3. Molecular Basis of Disease
3.1 Oxidative Stress
S3.1-1
The role of antioxidants in the cytotoxicity of
chemotherapeutic drugs
T. Ozben, H. Akbas, I. Akan, S. Akan and M. Timur
Department of Biochemistry, Faculty of Medicine, Akdeniz
University, Antalya, Turkey. E-mail:
A number of drugs used in cancer chemotherapy induce oxidative
stress by generation of oxygen free radicals (ROS) which might
be an alternative mechanism for their cytotoxic effect via indu-
cing apoptosis. In order to clarify the roles of antioxidants in
chemotherapy, we investigated Quercetin (3,3’,4’,5,7-pentahyd-
roxyflavone) and N-acetylcysteine (NAC) in different cell types
treated with anticancer drugs. We studied cytotoxic activity of
Topotecan alone and/or in combination with Quercetin in two
human breast cancer cell lines, MCF-7 and MDA-MB-231. We
also investigated the effect of NAC on MRP1-mediated doxoru-
bicin and vincristine cytotoxicity in Human Embryonic Kidney
(HEK293) and its MRP1 transfected (293MRP) cells. The viabil-
ity of the cells was measured using the colorimetric MTT
(3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay. Intracellular ROS was measured using fluorometric 2’,7’-
dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Our
data indicated increased oxidative status in MCF-7 and MDA-
MB-231 cells exposed to Topotecan. Treatment with Quercetin
did not inhibit ROS generation, and enhanced cytotoxicity of
Topotecan in both cells. In contrast, NAC enhanced resistance
against doxorubicine and vincristine in MRP1 overexpressing
cells. We conclude that Quercetin and NAC may have diverse
effects in the cytotoxicity of chemotheurapeutic drugs depending

After H202-induced oxidative stress, MsrA-overexpressing cells
exhibit lower protein oxidative damage than control cells indica-
ting that MsrA may play an important role in cellular defences
against oxidative stress by limiting oxidative damage to proteins.
S3.1-3
Role of oxidative stress in progressive kidney
failure
A. Tomasi
1
, S. Uggeri
2
, S. Beergamini
3
, L. Della Casa
3
,
A. Albertazzi
2
, L. Lucchi
2
and A. Iannone
1
1
Department Laboratory Medicine, Universita
`
di Modena e Reggio
Emilia, Modena, Italy,
2
Division of Nephrology, University
Hospital, Modena, Italy,

The role of the human mismatch repair
system in the processing of modified
nucleotides
N. Mojas, F. Fischer, M. Lopes, P. Cejka and J. Jiricny
Institute of Molecular Cancer Research, University of Zurich,
Zurich, Switzerland. E-mail:
The mismatch repair (MMR) system has evolved to correct
errors of DNA replication and to prevent illegitimate recombina-
tion events. However, MMR is also involved in DNA damage
signalling. In MMR-proficient cells, methylating agents such as
MNNG activate a cell cycle arrest in the second G2 phase after
treatment. The signalling cascade, which is triggered by the ATR
and Chk1 kinases, is not activated in MMR-deficient cells. We
show that the lesions triggering the cascade are not ‘mismatches’
arising through DNA modification by the methylating agents.
Nor are these lesions generated during DNA replication. Our evi-
dence suggests that the cell-cycle-arresting lesions are generated
through unsuccessful attempts by the MMR system to repair
mehylation damage, which leads to the generation of recombino-
genic intermediates. DNA recombination enables the cells to
cross the mitotic boundary, but the damage that remains kills the
cells during the subsequent cell cycle. Thus, in the absence of
recombination, the cells become hypersensitive to killing by
methylating agents and arrest already in the first G2. We are
currently searching for the structures of the recombination inter-
mediates that lead to cell death. We are also investigating the
role of the MMR system in the cytotoxicity of 5-fluorouracil
(FU), which has been reported to kill MMR-proficient cells more
efficiently than MMR-deficient ones. In our hands, isogenic
MMR-proficient and -deficient cells are equally sensitive to FU.

body maturation, but also for production and functions of
immunologically important cell types. The immunological imbal-
ances in the Ung-deficient mice may be central in the develop-
ment of lymphomas in a background of generalized lymphoid
hyperplasia.
S3.2-3
New complexities in base excision rep air and
relevance to human pathology
E. Dogliotti
Department of Environment and Primary Prevention, Istituto
Superiore di Sanita
`
, Rome, Italy. E-mail:
Base excision repair (BER) is the major mechanism for repair of
oxidative DNA damage. Xeroderma pigmentosum (XP) C is
involved in the recognition of a variety of bulky DNA distorting
lesions in NER of the genome overall. We show that XPC plays
an unexpected and multifaceted role in cell protection from
oxidative DNA damage. We provide the first in vivo evidence
that XPC is involved in the repair of 8,5’-cyclopurine 2’-deoxynu-
cleosides and of the mutagenic oxidized DNA base 8-hydroxy-
guanine (8-OH-Gua). By in vitro reconstitution experiments we
uncover a new role of XPC as co-factor in 8-OHGua cleavage by
the DNA glycosylase OGG1. XPC complex, by participating to
BER of 8-OH-Gua, might contribute to cancer prevention. A
provokative hypothesis has recently emerged in which BER
enzymes, more than controlling the integrity of the genome, may
create unintended consequences. Adaptive increases in BER
enzymes have been shown to generate microsatellite instability in
chronic inflammation. We detect up-regulation of BER genes in

