Therefore, we investigated the expression of the two estrogen receptors, ERa and ERb and their influence on regulation of GLUT4, and its associated structural protein, caveolin-1, in mous
Trang 11 Signal Transduction
1.1 Integration of Metabolism and Survival
S1.1-1
Regulation of metabolism by estrogen
signaling
J Gustafsson, R Rodrigo and W Warner
Department of BioSciences and Nutrition, Novum, Karolinska
Institutet, Stockholm, Sweden
E-mail: jan-ake.gustafsson@mednut.ki.se
Estrogen is known to influence glucose homeostasis but the role
of estrogen receptors in muscle glucose metabolism is unknown
Therefore, we investigated the expression of the two estrogen
receptors, ERa and ERb and their influence on regulation of
GLUT4, and its associated structural protein, caveolin-1, in
mouse muscle ERa and ERb are co-expressed in the nuclei of
most muscle cells and their levels were not affected by absence of
estradiol (in aromatase knockout, ArKO, mice) GLUT4
expres-sion on the muscle cell membrane was not affected by loss of
ERb but was extremely reduced in ERa–/–mice and elevated in
ArKO mice Upon treatment of ArKO mice with the ERb
agon-ist, DPN, GLUT4 expression was reduced Caveolin-1 expression
was higher in ArKO mice and lower in ERb–/–and ERa–/–mice
than in WT littermates GLUT4 and caveolin-1 were colocalized
in WT and ArKO mice, but not in ERb–/– and ERa–/– mice
Thus, ERa is necessary for GLUT4 expression while ERb has a
suppressive role Both ERb and ERa are necessary for optimal
caveolin-1 expression Taken together, these results indicate that
reduction in GLUT4 is a plausible explanation for the insulin
resistance observed in ERa)/) mice and that colocalization of
caveolin-1 and GLUT4 is not an absolute requirement for muscle
glucose metabolism
S1.1-2
Nutritional sensing in the MTOR/S6K1
pathway in the development of obesity and
diabetes
G Thomas
Genome Science, Genome Reserach Institute, Cincinnati, OH,
USA E-mail: thomasg4@uc.edu
Earlier we showed that nutrient overload induces S6K1
activa-tion, which suppresses insulin-induced class 1 PI3K signaling
leading to insulin resistance Counter to the prevailing view, our
recent studies show that the nutrient, e.g amino acid (AA), input
to S6K1 is not mediated by the tumor suppressor TSC1/TSC2 or
its target the oncogene Rheb In the absence of TSC1/2, we find
S6K1 is activated and refractile to insulin, but still regulated by
AAs However this is not the case for Rheb, as siRNA
knock-down of Rheb protein levels blocks both the insulin and AA input to S6K1 Nonetheless, withdrawal of AAs has no effect on Rheb-GTP levels, but triggers S6K1 inactivation, indicating Rheb-GTP is necessary but not sufficient for AA induced S6K1 activation This suggested the AA input to S6K1 is on a parallel pathway to the TSC1/2-Rheb axis That wortmannin, a class 1 PI3K inhibitor, blocks AA-induced S6K1 activation but AAs do not induce PKB activation, suggested a novel wortmannin sensi-tive signaling component mediated the AA input to S6K1, which
we identified as class 3 PI3K, hVps34 In brief, ectopic expression
of hVps34 drove S6K1 activation, an effect blocked by hVps34 siRNAs Moreover, AAs increase hVps34 activity and the pro-duction of PI3P, which serves to recruit FYVE or Px containing proteins to endosomes to build signaling platforms Consistent with this, S6K1 activation is attenuated by ectopic expression of
a dominant interfering cDNA containing two FYVE domains
S1.1-3 Regulation and function of the WNK1 and WNK4 protein kinases
D Alessi MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK E-mail: d.r.alessi@dundee.ac.uk
The WNK family of protein kinases were originally identified as enzymes that lack a conserved Lys residue normally found in subdomain II of the catalytic domain Subsequent studies, identi-fied mutations in the genes encoding WNK1 and WNK4, in fam-ilies with an inherited hypertension and hyperkalaemia disorder, called PHAII/Gordon1s syndrome WNK isoforms are large pro-tein kinases (WNK1-2382 residues, WNK4-1243 residues), in which the catalytic domain is located at the N-terminus Apart from two putative coiled-coil domains, the remainder of the WNK polypeptides possess no obvious structural features Muta-tions in the WNK1 gene found in PHAII subjects, are deleMuta-tions
