associations. Identification of laryngeal cancer-related genes using
high resolution array-CGH technique K. Szyfter1,2, M. Giefing1, M. Jarmuż1,3, M. Kostrzewska-Poczekaj1, K. Pelińska1, D. Brauze1, E. Baczyńska1, R. Grenman4., R. Siebert5 1Inst. of Human
Genetics, Polish Academy of Sciences, Poznań , Poland; 2Dept. of
Otolaryngology Medical Univ., Poznań, Poland; 3Dept. of
Hematology, Medical Univ., Poznań, Poland; 4Clinic of
Otolaryngology, Univ. of Turku, Finland; 5Inst. of Medical
Genetics, Kiel Univ., Germany. Tobacco smoking and
alcohol abusing are the dominant causable factors in laryngeal cancer. However,
as only a fraction of exposed persons develops laryngeal cancer, a significance
of genetic factor is to be studied. Further, a progression of cancer also seems
to be associated with expression of specific oncogenes and tumor suppressor
genes (TSG). Recently, molecular cytogenetics and biology provide promising
techniques to identify an involvement of particular genes not considered yet in
a given type of cancer. It was assumed that highly
amplified region contain oncogenes, whereas chromosome deletions indicate for
tumor suppressor genes. The study was done on 24 cell lines derived from
laryngeal cancer with variable characteristics concerning TNM, grading,
survival time, treatment etc. To get orientation in genome changes related to
cancer the cells were analysed by classical cytogenetics, FISH technique and
array-comparative genomic hybridization. Gene copy number and gene expression
were examined for the selected candidates. An involvement of three known
genes in laryngeal cancer was reinvestigated to attribute them to particular
stages of cancer progression. Tumor suppressor gene Rb [chromosome
localisation 13q14] seems to promote metastasis to the adjacent lymph nodes. TP16
[9p21] deletion was observed only in early tumors. Oncogene CCND1
[11q13] amplification was observed in late stages and is connected with a short
survival. Two other potential oncogenes (FGF3 and FGF4) amplified
in the same regions are not expressed. However, 5 other genes harboured in the
same region including cortactin are highly amplified and expressed. Next, for GNG7
[19p13] a TSG activity not reported yet was proven and shown to be correlated
with tumor undifferentiation. Homozygotic deletion in 3p17 region indicates for
TSG activity of SLC6A6 and GRIP2 genes. Oncogenic function of CRKl,
MAPK-1 [22q11-12], PGCP and SDC2 [ 8q22.1] is still under
study. Altogether, 5 TSG and 17
oncogenes have been studied until now. Some of them were linked to progression
of laryngeal cancer. At least two novel genes could serve as progression
markers.Genes
involved in cancer progression could be further studied in respect to targeted
therapy. Session II: Functional Genomics in Cancer Research MODELING AND EXPERIMENTAL
TESTING OF CELL CYCLE REGULATION VIA THE ERBB PROTEIN AND miRNA NETWORK IN
BREAST CANCER Stefan Wiemann, Anja Schwäger,
Jitao Zhang, Heiko Mannsperger, Ulrike Korf, Özgür Sahin Division Molecular Genome
Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, 69120
Heidelberg, Germany. Overexpression and
mutation of transmembrane ERBB tyrosine kinases are adverse prognostic markers
in several cancer entities. A causative relation of ERBB signaling with cancer
development and progression has been established and, therefore, molecules of
the respective pathways are common targets of antibody and small molecule
therapies. In breast cancer, ERBB2 is targeted by the monoclonal antibody
trastuzumab, however, frequently observed de-novo resistance to this drug
requires a thorough understanding of the ERBB signaling network in order to
improve prognosis and therapeutic outcome. To this end, we combined
computational simulations, experimental testing, and reverse engineering of the
ERBB protein and miRNA interaction network in a breast cancer cell system. We
first established the technologies and bioinformatic means required to
quantitatively analyze ERBB signaling that links extracellular growth-factors
with the cell cycle. To this end, we connected ERBB signaling with G1/S transition
via two major cell signaling pathways and two key transcription factors, to
model an interaction network that allows for the testing of perturbations and
the prediction and analysis of induced effects on the phenotype. Individual
components were then systematically knocked down in the system and effects on
G1/S transition were recorded employing quantitative proteomic and molecular
assays. Based on this quantitative data, the original literature-based network
could be refined and extended. Additional protein and miRNA components as well
as novel connections could be integrated based on experimental validation also
of miRNA-gene interactions and the identification of feedback and feed forward
loops regulating the signaling network in proliferation as well as in cell
migration and invasion. While our understanding of ERBB-signaling is still far
from being complete, our data already suggests several proteins and one miRNA
(family) as potential novel targets for therapy. Identification
of a phosphatase regulating insulin signaling Aurelio Teleman German Cancer Research Center
(DKFZ, Heidelberg, Germany. Insulin signaling
is an important regulator of organismal metabolism, tissue growth, and aging in
animals. The intracellular insulin signaling pathway consists of a large number
of kinases that activate each other. Since insulin signaling is a homeostatic
signaling pathway, it needs to be both activated and inactivated. Phosphatases
responsible for inactivating the insulin pathway remain to be characterized. We
will present data on the identification and characterized of a phosphatase
responsible for dephosphorylating and inactivating an important component of
the insulin pathway, S6 Kinase (S6K). Pyrosequencing-based
DNA methylation profiling of Fanconi anemia/BRCA pathway genes in head and neck
squamous cell carcinoma Szaumkessel M.1, Richter J.2,
Toennies H.2, Giefing M.1, Jarmuż M.1,
Kiwerska K.1, Grenman R.4, Siebert R.2,
Szyfter K.1,3 1Institute of Human
Genetics, Polish Academy of Sciences, Department of Environmental Mutagenesis,
Poznan, Poland; 2Institute
of Human Genetics, University Hospital Schleswig-Holstein Campus Kiel,
Christian-Albrechts University, Kiel, Germany; 3Department
of Otolaryngology, University of Medical Sciences, Poznan, Poland; 4Department of
Otorhinolaryngology, Head and Neck Surgery and Department of Medical
Biochemistry, Turku University Central Hospital and Turku, Finland. Fanconi anemia (FA) is a complex
of heterogenous genetical and phenotypical recessive disorders, characterized
by numerous congenital malformations. The syndrome is featured by
hypersensitivity to DNA crosslinking agents, increased chromosomal breakage,
and defective DNA repair. Moreover, FA patients predispose to both hematologic
malignancies and solid tumors, in particular acute myeloid leukemia (AML) and
squamous cell carcinomas (SCC). Chromosomal instability in FA as the result of
defect/mutation in system maintaining genome integrity leads to high
susceptibility to cancer development, with a very high risk of developing
aggressive forms of head and neck squamous cell carcinoma, especially in young
patients. Therefore the Fanconi/BRCA pathway genes are considered as
‘caretaker’ tumor-suppressor genes and suggested to be the targets of
inactivation in head and neck squamous cell carcinomas. The aim of the study was to investigate the methylation profiles of
Fanconi/BRCA pathway genes in the head and neck squamous cell carcinomas (cell
lines and primary tumors) to assess a potential involvement of methylation
mechanism in carcinogenesis regulation. The study group consisted of 13 head and neck squamous carcinoma cell
lines and 64 cases of larynx primary tumors. As a control group peripheral
blood DNA from 10 men and 10 women was included, as well as DNA extracted from
buccal swabs (5 men and 5 women). The basic technique used for methylation
profiling was pyrosequencing. Most of the Fanconi genes (BRIP1, FANCC, FANCD2,
FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, PALB2) showed no methylation or very
low methylation level suggesting that methylation is not the mechanism for
their deactivation. In turn FANCB (located on X chromosome) demonstrated
differences in methylation levels but only in respect to gender (women with
higher level than men). FANCA gene demonstrated lower methylation in most of
HNSCC samples, in comparison to controls, where a total hypermethylation was
observed. BRCA1 revealed hypomethylation in 3/13 (23%) cell lines and in 2/64
(3%) primary larynx cases. Moreover, BRCA2 gene appeared to be hypermethylated
in HNSCC cell lines, simultaneously displaying lower methylation level in
primary larynx samples but still higher than control group. BRCA2 is shown to
be deactivated in HNSCCs, where methylation appears to be its mechanism. The work was supported by the Federation of European Biochemical
Societies. GENOME-WIDE FUNCTIONAL SCREENS
TO IDENTIFY NOVEL FACTORS IN CANCER PATHWAYS Sandra Steinbrink and Michael
Boutros German Cancer Research Center (DKFZ), Dep. Signaling and Functional
Genomics, D-69120 Heidelberg, Germany. e-mail: s.steinbrink@dkfz.de A limited number of
highly conserved signaling pathways control cellular homeostasis, growth and
development of almost any organism. These pathways control various biological
functions and their dysregulation can cause developmental defects, aberrant
cell growth and cancer. With the availability of the human genome sequence, a
systematic analysis of genes implicated in signaling pathways is now possible.