DNA damage repair and the connection with
cancer and aging
J. Hoeijimakers
1
, J O. Andressoo
1
, L. Niedernhofer
2
,
I. van der Pluijm
1
, K. Diderich
1
, A. Lalai
1
, H. de Waard
1
,
G. Garinis
1
, J. Mitchell
1
, R.B. Beems
3
, H. van Steeg
3
and
G.T.J. van der Horst
1
1

are associated with an extreme clinical heterogeneity, ranging
from XP to XP combined with CS and TTD. Defects in the
NER and crosslink repair endonuclease, ERCC1/XPF, cause XP
or XP with multi-system dysfunction. Mouse models have provi-
ded important insights into the impact of the NER sub-pathways
on human health and the complex genotype-phenotype relation-
ship. XPD
TTD
mice, with a partial defect in both global and
TCR are only moderately cancer-prone, but exhibit wide spread
premature ageing. XPD
XP/CS
mutant mice are highly predisposed
to cancer, with a milder ageing phenotype. Complete repair defi-
ciency in TTDxXPA mice aggravates many premature ageing
symptoms, reducing life span to ~3 weeks. Mutations in the
ERCC1 gene induce a distinct set of accelerated ageing features,
with a rate of onset depending on the severity of the mutation.
The correlation between repair defect severity and clinical mani-
festation provides strong evidence for the DNA damage theory
of ageing. We propose that endogenous oxidative lesions com-
promise transcription, inactivate genes, and trigger apoptosis/sen-
escence inducing aging. Very cytotoxic interstrand cross-links
may also cause cell death, senescence and features of ageing. In
contrast, lesions or defects in genetic stability mechanisms caus-
ing enhanced levels of DNA damage-induced mutagenesis corre-
late with increased carcinogenesis. Various single and double
mutant mouse models including conditional mutants have been
generated and microarray analysis performed to study these pro-
cesses in a controlled, systematic manner.

3.4 Diabetes, Obesity and Metabolic Syndrome
S3.4-1
Integration of inflammatory and metabolic
signals in obesity and diabetes
G. Hotamisligil
Department of Genetics and Complex Diseases, Harvard School of
Public Health, Boston, MA, USA.
E-mail:
Obesity is associated with chronic, low grade, inflammatory
responses in metabolically active sites. This hightened inflamma-
tory status is among the key links between obesity and other
associated pathologies, i.e. type 2 diabetes. Recent studies in
our group demonstrated the involvement of the inflammatory
kinase JNK and endoplasmic reticulum (ER) stress as events
involved in both sensing and relaying stress signals and disturb-
ing metabolic homeostasis. Obesity generates conditions that
increase the demand on the ER and leads to strong activation
of JNK in insulin sensitive sites. We have shown that, in both
cultured cells and whole animals, experimentally induced ER
stress leads to IRE1±-dependent activation of JNK, serine
Abstracts
19
phosphorylation of insulin receptor substrate 1 (IRS1), and
inhibition of insulin action. Hence, we conclude that JNK acti-
vation and ER stress, integrates inflammatory and metabolic
responses, plays a crucial role in development of insulin resist-
ance and diabetes in obesity and present new targets for the
treatment of these disorders. Recently, we developed JNK- or
XBP-1 genetic gain- or loss-of function mouse models as well
as chemical tools to alter the functional capacity of ER and

cytokines, especially focusing on adiponectin is discussed with
respect to cardiovascular diseases.
S3.4-3
Nuclear receptors as therapeutic targets to
modulate the metabolic synd rome
B. Staels
UR545, Dpt d’Athe
´
roscle
´
rose, Institut Pasteur de Lille and Faculte
´
de Pharmacie, Universite
´
de Lille II, Lille, France.
E-mail:
Cardiovascular disease is significantly increased in patients with
the metabolic syndrome and type 2 diabetes. A clustering of risk
factors, including dyslipidaemia, insulin resistance, hypertension,
inflammation and coagulation disorders result in an increased
risk for cardiovascular events in these patients. The Farnesoid X
Receptor (FXR) and peroxisome proliferator-activated receptor
(PPAR) alpha are members of the nuclear receptor superfamily.
Whereas PPARalpha is activated by fatty acids, FXR has
recently been identified as a bile acid-activated nuclear receptor.
FXR not only controls bile acid synthesis, conjugation and trans-
port, but also lipid and glucose metabolism. Activation of PPAR-
alpha represents one important pathway that influences vascular
function both directly and indirectly. PPARalpha activation
induces beneficial effects not only on lipid metabolism, but also