in intron-1, which elevate the expression of the WNK1 protein, indicating that hypertension could result from increased expres-sion of WNK1 Consistent with this notion, mice lacking one allele of WNK1, had lower blood pressure Thus far, the muta-tions in the WNK4 gene found in PHAII subjects, lie distal to both of the putative coiled-coil domains Little is known about the molecular mechanism by which WNK isoforms regulate cel-lular processes In my talk I will present our recent results that indicate that the WNK protein kinases are activated by osmotic stress and phosphorylate and activate protein kinases of the STE20 family, termed STE20/SPS1-related Proline-Alanine-rich Kinase (SPAK) and the Oxidative Stress Response kinase-1 (OSR1)
Trang 21.2 Integration of Defence and Survival
S1.2-1
Immuno-receptors recognition of antigens: the
T-cells case
D Gakamsky and I Pecht
Immunology, Weizmann Institute of Science, Rehovot, Israel
E-mail: israel.pecht@weizmann.ac.il
Resolution of the interaction mechanism between the T-cell
recep-tor (TCR) and its ligands, MHC peptide (MHCp) complexes, is
still a central problem in immunology These are characterized
rel-atively high specificity and modest affinities 3-D structures of
TCR-MHCp complexes revealed marked changes are observed,
primarily in the TCR, upon ligand binding the Moreover, both
time course and affinity measurements of TCR-MHCp interactions
obtained using the Surface Plasmon Resonance (SPR) method
established the 0.1–100 mM affinity range for the interactions of
the different TCR-MHCp couples examined These were
inter-preted to be a result of relatively slow association (103–105/M/s)
and fast complex dissociation (0.1–100/s) rate constants However,
practically all values for the association rate constants were
calcu-lated by assuming the operation of a reversible, single step reaction
mechanism This conflicts with the crystallographic evidence for
conformational changes taking place in the TCR upon interaction
with its ligands, and calls for considering a more complex
mechan-ism for MHCp-TCR interaction Indeed, an in-depth analysis of
kinetic data obtained by SPR as well as an independent, FRET
based study of TCR-MHCp interactions shows that these fit well
two different, though related mechanisms, both involving
conform-ational transitions coupled to the binding process
S1.2-2
Amplification and control of B cell antigen
receptor signaling
M Reth and Y Kulathu
Institute for Biology III and Max-Planck-Institute for
Immunobiology, Freiburg, Germany
E-mail: reth@immunbio.mpg.de
Signal transduction from the B cell antigen receptor (BCR)
involves not only the activation of BCR-proximal protein
tyro-sine kinases (PTK) like Syk and Lyn, but also the inhibition of
protein tyrosine phosphatase (PTP) like SHP-1 through the
oxi-dation of its catalytic active cysteine For signaling it is therefore
important that the BCR is localized to areas of high production
of oxidants and low PTP activity Under these condition Syk is
released from auto-inhibition by binding to the BCR and can
amplify the BCR signal through ITAM phosphorylation The
activation of Syk and its release from auto-inhibition involves at the same time a conformational change and its translocation to the plasma membrane via binding of its tandem SH2 domain to phosphorylated ITAMs To uncouple these two important events
in Syk activation, we have targeted Syk permanently to the mem-brane and tested a panel of Syk mutants for their function These data show that several parts of the Syk molecule are involved in the auto-inhibition control Once resuming an open conforma-tion, Syk can recruit other signalling molecules thus facilitating their phosphorylation and the propagation of the BCR signal These studies, which are conducted in a novel signalling pathway reconstitution system with S2 Schneider cells, give a more detailed inside into the mechanistic aspect of BCR signaling
S1.2-3 Temporal regulation of cytolytic T cell programming
G Verdeil, J Chaix, A Guimezanes, N Auphan-Anezin and
A Schmitt-Verhulst Centre d’Immunologie de Marseille-Luminy, CNRS-INSERM-Univ de la Me´diterrane´e, Marseille, France