Approaches such as high-throughput RNA interference (RNAi) screens have proven
to be a valuable tool for the functional analysis of biological processes and
pathways in C. elegans and Drosophila and in recent years as well in cultured
mammalian cells. The further modification of such pathways with compounds of
low-molecular weight, so called small molecules, enables the functional
interference on a level that can be used to target hyperactivated signaling
pathways in the treatment of diseases such as cancer. We combine systematic
analysis of signaling pathways using genome-wide RNAi screens with
high-throughput compound screenings. The screens can be used to identify novel
targets and lead substances interfering with these targets. Furthermore, we
developed computational methods to analyze complex screening data sets and to
predict gene function by phenotypic similarity as well as possible mechanism of
action and side effects of small molecules.
In a genome-wide siRNA approach we systematically screened for modifiers of
cellular growth and survival by applying the death-inducing ligand TRAIL
(TNF-related apoptosis-inducing ligand) to human cervix carcinoma cells (HeLa).
Our analysis identified several novel genes that upon an RNAi-mediated
knockdown resulted in resistance of different human cancer cell lines to
apoptosis induction by TRAIL and in their enhanced clonogenic survival. Our
data gives insight into the complexity of signaling pathway integration and
regulation. The example of RNAi screens combined with therapeutic molecules
such as TRAIL, demonstrates the power of the approach to analyze synthetic
interactions and identify potential side effects and markers of treatment resistance. New
mathematical approaches in analyses of genomic and proteomic data Joanna Polanska Institute of Automatic
Control, The Silesian University of Technology, Gliwice, Poland. The rapid growth of
technology, we have been observing in last decade, allows for collecting a huge
amount of data in one experiment. Such massive information cannot be analyzed
without a help of data mining algorithms. Mathematical modeling is one of
these. It is especially useful while the experimental conditions are
multifactorial and the sample size is too small to obtain the satisfactory
power of statistical testing. We propose to use the modified
Gaussian mixture model (GMM) for the analysis of time-course and dose-response-pattern
DNA microarray data. The dataset consisted of the Human fibroblasts cells’
expression profiles obtained from three different subjects, irradiated five
different doses and measured at three time points (45 Affymetrix DNA microarray
HG-U133 Plus 2.0 in total). According to EURATOM standards, the applied
irradiation doses (0mGy, 50mGy, 100mGy, 200mGy, 2Gy) are classified, excluding
2Gy, as the “low dose” and the response signal is very low, hardly to be
separated from the measurement noise. The application of GMM methods allows the
unsupervised grouping of responses of cells to such low dose irradiation. The procedure for data
analysis involved the following steps: data preprocessing (reannotation,
normalization, filtration); grouping expression levels; functional description
of obtained groups (GO ontology, KEGG Pathway Analysis). The results of
grouping genes according to their patterns of expressions led to highly
statistically significant gene clusters. This suggests that the proposed
techniques lead to development of useful bioinformatic methods of the analysis
of multifactorial-course microarray data. As a result of analyses of patterns
of expressions, we observe the decrease of expression level for the genes
involved in cell cycle and mitosis, and DNA replication processes. Analogous methodology was
successfully applied to ChIP-on-chip data on the heat shock response of
mouse spermatocytes. Gaussian mixture modeling is
also a very useful tool for defining spectral features of MALDI ToF proteomic
mass spectra. Spectral features, computed as convolutions of Gaussian masks
with spectral signals, have been proven to be more efficient than commonly
applied spectral peaks, in the aspect of specificity and sensitivity of
classifiers. This work was supported by the
European Program FP6 – 036452, GENEPI-lowRT Thyroid
carcinoma as a model for gene expression profiling of cancer Barbara Jarząb Department of Nuclear Medicine
and Endocrine Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute
of Oncology, Gliwice Branch, Gliwice, Poland. Thyroid cancer
constitutes a good model for investigation of relations between the cancer
transcriptome and the disease characteristics. The rare histotypes, anaplastic
thyroid cancer (ATC) on one side and medullary thyroid cancer (MTC) on the
other, are subjects of intensive studies on the level of both gene expression
as well as array-CGH studies and the most important results will be presented
briefly. The main subject of the talk will be differentiated thyroid cancer.
The two DTC histotypes, papillary (PTC) and follicular (FTC), are different
diseases from the molecular point of view, while their clinical course is quite
similar. Despite the fact that they both arise from the same follicular
cell, their gene expression profiles differ very distinctly by the
expression of more than 1000 genes. In fact, the very distinct gene signatures
may also be attributed to the main molecular subtypes of PTC: there is a large
difference in gene expression between BRAF-positive and BRAF-negative PTCs, the
RET-PTC-related PTC is characterized by a distinct gene expression profile as
well. In PTC, many tumor and
patient-related features are correlated with the changed expression of multiple
genes. Among patient-related features, the sex signature is very distinct,
while the effect of age, also present, is less visible. Among tumor-related
factors, the grade is the most significant one, while metastatic signature has
not been well defined until now. In FTC, the very distinct gene signature is
related to the oncocytic type of tumors. In both types of
differentiated thyroid cancer (DTC), molecular markers of poor prognosis
are intensively looked for. The issue of BRAF mutation and its prognostic input
for the management of PTC patients will be discussed, completed by
microarray-based approaches to support the analysis. Also, the significance of
NIS expression for the prognosis of DTC will be analyzed in comparison to other
molecular data available. Another very interesting issue
from the clinical point of view is the molecular support for the differential
diagnosis between benign and malignant follicular tumors. A survey of
published attempts to solve this question will be presented, completed by own
experience in this field. The molecular diagnosis of thyroid nodules will be
evaluated in comparison to routine diagnostic procedures. FUNCTIONAL GENOMICS
AND PROTEOMICS IN PANCREATIC CANCER RESEARCH Andrea Bauer1,
Nathalia Giese2, Markus Büchler2 , Jörg D. Hoheisel1
and other collaborating partners withing the German NGFN PaCaNet group
and the European MolDiagPaCa consortium 1Division of Functional
Genome Analysis, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 2Department of
Surgery of the University of Heidelberg, Im Neuenheimer Feld, 69120 Heidelberg,
Germany.
