Tài liệu Báo cáo khoa học: The role of antioxidants in the cytotoxicity of chemotherapeutic drugs pptx

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

on their other pharmacological properties which may predomin-
ate their antioxidant effects.
S3.1-2
Oxidized protein degradation and repair in
ageing and oxidative stress
B. Friguet
Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement,
Universite
´
Denis Diderot – Paris 7, Paris, France.
E-mail:
Cellular ageing is characterized by the accumulation of oxidatively
modified proteins and oxidized protein buildup with age may be
due to increased protein damage and/or decreased elimination of
oxidized protein. Since the proteasome is in charge of protein turn-
over and removal of oxidized protein, its fate during ageing has
received special attention, and evidence has been provided for an
age-related impairment of proteasome function. In fact, depending
on the cellular system investigated, the loss in proteasome activity
observed during ageing and upon oxidative stress appears to be
due to either or both: (i) decreased proteasome expression and con-
tent, (ii) inactivation upon modification of proteasome subunits
and (iii) formation of inhibitory proteins. However oxidized pro-
teins can be eliminated through degradation but also repair. Oxid-
ized protein repair is limited to the reversion of few modifications
such as the reduction of methionine sulfoxide by the methionine
sulfoxide reductase (Msr) system. We have previously shown that
Msr activity is impaired during ageing. To analyse the relationship
between oxidative stress, protein oxidative damage and Msr, MsrA
has been overexpressed in immortalized WI-38 human fibroblasts.

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,

3
Department of Biomedical Sciences,
Modena, Italy. E-mail:
The leading cause of morbidity and mortality in patients with
end stage renal disease (ESRD), who are maintained on regular
dialysis treatment, is cardiovascular disease. Increased free radical
production and oxidative stress promote the atherosclerotic pro-
cess. The association between haemodialysis and activation of
circulating monocytes, both spontaneous and endotoxin-induced
release of TNF-alpha are well known. In our research spanning
many years, as well as in many other laboratories, there has been
a massive attempt to identify reliable biomarkers of oxidative
stress. Initially our attention was drawn on direct markers of oxi-
dative stress such as malondialdehyde (MDA) and conjugated
dienes. More recently, high plasma level of homocysteine, which
is considered a putative cardiovascular risk factor by inducing
endothelial dysfunction and serum C-reactive protein (CRP),
which contributes to monocytes recruitment in the atherosclerotic
lesion have been adopted as possible markers of atherosclerotic
progression in ESRD patients. It has been postulated that ESRD
patients have also a decreased ability to withstand oxidative
stress due to a reduced antioxidant capacity, which was also
investigated as a putative biomarker. Recently, we have tried to
define novel markers of oxidative stress applying proteomic pro-
filing technologies, employing both 2-D gel electrophoresis - mass
spectrometry and high throughput SELDI technology.
Abstracts
17
3.2 DNA Damage Processing
S3.2-1

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.

S3.2-2
Base excision repair proteins in cancer
prevention and acquired imm une responses
H. E. Krokan, M. Akbari, J. Pen
˜
a Diaz, B. Kavli, M. Otterlei,
M. Ericsson, H. Aarset, S. Andersen and G. Slupphaug
Department of Cancer Research and Molecular Medicine,
Norwegian University of Science and Technology, Trondheim,
Norway. E-mail:
Base excision repair repairs DNA by removing damaged bases
and filling in the gap with correct nucleotides. Uracil-DNA gly-
cosylase encoded by the UNG-gene is also required for somatic
hypermutation and class switch recombination. Furthermore,
Ung-deficient mice have lymphatic hyperplasia and several-fold
increased risk of developing B-cell lymphomas. Humans deficient
in UNG2 activity display the hyperIgM syndrome (HIGM) phe-
notype, including B-cell hyperplasia. We find that lymphomas in
mice are of follicular (FL) and diffuse large B-cell type (DLBCL)
type. All FLs and 75% of the DLBCLs were monoclonal while
25% were biclonal. Monoclonality was also observed in hyperpl-
asia, and could represent an early stage of lymphoma develop-
ment. Lymphoid hyperplasia was observed as a significant
increase of splenic B-cells. Furthermore, loss of Ung also causes
a reduction of T-helper cells, and 50% of the young Ung–/– mice
investigated have no detectable NK/NKT-cell population in their
spleen. In addition, production of the cytokines interferon c,
interleukin-6 and interleukin-2 is clearly different in wild type
and in Ung-deficient mice. Thus, Ung-proteins have important
functions in the immune system, not only in the process of anti-