E-mail: verhulst@ciml.univ-mrs.fr Naı¨ve CD8 T cells may develop into cytolytic effectors following engagement of their clonally expressed antigen receptor (TCR), thus committing the T cells to a program that includes clonal expansion, differentiation, and long-term survival Incomplete differentiation has been reported, however, in particular for weak tumor antigens To understand the molecular basis for the plasti-city in CD8 T cell differentiation, we analyzed the response of monoclonal naı¨ve CD8 T cells to antigenic ligands of different affinity A transient versus stable pattern of gene expression was induced by weak versus strong TCR agonists, respectively For these transcripts, including those of the IL-2 receptor (IL-2R), cytolytic effector molecules (granzyme B, perforin) and TNF-R family members (GITR, OX40, 4-1BB), expression was sustained upon addition of IL-2, an effect mimicked by expression of an active form of STAT5 Epigenetic modifications of histones asso-ciated with relevant genes were delayed in response to weak agonists Addition of IL-2 failed to shorten this delay, but induced STAT5 binding to granzyme B and 4-1BB promoters A delayed and weak ERK1/2 activation in response to weak TCR agonists was correlated with a delayed onset of proliferation We thus identified the TCR, IL-2R and members of the TNF-R fam-ily, as sequential contributors to the proliferation, differentiation and survival of effector CD8 T cells, involving ERK, STAT5 and NF-kB signaling pathways, respectively
1.3 Rhythmic Signals: The Setting of Biological Time
S1.3-1
An ultradian clock in yeast: metronome for
intracellular coherence
D Lloyd1and D Murray2
1Microbiology Group (Biosi 1) Cardiff University, Cardiff, Wales,
UK,2The Systems Biology Instititue, Shinanomachi Research
Park, Keio University School of Medicine, Shinjuku-ku, Tokyo,
Japan E-mail: lloydd@cf.ac.uk
Self-synchrony of a densely-populated culture of Saccharomyces
cerevisiaein aerobic continuous culture under controlled
condi-tions of stirring, temperature and pH reveals an ultradian (with a period of about 40\,min) clock The high amplitude respiratory oscillation is robust in that it can be maintained over extended times(up to months) and its period is compensated for tempera-ture and nutrtional changes Extensive studies of metabolism and micromolecular synthesis indicate that an oscillatory alternation between oxidative and reductive states underlies the observed outputs (mitochondrial respiratory activity, nicotinamide indica-ated by nucleotides reduced glutathione content, mitochondrial inner membrane potential and levels of transcripts) Ultra-structural changes also occur during redox state transitions;
Trang 3mitochondrial matrix volume indicates energization states We
suggest that this timekeeping mechanism serves as the universal
coordinating time-base upon which all intracellular reactions,
pathways, processes and events are dependent for synchronous
coordination and coherence Circadian control systems that
match internal activities to environmental changes may use
ultra-dian rhythyms as their bases The observables of the culture
pro-vide us with a unique insight into the temporal organization of
the living state in every cell and at all times
Lloyd D and Murray D B Ultradian metronome: timekeeper
for orchestration of cellular coherence TIBS 30: 373–377
S1.3-2
The mammalian circadian timing system: from
cyclic transcription to rhythmic physiology
B Kornmann, F Gachon, H Reinke, J Ripperger,
G LeMartelot and U Schibler
Department of Molecular Biology and NCCR Frontiers in
Genetics, University of Geneva, Geneva, Switzerland
E-mail: ueli.schibler@molbio.unige.ch
Circadian pacemakers were originally believed to exist only in a
few specialized cell types, such as SCN neurons However, in
recent years, this view has been challenged by the discovery that
self-sustained and cell-autonomous circadian clocks are operative
in most peripheral organs We are studying how such peripheral
oscillators can drive overt cycles in physiology The PAR basic
leucine zipper (PAR bZip) proteins DBP, HLF, and TEF serve
as clock output regulators Their expression is governed
directly by cellular circadian oscillators In turn, the rhythmic
accumulation of these proteins results in daily cycles of target
gene expression Transcriptome profiling studies with PAR bZip
deficient knockout mice revealed that DBP, HLF, and TEF
regu-late primarily the transcription of genes involved in hepatic,
renal, and intestinal detoxification and genes of the innate and
adaptive immune system REV-ERBalpha, a nuclear orphan
receptor acts as a strong transcriptional repressor when bound to