Our research aims at the development and immediate application of new
technologies for an analysis, assessment and description of both the
realisation and regulation of cellular function from genetic information.
Analyses on tumour material are at the centre of attention with an emphasis on
pancreatic cancer. Parallel studies at a global level are under way on the
epigenetic modulation of gene promoters, variations in transcription factor
binding, changes of transcript levels of coding and non-coding RNAs, on the
actual protein expression and the intensity of protein interactions. From the
resulting data, we aim at an understanding of cellular regulation and its
biological consequences. In combination with clinical facts, the knowledge is
used for the creation of means of early diagnosis, accurate prognosis and the
analysis of treatment results as well as the establishment of new therapeutic
approaches.
A more recent line of work aims at an in vitro implementation of complex
biological processes. Motivation is a utilisation for the production of
molecules and the establishment of artificial molecular systems. Cell-free
biosynthetic production will become important for many biotechnological and
pharmacochemical challenges ahead. http://www.dkfz.de/funct_genome/ Prognostic
and predictive factors in ovarian cancer- results of verification of DNA
microarray data Kupryjańczyk J., Ludwig-Gałęzowska A., Szafron Ł., Felisiak A., Rembiszewska A., Rzepecka I., Moes J., Lisowska K., Rubel T. Department of Molecular
Pathology, Institute of Oncology, Warsaw; Department of Tumor Biology,
Institute of Oncology, Gliwice, Poland. We aimed to verify
gene expression data obtained previously in 71 ovarian carcinomas (32 treated
with platinum-cyclophosphamide - PC, 39 treated with taxane-platinum - TP) with
the use of cDNA oligonucleotide microarrays (HGU 133 Plus 2.0, Affymetrix).
Statistical analyses (GC-RMA normalisation, test T-student, Mann-Whitney
U-test, Benjamin-Hochberg procedure, false discovery rate - FDR<5% or p
value <0.001) revealed relatively small numbers of genes (from 0 to 39,
depending on the analysis; fold change >1.5 or <0.67) differentiating
chemotherapy-sensitive and -resistant carcinomas, as well as those
differentiating short and long disease-free or overall survival in the both
chemotherapy groups. First, the results were verified at mRNA expression levels
with the use of real-time RT-PCR on the same (PC-treated) or enlarged group
(TP-treated) of ovarian carcinomas. Approximately one-third of the verified
genes were confirmed as to their clinical significance in this verification.
Next, clinical importance of selected genes was studied on large clinical
groups of ovarian cancer patients; these analyses were performed at protein
levels with the use of immunohistochemical detection. Among genes
differentiating the sensitivity to chemotherapy were TNFSF13, BCAP29,
TMEM106C, FAF1 and survivin, while among those differentiating survival are
ING1, PTPN2, PCID2, VGLL1 and survivin. Similarly
to our previous studies, TP53 status determined the clinical significance of
some of these genes. On the basis of our results and data from the
literature, it appears that the clinical endpoints in ovarian cancer patients
are modified by subtle changes in the expression of many proteins (in addition
to strong clinical factors), rather than by a stable gene expression profile. Supported by the grant
2361/B/PO1/2008/34 from the Polish Committee of Scientific Research. Potential
and challenges of microarray data analyses for predicting oncogenic signaling
in colon tumors Magdalena Skrzypczak, Krzysztof Goryca, Tymon Rubel, Jerzy Ostrowski Department of
Gastroenterology, Medical Center for Postgraduate Education and Cancer
Center-Institute, Warsaw, Poland. An individual cell
phenotype is a resultant of the sum of cell-specific, developmental
stage-specific, and metabolism-related changes in gene expression selectively
provided at the time and grouped into signaling and metabolic pathways. While
cancer development results from series of somatic “driver” mutations and
multiple epigenetic changes leading to growth advantage of a specific cell
population over its neighbors, cancer complexity at the gene level is likely
reduced to a limited number of alterations within signaling pathways. With an introduction of
high-density DNA microarrays, modern biology provides a great insight into the
overall status of a cell. However, the comparative analyses of microarray-based
gene expression profiles relating to carcinogenesis exhibit rather weak
consistent overlap. These divergences may result from technical reasons,
including a usage of different microarray platforms, different sample
collection methods and different analytical algorithms. Since single standard
protocol for microarray data has yet not been identified, gene expression
profiles still allow rather draft and mostly indirect assumptions on oncogenic
signaling. Colorectal cancer (CRC) arises
as multi-step process in which the morphologic counterpart of molecular
alterations leads to progressive cytological and architectural derangement
recognizable as the adenoma-carcinoma sequence. To clarify the oncogenic
pathway alterations underlying the colorectal adenoma-carcinoma sequence, we
designed an integrative genomics approach. Studies were performed on samples of
normal mucosa, adenomas and carcinomas obtained during surgery or colonoscopy.