dicarboxylic acids (MEDICA compounds) are incapable of being
esterified into lipids or b-oxidized. MEDICA-CoA specifically
binds to HNF4 with K
d
values in the nM range, and blocks its
transcriptional activity. MEDICA compounds induce hypolipide-
mia accounted for by a pronounced activation of plasma chylo-
microns and VLDL clearance complemented by robust inhibition
of liver VLDL production, transcriptional suppression of hepatic
glucose production, abrogation of non-alcoholic steatohepatitis,
activation of glucose uptake during hyperinsulinemic–euglycemic
clamp, and amelioration of ASCVD in animal models of the
Metabolic Syndrome.
S3.5-2
Why a metabolic syndrome?
P. G. Kopelman
Institute of Health, University of East Anglia, Norwich, UK.
E-mail:
The global prevalence of obesity confirms that it has become a
major threat to public health. One in five children in Europe are
overweight. The rise in obesity in young people is paralleled in
adults – obesity rates in men vary from 10% to 27%, in women
up to 38%: at least 25% of adults in US have the metabolic syn-
drome. The patho-physiological consequence of increased body
Abstracts
20
fatness is predictable from an understanding of the relationship
between insulin resistance, systemic hyperinsulinaemia and even-
tual pancreatic islet cell decompensation. There is a close associ-
ation between increasing weight, intra-abdominal fat, impaired

disease) were incubated with modified lipoproteins, enzymatically
modified LDL (E-LDL) and mildly oxidized LDL (Ox-LDL),
lipid deloaded with apoA-I and HDL3 and stimulated with reti-
noids. We applied Affymetrix DNA-microarrays, TaqMan
RT-PCR and computational biology approaches and determined
the cellular lipid content and composition using ESI-MS/MS.
(i) 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid
(ATRA) induce a nuclear receptor network strongly upregulating
apolipoproteins, scavenger receptors, steroid-27-hydroxylase,
ABCA1 and ABCG1 resulting in a potent induction of lipid
efflux. (ii) E-LDL increases cellular free cholesterol and forma-
tion of cholesterol/sphingomyelin-rich rafts in macrophages,
whereas Ox-LDL causes accumulation and cell surface expression
of ceramide and a stronger binding of apoE. (iii) ABCA1,
syntaxin-13 and flotillin-1 operate in both loading conditions
with different response rates and downstream signalling involving
an inward rectifying K+-channel along the ABCA1/AP-3
secretory pathway.
3.6 Oncogenes and Tumor Suppressors
S3.6-1
Molecular pathogenesis of liv er ca ncer
M. Ozturk
Department of Molecular Biology and Genetics, Bilkent University,
Ankara, Turkey. E-mail:
Liver cancer (hepatocellular carcinoma; HCC) is one of the 10
most common cancers worldwide. The major etiology of liver
cancer is cirrhosis that is observed mostly in old people with a
chronic history of viral (HBV and HCV) and/or non-viral hepa-
titis. Liver cancer appears as a heterogeneous disease at the
molecular level, probably because of the fact that different etio-

RSK-2 in vitro. We have been studying the role of Fos phos-
phorylation in OS development and found that tumors do not
progress in the absence of RSK-2. We speculate that the effect
on tumor development is due to increased stability of phosphor-
ylated Fos protein. When the Fos–/– osteopetrotic mutant was
crossed into the p53 background, double mutant mice specifically
develop rhabdomyosarcomas (RMS). Re-expressing Fos in
Abstracts
21
mutant muscle tumor cell lines induced apoptosis implying a
novel function of the oncogene Fos as a potential tumor suppres-
sor. Recent data regarding the target genes of Fos in OS and
RMS cells obtained by microarray analyses will be presented.
The functions of the Jun family members c-Jun and JunB have
been analyzed by conditional mutagenesis. Whereas c-Jun was
found to function as an oncogene in liver cancer, JunB acts as a
tumor suppressor gene in a mouse model of myeloid leukemias.
The combined inducible deletion of c-Jun and JunB in the skin
of adult mice causes a psoriasis-like phenotype strongly resem-
bling the human disease.
S3.6-3
Chromatin modifications and their function
C. Neilson, H. Santos-Rosa, A. Bannister and T. Kouzarides
Gurdon Institute, Tennis Court Road, Cambridge, UK
Covalent modifications of histones regulate many biological pro-
cesses, the most characterized of which is transcription. We are
interested in the molecular mechanisms by which these modifica-
tions function. Our focus recently has been the analysis of his-
tone methylation at lysines and arginines. We now find that one
site of lysine methylation on histone H3 is under the control of