RORE elements It generates circadian expression cycles of
BMAL1 and CLOCK, the two positive limb members of the
molecular oscillators, by periodically repressing the transcription
of these genes In addition, REV-ERBalpha governs the circadian expression of several enzymes and regulators of lipid metabolism Our genetic Rev-Erbalpha loss-of-function and gain-of-function studies suggest that circadian transcription in the periphery can
be governed by cell-autonomous and systemic cues
S1.3-3 Central and peripheral clocks in ontogeny
A Sumova, Z Bendova, M Sladek, R El-Hennamy,
K Laurinova, Z Jindrakova and H Illnerova Department of Neurohumoral Regulations, Institute of Physiology, Academy of Sciences of the Czech Republic
E-mail: sumova@biomed.cas.cz
In mammals, endogenous rhythmicity of the principal circadian clock located within the suprachiasmatic nucleus (SCN) is entrained predominantly by a light–dark cycle through pathways emanating from retinal photoreceptors Via separate pathways, non-photic cues may also reset the clock The SCN clock entrains circadian clocks in numerous peripheral tissues and controls rhythms of various body functions During ontogeny, the molecular mechanism responsible for generation of the circadian rhythmicity develops gradually from prenatal to postnatal period both in the SCN and in peripheral organs At the beginning, the maternal SCN sets the phase of the developing fetal and early postnatal clocks In altricial rodents, the external light–dark cycle starts to reset the SCN clock only after the first postnatal week However, full adjustment to the day-length, i.e to the photope-riod, is accomplished only around the time of weaning Appar-ently, maternal and photic resetting of the developing circadian clocks employ different and non converging signaling pathways While the photic entrainment pathways have already been roughly recognized, maternal entrainment pathways are still only suggested The data will summarize the current knowledge on mechanism of setting the time of the developing SCN and periph-eral clocks It appears that signaling from mothers and from the outside world to the newborn circadian clocks might be very complex
1.4 NF-jB Pathway in Normal Physiology and Disease
S1.4-1
The IKK complex-linking inflammation to
cancer
M Karin
Department of Pharmacology, University of California, San Diego
La Jolla, CA, USA E-mail: karinoffice@ucsd.edu
A link between inflammation and cancer has been suspected for
over two millennia, but its molecular nature remained ill defined
It has also been observed that certain bacterial (for instance
Heli-cobacter pylori) and viral (for instance HBV and HCV)
patho-gens are major risk factors for certain types of cancer, most
notably gastric and liver cancers We have postulated that
tran-scription factor NF-jB may be at the center of this nexus, as
NF-jB is activated in response to infection and inflammation
and in turn upregulates expression of anti-apoptotic and growth
promoting genes As there are several NF-jB transcription
fac-tors, we decided to inactivate the critical catalytic subunit of the
IjB kinase (IKK) complex, IKKb, as a way to inhibit activation
of most NF-jB forms While inflammation is a major factor that
contributes to the development and progression of CAC and
other inflammation-linked cancers and is estimated to be involved
in up to 20% of all human cancers, we asked whether inflamma-tion driven by NF-jB has an important role in other forms of cancer where chronic inflammation or infection do not precede tumor development To that end, we used a model of chemically-induced hepatocellular carcinoma (HCC) based on exposure of mice to a complete and potent carcinogen – diethyl nitrosamine (DEN) Heretofore, DEN administration, although resulting in pronounced cytotoxicity, was not found to trigger an inflamma-tion response
S1.4-2 NF-jB signalling in muscle regeneration
N Rosenthal, F Mourkioti, E Lara-Pezzi and M Pasparakis Mouse Biology Unit, EMBL-Monterotondo, Rome, Italy E-mail: rosenthal@embl.it
The adult mammalian body does retain the robust repair capa-city of the embryo into adulthood and gradually loses its regener-ative potential Our approach has been to intervene in the mechanisms at work in the mammalian response to damage or
Trang 4disease by reducing the impediments to effective regeneration of