The collections of cryostat sections prepared from tissue samples were
evaluated by the pathologists to control the relative cell type content, and
RNA was isolated from macro- and microdissected specimens. The measurements
were done using the Affymetrix GeneChip HG-U133plus2, and the data were
evaluated using pair-wise comparisons, clustering analyses and data
decomposition into SVD modes and ICA independent components, followed by
selection of potential alteration within signaling pathways. This presentation
will addressed the potential and challenges of a translation of
microarray-based gene expression profiling into the functional aspects of
carcinogenesis. Molecular
profiling of histopatologically normal prostate tissue adjacent to cancer Fiszer-Kierzkowska A.1, Małusecka E.1, Jarząb M.2, Gawkowska-Suwinska M.3, Rembak-Szynkiewicz J.4, Bobek-Billewicz B.4, Jarząb B.5, Maciejewski B.1 1Radiotherapy
Department, Maria Skłodowska-Curie Memorial Cancer Centre and Institute,
Gliwice Branch, Gliwice, Poland; 2Clinical Oncology
Clinic, Maria Skłodowska-Curie Memorial Cancer Centre and Institute, Gliwice
Branch, Gliwice, Poland; 3I Radiotherapy
Clinic, Maria Skłodowska-Curie Memorial Cancer Centre and Institute, Gliwice
Branch, Gliwice, Poland; 4Radiodiagnostics
Department, Maria Skłodowska-Curie Memorial Cancer Centre and Institute,
Gliwice Branch, Gliwice, Poland; 5Nuclear Medicine
and Endocrine Oncology Department, Maria Skłodowska-Curie Memorial Cancer
Centre and Institute, Gliwice Branch, Gliwice, Poland. Cancer begins with
multiple genetic alterations that sequentially transform a cell, or a group of
cells in a particular organ. As a result of this transformation, according to
field cancerization concept, genetically altered but histologically
normal appearing cells predate the development of neoplasia or coexist with
malignant cells. Prostate cancer is often multifocal, and it is likely that
multiple tumors arise from an organ which has been earlier genetically altered
by a particular carcinogen. Aim of our study was to identify molecular
signature of genetically changed but histologically normal prostate cells. In our study we performed a
comprehensive gene expression analysis on 45 human prostate biopsy samples
including prostate cancer tissue, prostate tissue adjacent to tumor and benign
prostatic hyperplasia, using U133 Plus 2.0 Affymetrix arrays. In
the first step of analysis genetic profiles of prostate cancer samples and
benign prostatic hyperplasia samples were compared. We have found 279 genes which
differentiate the groups, among them were genes found in other studies as
changed in prostate cancer: AMACR, hepsin, EZH2, which demonstrates that
microarray analysis of biopsy specimens gives similar results to the studies
performed using prostatectomy specimens. In the next step we compared the
genetic profiles of benign prostatic hyperplasia and normal-appearing prostate
tissue adjacent to cancer. We obtained 129 geneset differentiating those two
groups, and this difference was significant (p=0.013) according to the global
test of difference. Biocarta database analysis revealed that pathway:
“Chromatin Remodeling by hSWI/SNF ATP-dependent Complexes” seemed to be
particularly down-regulated in prostate tissue adjacent to cancer
(p<0.0001), with seven genes showing expression decrease (p<0.05). Genes
identified by us has yet to be validated by RT-PCR and immunohistochemical
analysis. Molecular changes in prostate
tissue adjacent to cancer found in our study appear to have potential utility
as early diagnostic markers. MALDI-TOF
MS-based Serum Proteome Pattern Analysis in Molecular Diagnostics of Breast
Cancer Pietrowska M.1, Behrendt K.1, Nowicka E.1, Walaszczyk A.1, Tarnawski R.1, Marczak Ł.2, Stobiecki M.2, Polańska J.3, Polański A.3, Widłak P.1 1Maria
Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice,
Poland; 2Polish Academy of
Science, Institute of Bioorganic Chemistry, Poznan, Poland; 3Silesian University
of Technology, Gliwice, Poland. Mass spectrometric analysis of
the blood proteome is an emerging method of clinical proteomics. The approach
exploiting multi-protein/peptide sets (fingerprints) detected by mass
spectrometry that reflect overall features of a specimen’s proteome, termed
proteome pattern analysis, have been already shown in several studies to have
applicability in cancer diagnostics. Here we aimed to identify serum proteome
patterns specific for the early stage breast cancer patients using MALDI-ToF
mass spectrometry. Blood samples were collected
before the start of therapy in a group of 92 patients diagnosed at stages I and
II of the disease, and in a group of age-matched healthy controls (104 women).
Serum specimens were purified and the low-molecular-weight proteome fraction
(2-10 kDa) examined by spectrometry after removal of albumin and other large
serum proteins, and then registered mass spectra were analyzed using new
bioinformatic tools created in our group. We identified the classifier
built of four spectral components that differentiated healthy persons and
breast cancer patients with ~85% specificity and sensitivity. Spectral
components (i.e., protein ions) that were the most frequent in such classifier
had approximate m/z value of 2866, 3579, and 2303 Da. The classifier
intentionally built of above three components showed 80% specificity and 88%
sensitivity. Interestingly, we have also observed significantly (p=0.0003)
increased level of osteopontin in blood of analyzed group of cancer patients.
However, the classifier built of osteopontin level showed 28% specificity and
88% sensitivity, and thus was outperformed by the classifier built of the most
frequent spectral components identified in serum by mass spectrometry. In
addition, we have identified several components whose levels in serum of individual
patients were significantly changed in the course of therapy. Our data clearly
indicates that MALDI-ToF-based serum proteome pattern analysis has an obvious
potential in diagnostics and monitoring of therapy of breast cancer patients. This work was supported by the
Ministry of Science and Higher Education, Grant 2 P05E 067 30. Session III: Bystander Mechanisms as Targets for Cancer Radiotherapy RADIATION-INDUCED NON TARGETED
EFFECTS OF LOW DOSES – WHAT, WHY AND HOW? Carmel Mothersill and Colin
Seymour McMaster University, Hamilton,
Ontario, Canada. Ever since the
grudging acceptance that non-targeted effects (NTE) can be measured in unirradiated
cells or distant progeny of irradiated cells, the discussion has raged about
the relevance of these effects. For the purposes of this presentation,
NTE are defined as effects not associated with DNA lesions due to energy
deposition in the cell showing the effect and so include genomic instability
and bystander effects. Obviously, it is important to consider relevance for
practical applications such as radiation protection and radiotherapy. To this
end this paper will review data from in vivo experiments, which address
questions about risk after medical and environmental exposures. However a major
area of interest is the intrinsic relevance of these mechanisms in biology.
Arguments will be made in this paper, that non-targeted effects (NTE) may call
into question not only radiation effects paradigms but may also have relevance
to wider mechanisms in cancer biology, population ecology and evolutionary
biology concerning process of selection, the transmission of heritable traits,
the relevance of “social” interactions between cells, organisms and populations
and the mechanism by which cells/organisms respond rapidly to environmental
stress. This paper will also argue that a key consequence of findings in NTE
biology is that at any given level of organization, from gene to ecosystem –
communication of stress signals and heritability of stress adaptations provide
the bridges linking one hierarchical level to the next and enable the rapid
propagation of change triggered by stress at one level, resulting in change at
a higher (or lower?) level. This addresses a major problem in evolutionary
biology because while the molecular mechanisms of natural selection are fairly
well understood a major knowledge gap exists in translating mutational drift at
the level of the individual cell to natural selection at the ecological level
where sociobiological factors are so important. The existence of the mechanism
discovered in the NTE field provides a glimpse of a major way that evolution
could be regulated through communicated signals between cells, individuals, and
populations, These control and optimize responses at the level of the
population and coordinate the emergence of exquisitely tuned systems which can
adapt rapidly to micro or macro environmental change. It is likely that
consideration of these mechanisms could also be of benefit in cancer biology
providing new insights into the regulation of cancer cell social groups and how
these interact with the host. Bystander Mechanisms as Targets for Cancer Radiotherapy Elisabeth Schültke Albert-Ludwigs-University
Freiburg, Germany. In collaboration with Carmel
Mothersill, Colin Seymour,Richard Smith, Jean Laissue, Hans Blattmann and Elke
Bräuer-Krisch When new
radiotherapy concepts are developed, the first question, of course, is whether
survival times can be increased, compared to already established therapy
concepts. Having established survival advantage, the next important task is to
investigate the new therapeutic concept for unwanted side effects and to identify,
where possible, the pathways of action. Since 2005, we have conducted
a series of studies using an experimental radiotherapy concept called microbeam
radiation therapy (MRT) at the biomedical beamline ID17 of the European
Synchrotron Radiation Facility (ESRF) in Grenoble (France). The concept of MRT
has been developed during the 1990s at the NSLS in Brookhaven (USA) and at the
ESRF. In the hospital setting, a broad beam in the MeV range is used for tumour
radiotherapy. For MRT, highly collimated synchrotron beam in the keV range is
split with the help of a special collimator into an array of small
near-parallel beams in the micrometer range. This creates a characteristic
irradiation profile and also permits the deposition of X-ray doses into the
tumour that can be higher by two orders of magnitude compared to the doses used
in hospital-based radiotherapy programs. This could prove a therapeutic
advantage especially where the target tumours are extremely radioresistant. Our research group has studied
survival, effects on new memory formation as well as bystander effects of MRT
in two small animal models of malignant brain tumour. Data from those studies
and the potential for transfer of MRT studies into a clinical trial phase will
be discussed. High-dose Lattice Radiation Therapy: Clinical, Physics and Biological
perspective of bystander effects Mansoor M. Ahmed Department of
Radiation Oncology, Sylvester Comprehensive Cancer Center, University of Miami,
Miami, FL 33136, USA. The
standard paradigm for ionizing radiation (IR) effects involved DNA damage with
dsDNA breaks as triggers for mutation, cell death and transformation. However,
a growing body of evidence reported non-targeted effects, including genomic
instability, gene induction, adaptive responses and low dose hypersensitivity.