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

association with microsatellite instability in an inflammation rela-
ted cancer (i.e. stomach cancer). Future research should address
the mechanistic basis of this phenomenon that might be of great
relevance in human pathology.
3.3 DNA Repair in Health, Disease and Aging
S3.3-1
DNA repair deficiencies in human premature
aging
A. Bohr
Laboratory of Molecular Gerontology, National Institute on Aging,
NIH, Baltimore, Maryland, USA. E-mail:
There are a number of human syndromes where the patients
appear much older than their actual chronological age. These
conditions are interesting model systems for the study of aging.
For many of these conditions, the responsible gene has been
cloned, identified, and characterized, thus enabling studies of its
molecular function. We are interested in the category of human
premature aging conditions that are defective in recQ helicases
and associated with genomic instability. These conditions include
Werner syndrome, Rothmund–Thompsons syndrome and others.
The molecular function of the recQ helicases will be discussed and
evidence presented that these proteins participate in an intergral
Abstracts
18
manner in the DNA repair process. Another premature aging
condition, also associated with DNA repair defects, Cockayne
syndrome, will also be discussed. These studies lend credibility to
the notion that aging is associated with defects in DNA repair
that then lead to genomic instability.
S3.3-2

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

MGC, CBG, Department of Cell Biology and Genetics, Erasmus
University, Rotterdam, The Netherlands,
2
University of Pittsburgh
Cancer Institute, Hillman Cancer Center, Pittsburgh, PA, USA,
3
RIVM, Bilthoven, The Netherlands
The physico-chemical constitution of our genes does not guaran-
tee life-long stability and proper function. A perplexing diversity
of lesions is continuously induced in our genes ranging from sin-
gle and double strand breaks, to chemical alterations of nucleo-
tides including adducts, as well as inter- and intrastrand DNA
crosslinks. They arise from ubiquitous, noxious exogenous agents
(UV- and X-radiation, chemicals), natural metabolites produced
by respiration (notably reactive oxygen species) and from intrin-
sic chemical instability of DNA. To protect the vital genetic
information from the deleterious effects of DNA injury a net-
work of genome care-taking mechanisms has evolved of which a
set of DNA repair systems constitute a key component. The
clinical impact of the DNA damage problem is still strongly
underestimated. Nucleotide excision repair (NER) removes a
wide range of helix-distorting lesions in a complex ‘cut and
patch’ reaction. There are two sub-pathways: global genome
NER critical for preventing mutations and transcription-coupled
repair (TCR) counteracting the cytotoxic effects of DNA injury.
Inherited NER defects are associated with sun (UV) hypersensi-
tive syndromes, including xeroderma pigmentosum (XP, highly
cancer-prone), and the severe neuro-developmental conditions
Cockayne syndrome (CS) and trichothiodystrophy (TTD). Muta-
tions in the multifunctional NER/TCR XPB and XPD helicases

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.

S3.3-3
DNA double strand repair and its relationship
to human disease
A. Jeggo
Genome Damage and Stability Centre, University of Sussex, UK.
E-mail:
DNA is constantly subject to damage. Cells utilise multiple DNA
damage response pathways to maintain genomic stability. A DNA
double strand break (DSB) represents a particularly critical DNA
lesion that can lead to cell death or carcinogenesis. DSBs are
introduced during immune development to generate genetic diver-
sity. Curiously, cells exploit the mechanism normally used to
maintain genomic stability after exposure to DNA damaging
agents to create diversity during immune development. This path-
way is DNA non-homologous end-joining (NHEJ), a process of
DSB repair. To date, three genetic diseases with defects in NHEJ
have been identified. As expected from the role of NHEJ in
immune development, such patients display immunodeficiency.
Additionally, since a DSB is induced following exposure to ion-
izing radiation, such patients and cell lines derived from them dis-
play marked radiosensitivity. LIG4 syndrome is a disorder
conferred by mutations in DNA ligase IV, the enzyme that effects
rejoining during NHEJ. LIG4 patients display pancytopaenia,
microcephaly and developmental delay. RS-SCID1 patients have
mutations in Artemis, a protein that processes DNA ends prior to
rejoining. More recently, a further disorder has been described,
which is associated with marked immunodeficiency and mild
developmental delay. The protein defective in this disorder is also
a NHEJ protein. The distinct clinical features of the patients will
be discussed in the context of the protein function.