skeletal muscle In one intervention, transgenic supplementation
of a locally acting Insulin-like Growth Factor 1 isoform
(mIGF-1) promotes efficient tissue repair of damaged skeletal and
car-diac muscle without scar formation, and prevents muscle atrophy
in heart failure In a second intervention, repression of the NFjB
inflammatory pathway by mIGF-1 in damaged muscle has
prompted studies in which mice lacking functional NFjB
signal-ling specifically in skeletal muscle exhibit increased muscle
regen-erative capacity In a second intervention, supplemental
expression of a naturally occurring, active calcineurin isoform
(CnAb1), which is normally induced transiently during skeletal
muscle regeneration and in response to mIGF-1 in atrophying
muscle, enhances repair and reduced fibrosis in response to
dam-age, and activates repressors of the NFjB pathway Taken
together, these observations support the feasibility of recapturing
embryonic regenerative capacity by modulating key signalling
pathways in the adult to restore injured or degenerating tissues
S1.4-3
The biology of NF-jB interactions with
chromatin
G Natoli
Department of Experimental Oncology, European Institute of
Oncology, Milan, Italy E-mail:
gioacchino.natoli@ifom-ieo-cam-pus.it
NF-jB is a family of rapidly activated transcription factors
con-served in multi-cellular eukaryotes, whose main role is to control
inducible transcription of most inflammatory genes, as well as genes involved in multiple steps of cancer development and pro-gression Overall, we know far less about the mechanisms regula-ting NF-jB activity in the nucleus than about signaling mechanisms responsible for its activation
Understanding NF-jB-regulated transcription is a crucial task to decode the logic of physiological and abnormal inflammatory responses and it should provide general paradigms about control
of inducible transcription in higher eukaryotes How are NF-jB:DNA transactions regulated? How are NF-jB-dependent genes with different functions expressed with kinetics that suit their function? How different microbes and inflammatory stimuli can induce transcriptional outputs that are plastically adapted to each input? Transcriptional specificity may represent a partic-ularly relevant task in the context of the anti-microbial response, since rapid and successful elimination of any pathogen will require the induction of a suitable transcriptional program In
my talk I will review some recent advancements in this field and their implications
1.5 Signaling and Cancer: Nuclear Receptor Connection
S1.5-1
Androgen action and prostate carcinogenesis
F Saatc¸iog˘lu
Division of Cellular and Molecular Biology, Department of
Biology, University of Oslo, Oslo, Norway
Androgens have critical roles in the development and
mainte-nance of the male reproductive system and important for
pro-gression of prostate cancer Using a simple screening protocol,
we have cloned androgen responsive genes that are also enriched
to prostate for expression The characterization of three of these
genes and the proteins that they encode will be presented:
Kallik-rein 4 (KLK4), six transmembrane protein of prostate 1
(STAMP1), and STAMP2 KLK4 belongs to the KLK family,
which includes Prostate Specific Antigen (PSA, or KLK3), a
secreted protein that is widely used as a diagnostic marker for
prostate cancer KLK4 is regulated by androgens and is highly
specific to prostate for expression Interestingly, KLK4 is the first
member of the KLK family that is intracellularly localized
KLK4 is predominantly expressed in the basal cells of the normal
prostate gland and overexpressed in prostate cancer STAMP1
and STAMP2 are six transmembrane proteins Whereas
STAMP1 is largely specific to prostate for expression, STAMP2
has a wider tissue distribution and its expression is exquisitely
regulated by androgens Live cell imaging using STAMPs fused
with green flourescent protein (GFP) show that STAMPs shuttle
between the trans-Golgi network (TGN) and the plasma
mem-brane suggesting that they may be involved in the
secretory/endo-cytic pathways STAMP1 is expressed exclusively in the epithelial
cells of the prostate and its expression is significantly increased in
prostate tumours compared with normal glands, whereas