One such non-targeted effect often observed is deleterious signaling exerted to
either neighboring or distal cells by radiation-induced damage cells. These
signals can be classified into two independent phenomena that are (i) Abscopal
effect and (ii) Bystander effect. This presentation focuses on the potential
utilization of bystander/abscopal effects in different therapeutic settings to
eliminate malignant cells while protecting the normal tissue in lung cancer.
Our media transfer experiments and analysis of serum samples from high-dose
GRID radiation-treated patients demonstrated that such high-dose
radio-inductive therapy caused the release of TNF-a, TRAIL and
ceramide as bystander factors mediating the killing of unirradiated tumor
cells. We found that reduced bystander response in A549 lung cancer cells was
due to activation of NFêB signaling by TNF-a, whereas, enhanced
response to IR-induced bystander signaling in H460 lung cancer cells was due to
release of TRAIL associated with nuclear translocation of PAR-4. Using the low
energy GRID for animals, high-dose Spatially Fractionated Grid Radiation
Therapy (SFGRT) caused regression of the neighboring untreated tumor and the
regression was significantly enhanced with addition of 2 Gy. This was evident
in both nude mice xenograft as well as in Lewis Lung Carcinoma (LLC) model in
C57Bl/6 mice. Further, in serum of SFGRT-treated patients, ceramide was
significantly induced in response to high-dose radiation of 15 Gy and targets
the endothelial cells of tumor microenvironment. Although, clinically,
relatively small in number of patients receiving SFGRT treatment either with
early ortho-voltage machines or more recently with MV X-rays, tumor regressions
were reported, leaving a perplexed paradox contradicting to the principle of
conventional radiation therapy, in which a total tumor volume irradiated with a
rather uniform dose is called for. Thus, this evidence strongly suggest that
SFGRT would induce a rapid and higher rate of tumor cell apoptosis in bulky
tumors. This leads to the new notion of using high-dose SFGRT as an induction
therapy to enhance therapeutic outcome of subsequent conventional radiation
therapy. It is not unreasonable to anticipate that such new approach may open a
new paradigm in radiation therapy, if the clinical efficacy proven positively.
Most importantly, the highest-dose regions of the SFGRT are superficial, and
often are outside of the tumor target itself. Unnecessary high dose exposure to
the surrounding normal tissue can be significantly reduced by reconfiguring the
SFGRT treatment into a 3D-SFGRT dose LATTICE, a new approach to spatially
fractionated radiation which takes advantage of modern-era technology. This
technique can also be used to place high-dose islands within the tumor target
only, not outside of the target. Using this technique, high doses of radiation
are concentrated at vertices within the tumor volume, with drastically lower
dose between vertices (peak-to-valley effect) and leaving anything outside of
tumor volume minimally exposed. Because more pronounced radiation dose peaks
and valleys are generated using LATTICE technique compared to SFGRT, it may be
more radiobiologically effective, with significantly less radiation dose to
adjacent normal tissues, and therefore should confer less additional toxicity.
This new concept is adopted in clinical settings as well as in animal models.
Further, current studies are in focus to study the synergizing effect of
high-dose radiation-induced bystander factors such as TNF-a and TRAIL with standard
chemotherapeutic drugs (such as Taxanes, Carboplatin and 5-FU), signaling
agents (such as TKIs, proteosome inhibitors and 2-Deoxy-Glucose) and natural
herbal agent such as Curcumin. Haemotologic toxicity of radioimmunotherapy Wojciech Jurczak Jagiellonian
University, Cracow, Poland Background:
Radioimmunotherapy (administration of 90Y-Zevalin) is registered by
FDA for Follicular Lymohoma Pts (FL) refractory/ relapsing to previous
Rituximab containing regimens. In Kraków University Hospital, there were 101
radioimmunotherapy procedured performed, 85 either as monotherapy or
consolidation to previous chemotherapy regimens: 20 in FL, 53 in MCL (Mantle
Cell Lymphoma) and 12 in DLBCL(Diffuse Large B cell Lymphoma). Further 16
procedures were elements of autologous transplant (ASCT) conditioning (3 –in
MCL, 13 in DLBCL). A short clinical background and our clinical results
will be presented Methods: Patients were
given 250 mg/m2 of R followed 1 wk later with a second dose of R +
Zevalin (11 or 15 MBq = 0.3 or 0.4 mCi/Kg based on PLT count; maximum dose = 32
mCi). Cytopenia was assessed with full blood counts every wk until
recovery. In a subset of patients trephine biopsies were performed before
Zevalin dosing and at 2 and 6 wks after Zevalin to assess marrow cellularity
and stromal cells.At the same time points, mantle cell (CD19+CD20+CD5+CD23-)
infiltration of BM was assessed by flow cytometry; myeloid clonogenic capacity
(CFU-GM, BFU-E, CFU-GEMM, CFU-Meg) was evaluated by cell culture; and BM stroma
function was measured by assaying 5 cytokines: GM-CSF, EPO, TPO, IL-3, and SCF.