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

JNK activity in whole animals to demonstrate the causal link
between these pathways and metabolic disease. Recent activities
in these areas and identification of cellular and molecular tar-
gets and development of new genetic models are underway and
will be discussed here.
S3.4-2
The metabolic syndrome and adipocytokines
Y. Matzuzawa
Department of Internal Medicine and Molecular Science, Graduate
School of Medicine Osaka University, Osaka, Japan
Visceral fat accumulation plays crucial roles in the development
of cardiovascular disease as well as diabetes mellitus, hyperlipide-
mia and hypertension and so-called metabolic syndrome. Adipo-
cyte functions has been intensively investigated in the past
10 years, and adipocytes have been revealed to act as endocrine
cells which secrete various bioactive substances.(adipocytokine)
Among adipocytokines, tumour necrosis factor-a, plasminogen
activator inhibitor type 1, heparin binding epidermal growth fac-
tor-like growth factor and visfatin (newly found in visceral fat)
are produced in adipocytes as well as other organs, and may con-
tribute to the development of vascular diseases. On the contrary
to these adipocytokines, adiponectin, an adipose-tissue-specific,
collagen-like protein, has been noted as an important antiathero-
genic and antidiabetic protein, or as an anti-inflammatory pro-
tein. The functions of adipocytokine secretion might be regulated
dynamically by nutritional state. Visceral fat accumulation causes
dysregulation of adipocyte functions, including oversecretion of
TNF-a, PAI-1 and heparin HBEGF and hyposecretion of adipo-
nectin, which results in the development of a variety of metabolic
and circulatory diseases. In this review, the importance of adipo-

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

influences glucose homeostasis, endothelial function and vessel
wall inflammation. PPARalpha agonists in clinical use, such as
fibrates, may alter the process of atherosclerosis, especially in
subjects with the metabolic syndrome and type 2 diabetes. This
presentation will highlight the molecular mechanisms of FXR
and PPARalpha action in the prevention and treatment of the
metabolic syndrome and atherosclerosis.
3.5 Lipid Related Disorders and Atherosclerosis
S3.5-1
Treatment of the metabolic syndrome by
targeting hepatocyte nuclear factor-4a by
MEDICA
R. Hertz and J. Bar-Tana
Department of Human Nutrition and Metabolism, Hebrew
University Medical School, Jerusalem, Israel.
E-mail:
Dyslipoproteinemia, NIDDM and essential hypertension often
coexist, and synergistically promote ASCVD. The Metabolic Syn-
drome that comprises these diseases is driven by nutritional sat-
urated fat. Transcriptional modulation of liver genes involved in
lipoprotein production and their plasma clearance (apoB, apoC
III,MTP), hepatic glucose production (G6Pase, PEPCK), blood
coagulation and others is dominated by hepatocyte nuclear fac-
tor-4a (HNF4). Activation/suppression of HNF4 transcriptional
activity by its acyl-CoA ligands as function of their chain length,
unsaturation or extent of substitution, correlates with the repor-
ted effects of long chain fatty acid nutrients in the Metabolic
Syndrome context. Hence, HNF4 serves as cellular sensor for
long chain fatty acids, and targeting HNF4 may offer means for
targeting the Syndrome. Long chain methyl-substituted a,x-