STAMP2is highly overexpressed in a subset of the prostate ade-nocarcinoma specimens compared with normal prostate epithelial cells Ectopic expression studies indicate that these genes may be involved in proliferation of prostate cancer cells Taken together, these data suggest that KLK4, STAMP1, and STAMP2 contri-bute to the normal biology of the prostate cell, as well as pros-tate cancer progression In addition to these data, the molecular mechanism of antiandrogen action will be presented, determined
by live cell imaging of GFP-tagged androgen receptor (AR) inter-acting with its target sites
S1.5-2 Transcription factor mobility and promoter progression
G L Hager, R L Schiltz, M Wiench, T Johnson, S John,
A Nagaich and Y Qiu Laboratory of Receptor Biology & Gene Expression, NCI, NIH, Bethesda, MD, USA E-mail: hagerg@exchange.nih.gov The classical view of nuclear receptor action postulates the static binding of liganded receptors to the promoter We discovered, however, that nuclear receptors interact dynamically with regula-tory elements in living cells, and have proposed the hit and run hypothesis for receptor function We have also observed that steroid receptor responsive promoters move through a complex series of activity states, a phenomenon we term promoter pro-gression Genome-wide profiling of glucocorticoid receptor (GR) regulated loci reveals several classes of response, including genes that are transiently activated and genes that are transiently repressed Thus receptor action either leads to a series of events
Trang 5programmed into each promoter, or the receptor and/or
associ-ated factors are subject to a time dependent modification of their
activity states We have discovered that HDAC1 is a coactivator
for GR induction of MMTV A sub-fraction of HDAC1 present
in a complex with GR becomes acetylated upon hormone
treat-ment This acetylated form of HDAC1 appears in the GR
com-plex when MMTV transcription is inhibited, indicating that
acetylation of HDAC1 plays a repressive role on MMTV
tran-scription These findings suggest that a critical component of the
GR responsive transcriptional apparatus requires deacetylation
by HDAC1 for activity, and inactivation of the enzyme during
the induction cycle inhibits this process, thus shutting down the
promoter after a period of transient activation
S1.5-3
Analysis of steroid hormone receptor function
by gene targeting
G Schu¨tz
Molecular Biology of the Cell I, German Cancer Research Center,
Heidelberg, Germany E-mail: g.schuetz@dkfz.de
Germline and somatic gene targeting of genes for steroid
hor-mone receptors allows the characterization of their functions as
well as their molecular modes of action For the glucocorticoid receptor (GR) multiple modes of action have been identified The receptor activates expression of genes by binding as a dimer to glucocorticoid response elements (GRE) as well as by interaction
as a coactivator with DNA-bound Stat5 The receptor is able to repress expression of genes by protein–protein interaction and by binding to negative GREs Cre/loxP-mediated generation of somatic mutants of the mineralocorticoid receptor (MR) circum-vents the early lethality observed after germline inactivation Inactivation of MR in the forebrain leads to impaired hippocam-pal-dependent learning, but the limbic MR is dispensable for the maintenance of basal hypothalamic–pituitary–adrenal axis activ-ity The mechanisms underlying the critical actions of estrogen in the secretion of the gonadotropin-releasing hormone (GnRH) are unknown A neuron-specific ERa mutation in the forebrain leads
to infertility and loss of the positive feedback effects of estrogen upon GnRH neurons As GnRH neurons do not express ERal-pha, these results indicate that ERalpha-expressing neuronal aff-erents to GnRH neurons are critical for the preovulatory GnRH/
LH surge These genetic approaches not only reveal novel neural functions of these regulatory molecules in gene expression, but also unprecedented modes of their activity
1.6 Cell Surface Receptors and Downstream Targets
S1.6-1
Small GTPase signalling pathways in tumour
biology
C J Marshall
Oncogene Team, Cell and Molecular Biology, Institute of Cancer
Research, London, UK E-mail: chris.marshall@icr.ac.uk
Small GTPases of the Ras, Rho and Ral families play important
roles in tumour biology Genetic alterations to small GTPases
underscore this role Ras is mutated in some tumours while wild
type RhoA and RhoC are over-expressed particularly in more