Results were compared between MCL and FL patients who received Zevalin alone
following relapse after chemoimmunotherapy. Results: The level
of neutro- and thrombocytopenia was greater when fludarabine was given before
Zevalin than in patients who received only Zevalin. Patients given fludarabine
pretreatment experienced a 2- to 4-fold greater degree of clonogenic capacity impairment
compared with those that received only Zevalin, except CFU-Megs, whose profiles
were similar in both groups. Seven- to 10-fold ¯ in myeloid stem
and progenitor cells in response to FCM à RIT were
associated with ¯ in PMNs and PLTs. Stromal cells experienced
minimal, transitory (fully reversible by 2 wks) impairment, suggesting a
negligible adverse effect of RIT on BM stroma. Compared
with pre-Zevalin values, GM-CSF levels ¯ 2 fold
at wk 4, and TPO and IL-3 ¯ 30% and 3-fold, respectively, at wk 2. EPO levels ¯ 3 fold during the first 4 wks, and paralleled
decreases in BFU-Es. Conclusions: Delayed pattern of
cytopaenia after radioimmunotherapy is not characteristic for any chemotherapy
regimens. It depends from the diagnosis (pattern of infiltration of BM by
lymphoma) and the intensity of preceeding therapy. It is most likely secondary
to the stem cell damage, as indicated by clonogenic capacity studies,
lack of impairement of the stroma cell assessment and lack of any additional
perturbations in transplanted patients with the stem cells reinfused. What
are non-targeted radiation effects? Colin Seymour and Carmel
Mothersill McMaster University, Hamilton,
Ontario, Canada Non targeted
effects suggest an unpredictable response, as opposed to the predictable
response of target theory. Experimental data though produces fairly robust
responses, which are reproducible even if they do not follow a direct dose response
relationship. For non targeted effects to be
exploited in therapeutic situations a basic understanding of mechanisms is
required. Even if a response is reproducible the underlying mechanisms may be
unclear. In this paper we will examine
potential mechanistic theories in terms of our fish model which uses water
borne transmissible factors from irradiated to non irradiated fish. Low
LET radiation and bystander factor damage to mammalian mitochondria Sharon Nugent2, Carmel Mothersill3, Colin Seymour3,
Brendan McClean4, Fiona Lyng2 and James Murphy1 1Mitochondrial
Biology & Radiation Research Group, IT Sligo, Ireland; 2Radiation & Env.
Science Centre, DIT, Dublin, Ireland ; 3Medical Physics
& Applied Radiation Sc., McMaster University, Hamilton, Canada; 4St. Luke’s
Hospital, Dublin, Ireland. Radiation damage
incurred by nuclear DNA is well documented and interest is increasing in the
properties of “bystander” factors though its effects and those of direct low
LET irradiation on the mitochondria, and more particularly mitochondrial DNA
(mtDNA) are less well understood. The current study characterised the
mitochondrial response to both direct irradiation and bystander factors in
human keratinocytes (HPV-G) and Chinese hamster ovarian cells (CHO-K1). Cells were exposed to either g radiation (0Gy,
5mGy, 0.5Gy or 5Gy) or Irradiated Cell Conditioned Medium (ICCM) and analysed 4
to 96 hours post exposure. MtDNA damage analysis included mutation and deletion
analysis. Mitochondrial (dys)function was analysed by a range of approaches
including polarography, in organello mitochondrial protein synthesis and
kinetics of the oxidative phosphorylation (OXPHOS) enzyme complexes.
Mitochondrial mass was determined using MitoTracker FM. Results demonstrate mtDNA
damage in HPV-G mtDNA was induced as early as 12 hours post direct exposure 24
hours post exposure to ICCM. Furthermore, low dose exposure appeared most
potent in inducing the mtDNA4881 deletion. Significant increases in
mitochondrial mass were observed after exposure to both direct radiation and
ICCM in both cell types and mitochondrial dysfunction varied greatly post
exposure and was non-uniform between OXPHOS enzyme complexes Findings show mitochondria are
prone to even very low-level ionizing radiation-induced stress and medina
damage load may accumulate with time post exposure. The multi-heterogeneous
medina population of cells represents a sensitive barometer for low-level
radiation exposure damage. Targeting
transcription-coupled repair as an anti-cancer strategy Bruce McKay Cancer
Therapeutics Program, Ottawa Hospital Research Institute, Ottawa, ON, CA. Many cancer
therapeutics and drugs undergoing clinical trials for cancer treatment elicit
their anti-neoplastic effects by inducing DNA damage. There is considerable
interest in the development of strategies to target specific DNA repair
pathways to increase the efficacy of these agents. We have used RNA
interference to target several proteins involved at specific steps in
transcription-coupled subpathway of nucleotide excision repair (TC-NER).
Notably, Cockayne syndrome (CS) group B protein (CSB) is an ATP-dependent
chromatin remodeling protein of the SWI2/SNF2 family that plays an essential role
in the rate limiting step of the transcription-coupled subpathway of nucleotide
excision repair (TC-NER). RNA interference against CSB resulted in a marked
increase in the sensitivity of a variety of tumour cell lines to cisplatin
and this was independent of p53 and DNA mismatch repair. These
genetic alterations, common in cancer, have been linked to cisplatin resistance
in a variety of model systems and may contribute to resistance in the
clinic. Our results suggest that targeting this DNA repair pathway
may represent an effective anti-cancer strategy even in drug resistant tumours. Characterisation
of the hematopoietic stem cells using mathematical
models Anna Marciniak-Czochra ResearchGroup LeaderCenter of
Mathematical Modeling and Simulation in Biosciences (BIOMS), Interdisciplinary
Center for Scientific Computing Institute of Applied Mathematics BIOQUANT,
University of Heidelberg, Heidelberg, Germany. Stem cells
behaviour is an important field of research with promising clinical
impacts. Due to the revolutionary new technologies of
biological data collection, an enormous amount of information on specific
factors and genes responsible for cell differentiation is available.
However, the mechanisms controlling stem cell self-renewal, maintenance
and differentiation are still poorly understood and there exists no
general characterisation of stem cells based on measurable cell
properties. We address these problems with the help of new mathematical
models. Results of numerical simulations are compared with the
experimental data obtained from patients with multiple myeloma after
high-dose chemotherapy and stem cells transplantation. It leads to the
conclusion that the regulation of the asymmetry of cell divisions is
significantly more efficient that the regulation of the
proliferation rates. Moreover, analysis of the model equations leads to a
generalization of the concept of self-renewal potential, which might be
helpful to define the stem cell compartment. Crosstalk
between p53 and nuclear factor-kB systems: pro- and anti-apoptotic functions of
NF-kB K. Puszyński1, R. Bertolusso2, T. Lipniacki3 1Institute of
Automatic Control, Silesian University of Technology, Akademicka 16, 44-100
Gliwice, Poland; 2Baylor College of
Medicine, Houston, Texas, USA; 3Institute of
Fundamental Technological Research, Swietokrzyska 21, 00-049 Warsaw. Nuclear factors p53
and NF-kB control many physiological processes including cell cycle arrest, DNA
repair, apoptosis, death, innate and adaptive immune responses, and
inflammation. There are numerous pathways linking these systems and there is a
bulk of evidence for cooperation as well as for antagonisms between p53 and
NF-kB. In this theoretical study, the authors use earlier models of p53 and
NF-kB systems and construct a crosstalk model of p53-NF-kB network in order to
explore the consequences of the two-way coupling, in which NF-kB upregulates
the transcription of p53, whereas in turn p53 attenuates transcript_ion of
NF-kB inhibitors IkBa and A20. We consider a number of protocols in which cells
are stimulated by tumour necrosis factor-a (TNFa) (that activates NF-kB
pathway) and/or gamma irradiation (that activates p53 pathway). The authors
demonstrate that NF-kB may have both anti- and pro-apoptotic roles. TNFa
stimulation, preceding DNA damaging irradiation, makes cells more resistant to
irradiation-induced apoptosis, whereas the same TNFa stimulation, when preceded
by irradiation, increases the apoptotic cell fraction. The finding suggests
that diverse roles of NF-kB in apoptosis and cancer could be related to the
dynamical context of activation of p53 and NF-kB pathways. Can
blood cells be important in bystander effects? Bystander effects in
lymphoblastoid cells Joanna Rzeszowska1,2,
Joanna Łanuszewska1, Agnieszka Gdowicz1, Magdalena
Skonieczna2, Roman Jaksik2 1Department of
Experimental and Clinical Radiobiology, Maria Skłodowska-Curie
Memorial Cancer Center and Institute of Oncology, 44-100 Gliwice, Poland; 2System Engineering Group, Institute of Automatic Control, Silesian
University of Technology, 44-100 Gliwice, Poland. All tissues of
multicellular organisms are permanently in contact with cells of the blood,
including lymphocytes, macrophages and other cells of the immune
system. These cells are naturally programmed to produce signaling molecules and
molecules inducing oxidative stress, and to interact and influence the fate of
other cells. Because during anticancer therapy they always receive some dose of
radiation or drugs, their ability to communicate with other non-irradiated
cells and the processes which they induce i may be of importance for
therapy. We have studied the bystander effect in lymphocytes isolated
from peripheral blood and in lymphoblastoid cells. At different times after
direct irradiation or co-culture with irradiated cells, oxidative DNA damage
and DNA breaks, apoptosis, micronuclei, and clonogenic survival were assessed.