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

glucose tolerance and deleterious alterations in lipid profile that
are associated with alterations in pro-inflammatory markers
CRP, interleukin-6 and TNF-a. Unsurprisingly these changes are
clinically characterised by type 2 diabetes, hypertension and
accelerated coronary artery disease. The INTERHEART trial
from 52 countries confirms the detrimental impact of abdominal
obesity on rates and outcome from myocardial infarction. Of
particular worry is that these chronic diseases begin in young
people. The great expectation of new therapies to effectively treat
obesity has not been fulfilled. Thus a major challenge for 21st
century is the prevention of obesity, and the metabolic syn-
drome, through populations adopting healthier lifestyles with
health-promoting social marketing becoming an important
prevention tool.
S3.5-3
Transcriptional regulatory networks in the
ABCA1/AP3-pathway determine lipid
trafficking in macrophages
G. Schmitz, M. Grandl, G. Liebisch, A. Boettcher, E. Orso and
T. Langmann
Institute of Clinical Chemistry and Laboratory Medicine,
University Hospital Regensburg, Regensburg, Germany.
E-mail:
ABCA1 controls cellular cholesterol and choline-phospholipid
release and is required for the generation of pre-b-HDL. Our
goal was to characterize gene regulatory networks and ABCA1
associated lipid pathways in human macrophages in cardiovascu-
lar disease and under atherogenic and nutritional stimulation.
Macrophages from healthy apoE3 donors and patients with
monogenetic lipid disorders (ABCA1 deficiency, Niemann–Pick C

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-

logical factors contribute to the development of these cancers,
depending on geographical locations. The most shared genetic
alterations of liver cancers are mutations affecting p53, b-catenin
and Axin1 genes together with the epigenetic or mutational inac-
tivation of the p16INK4a gene. Most liver cancers also display
hTERT telomerase induction whose mechanisms remain elusive.
Gene expression profiling data show deregulated expression of
genes involved in proliferation, apoptosis, DNA repair, protein
synthesis and liver-specific functions. Taken together these
observations provide evidence for critical involvement of senes-
cence- and differentiation-regulating events, in addition to well
established cell cycle and apoptosis regulatory changes in liver
cancer. Among others, ‘p53-retinoblastoma tumor suppressing
network’ and ‘canonical Wnt-b-catenin signaling’ appear to play
a central role in hepatocellular carcinogenesis. New experimental
data incriminating senescence- and differentiation-related
molecular changes in the pathogenesis of liver cancer will also
be presented.
S3.6-2
AP-1 (Fos/Jun) and cancer
E. F. Wagner
IMP, Research Institute of Molecular Pathology, Vienna, Austria.
E-mail:
AP-1(Fos/Jun) proteins are prototypic oncogenes regulating cell
proliferation, differentiation and cell transformation in various
organs. We are investigating the specific functions of Fos pro-
teins, e.g. Fos, Fra-1 and Fra-2 in development and disease.
Transgenic mice expressing Fos develop osteosarcomas (OS),
whereas mice lacking Fos are osteopetrotic. It is well established
that Fos is phosphorylated in the C-terminus by kinases such as

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

proline isomerization. Prolines can exist in a -cis or -trans confor-
mation. Enzymes exist (peptidyl prolyl isomerases), which can
convert prolines from one conformation to the other. We have
found that a yeast protein FPR4 is an enzyme that can isomerize
specific prolines in histone H3 and that this isomerization affects
lysine methylation of H3. The cross-talk between proline isomeri-
zation and lysine methylation has a consequence in the regulation
of transcription of certain genes in yeast. These data define a
novel non-covalent histone modification that regulates gene
expression.
3.7 Intracellular Trafficking in Health and Disease
S3.7-1
The yeast vacuolar protein sorting pathway
as an experimental model for lysosomal
trafficking
J. Rytka
Department of Genetics, Institute of Biochemistry and Biophysics,
Warsaw, Poland. E-mail:
Compartmental organization of eucaryotic cells requires mecha-
nisms for precise sorting and distribution of proteins and metab-
olites to their target organelles. Due to impressive conservation
of intracellular traffic across phyla, a great deal of the current
knowledge on the mechanisms of integration of these processes
into the cellular interaction network has been acquired from
studies on Saccharomyces cerevisiae. A good example is vacuolar/
lysosomal transport, as the yeast vacuole is analogous to the
mammalian lysosome, and trafficking to both organelles is
remarkably conserved. Defects in vacuolar function lead to pro-
tein mislocalization, disturbances in ion homeostasis, affect such
complicated processes as osmoregulation and sporulation. Isola-