aggressive tumours As well as their individual roles it is
emer-ging that there are significant interactions between small GTPase
signalling pathways Interesting examples of such interactions are
emerging through studies on invasion/cell motility A number of
the transcriptional targets of ERK-MAP kinase signalling
down-stream of oncogenic Ras are involved in cell movement and
adhesion Others act through regulating the activation state of
Rho-family signalling pathways, for example by down-regulation
of ROCK or Rho activation and up-regulating Rac activation
S1.6-2
Signal transduction via receptors for PDGF
and TGF-beta – possible targets for tumor
therapy
C.-H Heldin
Ludwig Institute for Cancer Research, Uppsala University,
Uppsala, Sweden E-mail: c-h.heldin@licr.uu.se
Platelet-derived growth factor (PDGF) and transforming growth
factor-beta (TGF-beta) affect cell growth, survival and migration,
and have important functions during the embryonal
develop-ment PDGF isoforms exert their cellular effects via two
structur-ally similar tyrosine kinase receptors Since PDGF promotes cell
growth and survival, overactivity of the PDGF signaling pathway
is associated with diesease, e.g malignacies We have explored
the use of PDGF antagonists in tumor treatment, and found effi-cient inhibition of tumor growth in animal models of tumors dri-ven by autocrine PDGF production In addition, we have observed that inhibition of paracrine PDGF stimulation of stro-mal fibroblasts and vessel pericytes lowers tumor interstitial fluid pressure and tumor angiogenesis TGF-beta has a more compli-cated role in cancer; initially TGF-beta is a tumor suppressor through its ability to inhibit growth and to promote apoptosis of tumor cells At later stages, when tumor cells become insensitive
to the cytostatic effects of TGF-beta, TGF-beta has tumor pro-moter effects through stimulation of epithelial-to-mesenchymal transition of tumor cells, stimulation of angiogenesis and suppression of the immune system We are currently delineating the signaling pathways involved in the various cellular effects of TGF-beta, and exploring the possible use of TGF-beta antago-nists in tumor treatment
S1.6-3 New functions of focal adhesion kinase: a major Src effector involved in cancer
M Frame and B Serrels Beatson Institute, Glasgow, UK
E-mail: m.frame@beatson.gla.ac.uk FAK expression is elevated, often at the level of gene dosage/ amplification, in epithelial cancer cells We reported the first skin-specific conditional knockout of FAK using Cre-lox technol-ogy Skin carcinogenesis experiments revealed that FAK defici-ency leads to reduced incidence of papillomas and inhibition of progression to carcinomas Mechanistically, loss of FAK is asso-ciated with increased apoptosis in both keratinocytes in vitro and
in the skin in vivo, particularly in the hair follicle bulge region where the stem cell targets for tumorigenesis reside We have recently expressed putative gain-of-function and loss-of-function mutants of FAK as skin-targeted transgenes, and have found that elevated FAK expression can cause accelerated carcinogenesis
Trang 6and progression to carcinoma In addition to our in vivo
experi-ments, in vitro experiments have identified a new function of the
FAK FERM domain in regulating actin assembly via the Arp2/3
complex Specifically, the FERM domain binds and recruits the
Arp2/3 complex to peripheral adhesion sites Critically, Arp2/3 is
subsequently released from complex with FAK by
integrin-induced auto-phosphorylation of FAK-tyrosine-397, which lies adjacent to the FAK FERM domain Moreover, release of Arp2/3 is associated with recruitment of actin into filaments in the vicinity of focal adhesions These data suggest new ways in which integrin and Src signalling to FAK may contribute to the cancer phenotype
1.7 Signaling Through Ion Channels
S1.7-1
Voltage-gated sodium channel upregulation as
an accelerating factor in cancer cell behaviour
M.B.A Djamgoz
Divison of Cell & Molecular Biology, Imperial College London,
London, UK E-mail: m.djamgoz@imperial.ac.uk
Electrophysiological recordings from contrasting strongly versus
weakly metastatic human prostate cancer (PCa) and breast
can-cer (BCa) cells showed that the former specifically expressed
functional voltage-gated sodium channels (VGSCs) At mRNA
level, the upregulation was greater than 1000-fold Similar