Changes of the transcriptome were also studied by a microarray approach. All
our results suggest that lymphoblastoid cells express bystander effects, and
that after irradiation they release signals which may sometimes counteract the
induction of apoptosis and support the survival of neighboring cells. Session IV: Ion
Channels and Cancer Thermal
effects in the Ca2+-ATPase protein Miguel Rubi Departament de Física
Fonamental, Facultat de Física, Universitat de Barcelona, Spain. email: mrubi@ub.edu To know
the microscopic mechanism controlling energy transport in bio-molecules
constitutes a considerable challenge which could provide a basis to understand
complex biological processes. The time scale associated with the energy
relaxation throughout the molecule can give information about the kinetics of
bio-molecule reactions. Moreover, the pathways followed by the energy during
the relaxation process can provide important clues to understand basic
mechanisms of the molecule. In order to analyze these processes it is necessary
to know how perturbations occurring at a specific spot in the bio-molecule,
namely, binding of small molecules at receptor sites, may impart a
conformational change at a distant spot, lying several nanometers away. We
apply non-equilibrium thermodynamics and computer simulations to analyze
thermal effects in the Ca2+-ATPase. 1. Kjelstrup, S.;
Rubi, J.M.; Bedeaux, D. Physical Chemistry Chemical Physics 2005, 7,
4009–4018. 2. A. Lervik, F.
Bresme, S. Kjelstrup, D. Bedeaux, J. M. Rubi, preprint Ion channels are increasingly being linked
to cancer and tumour progression Walter Stühmer Max-Planck Institute of
Experimental Medicine, Göttingen, Germany. Here we describe a
voltage-gated, potassium selective channel (Eag1, Kv10.1) with novel electrophysiological
properties, whose normal physiological function is yet unknown but which shows
oncogenic transforming potential if expressed ectopicaly. Strikingly, the
expression of the human Eag1 is restricted to brain, but it is also present in
several tumour-derived cell lines. More importantly, the protein can be
detected in more than 72% of human tumour samples, while the corresponding
normal tissues are devoid of the channel. Experiments under in vitro
conditions have demonstrated decreased proliferation of Eag1-expressing cells
by inhibition of expression and/or function of this channel. This inhibition of
Eag1 is accomplished using RNA interference, functional anti-Eag1 antibodies,
or (unspecific) EAG1 channel blockers. We have also used in vivo models to
visualise the distribution of Eag1 in tumour-bearing mice using specifically
designed recombinant antibodies. We conclude that Eag1 is a widely
distributed tumour marker with diagnostic and therapeutic potential. Citrate
transporter from prostate epithelial cells Maria E. Mycielska Division of Cell &
Molecular Biology, Sir Alexander Fleming Building, Imperial College London,
South Kensington Campus, London SW7 2AZ, UK. Prostate gland is a
unique organ that produces and releases large amounts of citrate into prostatic
fluid (up to 180 mM). This is necessary to sustain sperm vitality and motility.
Importantly, citrate levels drop dramatically when prostate becomes metastatic.
Determination of the mechanisms of citrate synthesis and release in prostate
epithelial cells are therefore important for understanding crucial aspects of
male infertility and prostate cancer. Large amounts of citrate are
produced in prostatic cells because of the rate-limiting role of mitochondrial
aconitase which expression and activity is regulated by hormones and Zn2+.
Surprisingly, the way citrate is released from prostatic epithelial cells has
not been known until now. We used cDNA library screening and RACE PCR to
determine the molecular nature of the citrate release transporter from PNT2-C2
cells. We obtained a functional clone, expressed it in HEK cells and evaluated
the role of the newly cloned transporter in prostatic cells using siRNAs and
functional assays. We also produced an antibody for the novel transporter and
showed that the cloned protein co-localised with the plasma membrane of
prostatic cells. Streszczenia wykładów zgłoszonych na konferencję i nie wygłoszonych z powodów losowych (nieobecność wykładowców)
The latest advances in clinical genetics of tumours including gastric
cancer J. Lubiński International Hereditary Cancer Center Pomeranian Medical
University, Szczecin, Poland.
e-mail: lubinski@sci.pam.szczecin.pl In order to solve problem – be successful, it
is critical to work hard and wise, but also to be lucky. Polish society is a
lucky one for effective performance of studies on clinical–genetic
correlations, because Poland is relatively big country – with almost 40 mln population
additionally showing high level of genetic homogeneity. This is the main reason why in the field of
genetic–clinical correlations, at least in oncology, studies conducted in
Poland are frequently of unique international value. Historical milestone in our understanding of
genetic characteristic of Polish population were studies performed 10 years
ago, in which we sequenced BRCA1/BRCA2 genes in almost 70 families with strong
aggregations of breast/ovarian cancers. Gorski B. et al. showed that Poland is dominated
by BRCA1 mutations and, additionally, only 3 of them constitute around 90% of
all BRCA1 mutations in Poland. DNA test designed especially for Polish
population allowed to detect BRCA1 mutations a few dozen times quicker and
cheaper than in rich but genetically heterogenous Western countries. This is
why in our centre only we performed almost 200 000 of BRCA1 tests
detecting almost 5 000 carriers. This is the largest worldwide registry of
females with mutations and under surveillance of cancer genetic outpatient
clinics from almost all regions of Poland. Genetic homogeneity of Polish population has
been confirmed in studies of other genes associated with predisposition to
cancers. As a consequence, we noted very rapid progress in identification of genetic
markers for almost all sub-groups of tumours. In 2008, we published the first
panel of genetic markers covering more than 90% of breast cancers. This work
suggests directly for the first time that carcinogenesis of all tumours is
caused by both groups of features–environmental and genetic, however their
relative contribution in tumorigenesis of particular case can be variable
ranging from a few to a few dozen of per cent. In last years almost all genes
associated with monogenic high risk of cancers characterized by strong
aggregation of tumours such as i.e. BRCA1/BRCA2 (~80% risk of breast/ovarian
cancers), MSH2/MLH1/APC (~80% risk of colorectal cancers) or E-cadherin (~80%
risk of diffuse stomach and lobular breast cancers) have been identified (Tab.