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

proteins can result in inappropriate targeting and consequent cel-
lular malfunction. This will be illustrated by reference to muta-
tions in protein targeting motifs in cargo trafficking to/from the
plasma membrane and to lysosomes. An example of traffic
machinery in which abnormalities of function can result in dis-
ease is that for formation of multivesicular bodies on the endocy-
tic pathway. Components of this machinery have been implicated
in viral disease, cancer and interacting with spastin, a protein
encoded by a hereditary spastic paraplegia gene.
S3.7-3
How vesicular stomatitis virus hijacks the
endocytic pathway to infect cells
P. P. Luyet, I. Le Blanc and J. Gruenberg
Biochemistry Department, University of Geneva, Geneva,
Switzerland. E-mail:
Like other enveloped viruses, vesicular stomatitis virus (VSV)
infects cells through the endocytic pathway. VSV infection
requires transport beyond early endosomes, hence to multivesicu-
lar endosomes (MVBs), and then the transcriptionally active
nucleocapsid is delivered to the cytoplasm after low pH-mediated
fusion of the viral envelope with endosomal membranes. We
observed that VSV envelope fusion and nucleocapsid release into
the cytosol occur sequentially at two successive steps of the endo-
cytic pathway – and not concomitantly as it was thought. Our
results indicate that VSV envelope undergoes fusion with the
membrane of intra-endosomal vesicles contained within MVBs,
thereby releasing the nucleocapsid into the lumen of these vesi-
cles. Then, the capsid uses these intra-endosomal vesicles as a
Trojan horse to reach late endosomes, from where the nucleocap-
sid is delivered to the cytoplasm. This latest step occurs presuma-

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

mimified in vivo by using transgenic mouse models. In these
models, human tau, bearing some of the mutations found in
patients with a tauopathy, FTDP-17, was expressed. Addition-
ally, mutations on APP and/or PS-1 proteins will facilitate tau
phosphorylation by kinases like GSK3 in mouse models. Thus, a
transgenic mouse model overexpressing GSK3 was also charac-
terized. In some of these mouse models, a link between tau phos-
phorylation and tau assembly has been established. Finally, the
possible toxic effect of phosphotau or tau aggregates will be dis-
cussed.
S3.8-2
Alpha-synucleinopathies
M. G. Spillantini
Department of Clinical Neurosciences, Brain Repair Centre,
University of Cambridge, Cambridge, UK.
E-mail:
Parkinson’s disease (PD) and dementia with Lewy bodies (DLB)
are characterized neuropathologically by the presence of Lewy
bodies (LBs) which are intracytoplasmic filamentous inclusions.
Alpha-synuclein (alpha-syn) has been implicated in the pathogen-
esis of PD based on the presence of missense mutations and
abnormal copy number of its gene in familial cases as well as for
its presence in LBs. Alpha-syn constitutes also the major compo-
nent of the glial cytoplasmic inclusions found in multiple system
atrophy. These diseases are therefore grouped under the name of
‘alpha-synucleinopathies’. The mechanism of alpha-syn aggrega-
tion in PD and DLB is not yet clear. The presence of ubiquitin
in LBs and the association of mutations in the ubiquitin-protea-
some pathway with early-onset Parkinsonism, has led to the sug-
gestion that abnormalities in ubiquitin-dependent degradation

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-

sive ataxia. Ataxia mutations in the other genes are hypomor-
phic, as their complete loss function would not be viable.
Friedreich ataxia is caused by intronic trinucleotide expansions
which cause severely reduced expression of frataxin and subse-
quent mitochondrial and cytosolic Fe-S protein deficiency. We
have used conditional and inducible knock-out strategies to artifi-
cially recreate the partial frataxin deficiency in mouse models that
present with several features of the human disease.
Abstracts
23