upre-gulation of VGSC mRNA and protein was detected in human
biopsies Blocking VGSC activity with the highly specific
neuro-toxin, tetrodotoxin (TTX), suppressed the cells’ metastatic
behav-iours (MCBs) in vitro Thus, lateral motility (including
galvanotaxis), transverse migration, endocytic membrane activity,
adhesion and Matrigel invasiveness were modulated by 50%,
consistent with the notion that VGSC activity would potentiate
metastasis Semi-quantitative PCR measurements also revealed
that Nav1.7 and Nav1.5 were the culprit VGSCs in PCa and
BCa, respectively Importantly, in both cancers, the VGSC was
expressed in its neonatal splice form In the case of BCa, the
neo-natal splice variant differed from the adult in a string of seven
amino acids of unique sequence and a polyclonal antibody could
be raised to target the VGSC protein Blocking VGSC activity in
the human BCa MDA-MB-231 cell line with either the antibody
or siRNA, again, suppressed MCB by50% and eliminated the
TTX sensitivity, i.e the potentiating effect of VGSC It is
conclu-ded that VGSC expression/activity is a viable novel target for
clinical management of metastatic disease
S1.7-2
The role of EAG, a potassium channel, in
cancer
W Stu¨hmer
MBNS, Max-Planck-Institute of Experimental Medicine,
Go¨ttin-gen, Germany E-mail: wstuehm@gwdg.de
Ion channels are increasingly being linked to cancer and tumour
progression Here we describe a voltage-gated, potassium
select-ive channel (EAG or ether-a-go-go) with novel
electrophysiologi-cal properties, whose normal physiologielectrophysiologi-cal function is yet
unknown but which acts as an oncogene if expressed ectopicaly
One of the most characteristic properties of the channel, its ionic
selectivity, is modulated during cell-cycle transitions Strikingly,
the expression of the human EAG is restricted to brain, but it is
also present in several tumour-derived cell lines While normal tissue is not stained with EAG antibodies, over 75% of tumours tested showed positive staining Experiments under in vitro condi-tions have demonstrated decreased proliferation of EAG1-expres-sing cells by inhibition of this channel This inhibition of EAG1
is accomplished using RNA interference, functional anti-EAG1 antibodies, or (unspecific) EAG1 channel blockers We have used the eXplore Optix system to visualize the distribution of EAG in
an in vivo mice tumour model For this, a specific recombinant Fab fragment against EAG was labelled either with DsRed or Cy5.5 These IR-fluorescent antibody constructs were injected into immuno-suppressed mice carrying previously grafted MDA-MB-435 S human mammary carcinoma cells This enabled us to follow tumour localization as well as tumour progression We conclude that EAG is a widely distributed tumour marker with diagnostic and therapeutic potential
S1.7-3 Cyclic nucleotide-gated ion channels
A Menini, A Boccaccio and S Pifferi Neurobiology Sector, International School for Advanced Studies, SISSA, Trieste, Italy E-mail: menini@sissa.it
Ion channels activated by the binding of cyclic nucleotides are present in several types of cells Their main physiological role consists in translating changes in the concentration of cAMP or cGMP into a variation of the membrane potential Cyclic nuc-leotide-gated (CNG) channels were first discovered in the outer segment of retinal rods and shortly after in the cilia of olfactory sensory neurons In these sensory systems CNG channels medi-ate sensory transduction by conducting cationic currents carried primarily by sodium and calcium ions In olfaction the binding
of odorants to olfactory receptors in the cilia causes, via G pro-tein activation of adenylyl cyclase, the increase of intracellular cAMP, which directly opens CNG channels The increase in intracellular calcium entering through CNG channels has both inhibitory and excitatory feedback effects The opening of Ca-activated Cl channels increases the transduction current because olfactory sensory neurons maintain a high intracellular
Cl concentration, whereas calcium in combination with calmodu-lin exerts a negative feedback on CNG channels By using the whole-cell voltage-clamp technique to record the current, in combination with photolysis of caged cAMP or 8-Br-cAMP, we showed that the Ca feedback on CNG channels is the main molecular mechanism responsible for fast adaptation in olfactory sensory neurons