1). At present, investigations aimed to identify markers on moderate/low
cancers risk are the most frequently performed. Their identification seems to
be very important also from clinical practice perspective because these
markers: 1.
can interact – i.e. women carrying CHEK2 mutation and some BRCA2 variants can
be at 6-fold increased risk of breast cancer. 2.
can identify high risk persons if combined with family history – i.e. ~10 fold
increased risk of prostate cancer occurs in men–carriers of NBS1, CHEK2 or some
BRCA1 mutations if even one prostate cancer was diagnosed among
relatives. 3.
are identifying multiple site predisposition – i.e. CHEK2 mutations are
associated with increased risk of cancer of the: breast, ovaries, colon,
kidney, stomach, prostate and thyroid and decreased risk of cancers of the lung
and larynx and are associated with distinct clinical characteristics of cancers
– i.e. breast cancers in families with NOD2 or CDKN2A changes are characterized
by occurrence of microcalcifications and significantly increased risk already
at age 35 yrs, and cancers dependant on CHEK2 are ER(+) what suggests potential
value of tamoxifen in their chemoprevention. Spectacular progress was noted recently in
chemotherapy of breast cancers dependent on BRCA1. In 2007 Byrski T. et al.
published retrospective observation on the lack of effectiveness of taxans – in
9 out of 15 females – mutation carriers with breast cancers treated using AT
scheme remissions were not seen in neo-adjuvant therapy. Such results were in
accordance with observation of British researchers who found that cell lines
from breast cancers in BRCA1 carriers are resistant to taxans. These scientists
in the same publication reported high sensitivity of cancer cells lines to
cis-platinum. This is why, we launched clinical trial on cis-platinum
efficiency in treatment of breast cancers among BRCA1 carriers. Results of this
first completed clinical trial have been published in July 2008. In all 10
recruited patients, we observed clinical and pathologic remissions which was
complete in 9 of them. Recently, another paper by Byrski T. et al. has
been accepted for J Clin Oncol in which efficiency of different schemes of
neo-adjuvant therapies in BRCA1 carriers with breast cancers was compared
retrospectively. Frequency of complete remissions in monotherapy with
cis-platinum was above 80%, in AC scheme – 30% and using CMF or AT (taxans) –
8%. At present, we perform clinical trial on the use of cis-platinum for affected
BRCA1 carriers independently on cancer site thus including gastric cancer
patients with germline mutations. Table
1. Syndromes with
familial susceptibility to gastric cancers (GC)
Bystander
effects elicited by different radiation qualities - therapeutic
opportunities? Marie Boyd1, A. Sorensen1, S. Ross1, C.
Mothersill3,
and R. J. Mairs2 1Strathclyde
Institute for Pharmacy and Biological Sciences, University of Strathclyde,
Glasgow, Scotland; 2CRUK Beatson
Laboratories Glasgow, Scotland; 3McMaster
University, Ontario Canada. Radiotherapy is
currently utilised in the treatment of a significant number of cancers, however
its efficacy is hampered by limited dose administration due to normal tissue
toxicity. Several approaches are currently under consideration for improved
efficacy and one particularly promising approach is the utilisation of radiation
induced biological bystander effects ( RIBBE) for tumour cell kill. It has long
been accepted that when cells are irradiated, both direct effects and indirect
effects are consequences of such irradiations. It has become apparent however
that different radiation sources result in a variety of indirect (bystander)
effects that differ with respect to their magnitude and nature. In particular
RIBBE following irradiation of cells with targeted radionuclides conjugated to
alpha--beta- and auger emitters are quantitatively and
qualitatively different to those achieved following irradiation with external
beam sources. It appears that when cells are irradiated with the lower dose and
dose rate radiopharmaceuticals, RIBBE are more toxic to recipient cells and the
effect on recipient non-irradiated cells is more dependant on dose. When
pharmaceuticals are conjugated to high LET radiopharmaceuticals, recipient
cells succumb to toxic bystander effects in a dose dependant fashion, superior
to direct irradiation and with higher target cell dose this effect is perturbed
(U-shaped survival curve). Thus RIBBE following targeted radionuclide therapy
is a complex and novel phenomenon which offers a new tool for optimisation of
tumour cell kill if its mechanism and nature can be determined. Our studies are
aimed at investigating such effects to determine optimum radiopharmaceuticals
for maximisation of both direct and indirect effects. Our studies are
suggestive of a mechanism that involves several unique components including
generation of Reactive Oxygen and Nitrogen Species and complex cellular
signalling pathways. As it is likely that clinical translation of targeted
radionuclide therapy will involve combinations with radiosensitisers and other
chemotherapy drugs, it is vital to understand the RIBBE process at play in such
strategies to ensure that drug/radiation combinations are complementary to the
production of RIBBE so that their potential for eliciting cell kill in a tumour
specific fashion can be maximised. These indirect effects thus offer a novel
approach for cancer cell treatment utilising targeted radionuclide therapy. Pro-apoptotic,
pro-autophagy and proliferative effect of calprotectin Saeid Ghavami1,
Andrew J. Halayko1, Claus Kerkhoff 2 and Marek
Los3 1Department of
Physiology, Univ. Manitoba, Winnipeg, Canada; 2Institute of
Immunology, University of Muenster, Roentgenstr. 21, Muenster, Germany; 3Interfaculty
Institute of Biochemistry, Hoppe-Seyler-Str. 4, Univ. Tübingen, Germany. The complex formed
by two members of the S100 calcium-binding protein family, S100A8/A9, known
also as calprotectin, may exert pro-cell death effects, growth-promoting
activity, and immunomodulatory functions, depending on concentration, cell
type, and local micro-environment. An element of the complex, S100A9 gained
recently a significant attention due to its tumor protecting activity against
immune response. S100A8/A9 triggers proliferation at low micromolar
concentration, whereas at intermediate or high micromolar concentrations it is
toxic. Cell death induction by S100A8/A9 is independent of RAGE-mediated
signaling, but both proliferative effects and immunomodulatory properties
require RAGE. The underlying molecular mechanisms of cell death induction are
both programmed cell death I (PCD I, apoptosis), and PCD II (autophagy).
Treatment of cells with S100A8/A9 caused the increase of Beclin‑1
expression as well as Atg12-Atg5 formation. S100A8/A9-induced cell death was
partially inhibited by the specific PI3-kinase class III inhibitor,
3-methyladenine (3-MA), and by the vacuole H+-ATPase inhibitor,
Bafilomycin-A1 (Baf-A1). S100A8/A9 provoked the translocation of BNIP3, a BH3
only pro-apoptotic Bcl2 family member, to mitochondria. Consistent with this
finding, ΔTM-BNIP3 over-expression partially inhibited S100A8/A9-induced
cell death, decreased ROS generation, and partially protected against the
decrease in mitochondrial transmembrane potential in S100A8/A9-treated cells.
In addition, either ΔTM-BNIP3 over-expression or N-acetyl-L-cysteine
co-treatment decreased lysosomal activation in cells treated with S100A8/A9.
Our data indicate that S100A8/A9-promoted cell death occurs through cross-talk
of mitochondria and lysosomes via ROS. | ||||||||||||||||||||||||||||||||||||||||||||
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