tion and characterization of yeast mutants blocked at various sta-
ges of transport pathways are an invaluable method for
unravelling the molecular details of vacuolar and lysosomal traf-
fic. The activities of lysosomes are critical to many essential cellu-
lar processes. Such diseases as Huntington’s, Alzheimer’s,
Parkinson’s, tumor invasion and metastasis are all associated
with altered lysosomal trafficking. Our research concentrates on
Ccz1p, the yeast homolog of mammalian HPS4. The mutated
HPS4 protein is responsible for Hermansky–Pudlak syndrome
type 4.
S3.7-2
Sorting, targeting and delivery in mammalian
post-golgi pathways
P. Luzio
Cambridge Institute for Medical Research, University of
Cambridge, Cambridge, UK. E-mail:
The targeting and localization of proteins to the correct intracel-
lular organelle or cell surface domain is essential for their physio-
logical function. In the post-Golgi secretory and endocytic
pathways proteins are targeted to many locations, with vesicular
traffic being the major mechanism of transport between donor
and acceptor compartments. Disruption of membrane traffic
events is the molecular basis for many hereditary, autoimmune
and other diseases. In addition, several microbial pathogens sub-
vert normal membrane traffic and some viruses are able to hijack
membrane traffic machinery to avoid recognition by the immune
system and/or for the purposes of viral budding. In general,
membrane traffic defects in hereditary disease can be subdivided
into abnormalities of cargo or of traffic machinery. The former
are by far the most common. Mutations in cargo or machinery

bly via the back-fusion of the internal vesicles with the endosome
limiting membrane, in a process regulated by the endosomal lipid
Abstracts
22
lysobisphosphatidic acid under the control of Alix/Vps31p and
by the phosphatidylinositol-3-phosphate via Snx16. We propose
that proteins and lipids, which are transported from within late
endosomes to other cellular destinations, also use this intra-endo-
somal trafficking pathway. The functions of other effectors in
this pathway will be discussed.
3.8 Neurodegenerative Disorders
S3.8-1
Tau and tauopathies: tau phosphorylation and
tau assembly
J. Avila
Centro de Biologı
´
a Molecular ‘Severo Ochoa’, Facultad de
Ciencias, Campus de Cantoblanco, Universidad Auto
´
noma de
Madrid, Madrid, Spain. E-mail:
The role of tau protein in different tauopathies, mainly in Alzhei-
mer’s disease, will be commented. Tau, a microtubule associated
protein, can aberrantly polymerize, in phosphorylated form,
yielding the paired helical filaments found in the brain of Alzhei-
mer’s disease patients. Tau assembly can be reproduced in vitro
by mixing tau protein with polymerization-inducers like heparin
or Coenzyme Q0, being the assembly of phosphotau facilitated in
the presence of Co.Q0. Polymerization of tau has been also

and alpha-synuclein aggregation may be related processes in
inclusion formation. However, alpha-syn can be degraded by the
proteasome in a ubiquitin independent manner and not all LBs
contain ubiquitin. These results suggest that alpha-syn ubiquityla-
tion is not a primary event for alpha-syn fibrillization. This is in
agreement with the observation that transgenic mouse models of
alpha-syn accumulation show very little ubiquitin staining. At
present is clear that alpha-synuclein is linked to neurodegenera-
tion in PD but the mechanisms and significance of its aggregation
remain to be determined.
S3.8-3
Friedriech ataxia and other recessive ataxias
M. Koenig
IGBMC (CNRS-INSERM-ULP), Illkirch, Strasbourg, France
Progressive recessive ataxias are severe disabling neurodegenera-
tive diseases that affect the cerebellum and/or the spinocerebellar
and sensory tracts of the spinal cord. Thirteen genes causing
non-metabolic degenerative ataxias have been identified in the
recent years. The majority of them encodes for either mitochond-
rial or nuclear proteins. The nuclear proteins are ATM, MRE11,
tyrosyl-DNA phosphodiesterase 1 (TDP1), aprataxin and sena-
taxin, respectively. While the functions of ATM and MRE11 in
DNA double strand break repair and of TDP1 immediately
upstream of nucleotide excision repair are now well established,
the precise function of aprataxin and senataxin remain elusive.
The mitochondrial proteins are frataxin, ABC7, twinkle and
DNA polymerase gamma (POLGA). Twinkle and POLGA are
involved mitochondrial DNA maintenance and frataxin and
ABC7 are involved in Fe-S cluster biogenesis. Complete loss of
function of only ATM, aprataxin and senataxin results in reces-