deregulation of the activin/follistatin system in hepatocarcinogenesis

www.elsevier.com/locate/jhep

Journal of Hepatology 45 (2006) 673–680

Deregulation of the activin/follistatin system in hepatocarcinogenesisq

Michael Grusch1,*, Claudia Drucker1, Barbara Peter-Vorosmarty1, Natascha Erlach1,Andreas Lackner1, Annemarie Losert1, Doris Macheiner1, Wolfgang J. Schneider2,

Marcela Hermann2, Nigel P. Groome3, Wolfram Parzefall1, Walter Berger1,Bettina Grasl-Kraupp1, Rolf Schulte-Hermann1

1Department of Medicine I, Division: Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria2Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-gasse 9/2, A-1030, Austria

3School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, OX3 0BP, UK

Background/Aims: Activins A and E negatively regulate hepatic cell number by inhibiting cell replication and inducing

apoptosis. Follistatin and follistatin-like 3 bind activins and antagonise their biological activities. Aim of our study was to

investigate, whether activins and follistatins may play a role in hepatocarcinogenesis.

Methods: Expression levels of follistatin, follistatin-like 3, and activin subunits bA as well as bE were investigated in chem-ically induced rat and human liver tumours by real-time PCR and immunohistochemistry. In addition, the effects of follistatin

and activin A on DNA synthesis of normal as well as preneoplastic hepatocytes and hepatoma cells were analysed.

Results: Follistatin was overexpressed while both activin subunits were downregulated in the majority of rat and human liver

tumours. Follistatin-like 3 expression was low in normal but enhanced in malignant rat liver. In human normal liver, in con-

trast, it was abundantly expressed but downregulated in liver cancer. Administration of follistatin to normal and preneoplastic

hepatocytes stimulated. DNA synthesis preferentially in preneoplastic rat hepatocytes, whereas activin A repressed it.

Conclusions: The balanced expression of follistatins and activins becomes deregulated during hepatocarcinogenesis. The

sensitivity of preneoplastic hepatocytes to activin signals suggests the activin/follistatin system as promising target for ther-apeutic intervention.

� 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: Activin; Follistatin; Liver tumours; DNA synthesis; Primary hepatocytes

1. Introduction

Liver cancer accounts for more than half a milliondeaths per year world-wide [1] with rising incidence in

0168-8278/$32.00 � 2006 European Association for the Study of the Liver.

doi:10.1016/j.jhep.2006.06.014

Received 31 January 2006; received in revised form 23 May 2006;

accepted 27 June 2006; available online 28 July 2006q The authors who have taken part in this study declared that they

have no relationship with the manufacturers of the drugs involvedeither in the past or present. The authors received funding fromHerzfelder’sche Familienstiftung, which enabled them to carry outtheir study.

* Corresponding author. Tel.: +43 1 4277 65144; fax: +43 1 42779651.

E-mail address: [email protected] (M. Grusch).

Europe, Asia, and North America, due to the high prev-alence of liver cirrhosis, chronic hepatitis B and C infec-tions, alcohol disease, and obesity [2]. Curativetreatment by surgical resection, liver transplantation,or local ablation can be achieved only in a minority ofpatients. Chemotherapy success in case of liver cancerhas been disappointing without resulting in a significantimpact on patient survival [3]. Therefore, a better under-standing of the molecular events contributing to hepato-carcinogenesis is urgently needed in order to developadditional approaches for prevention and therapy.While hyperactivation of growth promoting signals iswell documented in liver carcinogenesis [4], escape fromgrowth-limiting signals may be equally important.

Published by Elsevier B.V. All rights reserved.

mailto:[email protected]

674 M. Grusch et al. / Journal of Hepatology 45 (2006) 673–680

Activins are members of the TGFb superfamily ofgrowth and differentiation factors [5]. They were discov-ered as gonadal stimulators of pituitary FSH secretionand subsequently found to have diverse regulatory func-tions in a wide range of tissues and organs [6]. Activinsare dimers of disulphide-linked b subunits. Four mam-malian genes coding for activin b-subunits, termed bA,bB, bC and bE, have been cloned [7–9]. Activin A, thehomodimer of two bA subunits, inhibits mitogen-in-duced DNA synthesis and induces apoptosis in hepato-cytes and hepatoma cells in vitro and in vivo [10–12].Therefore, it is considered a physiological inhibitor ofliver growth. Less is known about hepatic functions ofthe other b subunits. Expression of activin bE has beenreported to induce apoptosis in hepatoma cells andmouse liver cells and reduce regenerative DNA synthesisin mouse liver [13–15]. The function of activin bC

remains controversial (for review, see [16]), and activinbB transcripts are not expressed at significant levels inthe liver [17]. Thus, in the present investigation we focuson expression of the bA and bE subunits.

Follistatin (fst), a secreted monomeric protein structur-ally unrelated to activins, binds activin A with high affin-ity, thereby blocking its interaction with activinreceptors [18,19,9]. Infusion or adenovirus-mediatedoverexpression of fst leads to an increase in hepaticDNA synthesis in vivo, presumably by blocking the inhib-itory action of endogenous activin A [20,21]. Several fst-related proteins have been identified by the presence ofstructurally conserved fst domains. Of these, however,only follistatin-like 3 (fstl-3, also termed FLRG) seemsto share with fst the ability to antagonise activin A [22].

In the present study, we demonstrate in well-estab-lished rat models [23] and in human liver cancers thatthe balance between activins and their endogenousinhibitors fst and fstl-3 becomes deregulated duringhepatocarcinogenesis. At the same time (pre)neoplastichepatocytes are highly sensitive to activin A, suggestingthis growth- and differentiation-regulating system aspotential target for therapeutic interventions.

2. Materials and methods

2.1. Materials

Recombinant human activin A was from R&D Systems (USA),recombinant human fst 288 from the National Hormone and PituitaryProgram. Recombinant human activin C and recombinant humanactivin E monomer produced in Escherichia coli were supplied byBiopharm (Germany).

2.2. Animals and treatment

Male SPF Wistar rats were obtained from the animal facilities ofthe Institute for Biomedical Research at the Medical University ofVienna. Animals were kept under standardised conditions (Macroloncages, 20 ± 3 �C room temperature, 40–70% relative humidity). At

the age of four weeks animals were treated with N-nitrosomorpholine(NNM, Sigma, USA) by gavage. NNM was dissolved in PBS (pH 7.4)immediately before treatment and was given as a single dose of250 mg/10 ml PBS/kg body weight. For tumour promotion, animalsreceived phenobarbital (PB) at a dose of 50 mg/kg body weight withfeeding for 17 months. Animals were sacrificed by decapitation underCO2 narcosis. Tumour tissue and non-tumorous liver tissue were snapfrozen in liquid nitrogen for gene expression analysis, or immediatelyfixed for immunostaining in either buffered formalin (Lillie) or Car-noy’s solution. Presence of liver tumours was confirmed histologicallyon hematoxylin–eosin stained sections. Nafenopin-induced tumourswere from a previously described investigation [24]. All experimentswere performed according to the Austrian guidelines for animal careand protection.

2.3. Human liver samples

Liver samples of patients, all of whom had given informed consent,were obtained from the General Hospital Vienna. Samples were frozenin liquid nitrogen for RNA isolation or embedded in paraffin for rou-tine diagnosis and immuno-histochemistry. Two samples of normal liv-er and 11 samples of HCC were analysed by real-time PCR. Suspectedcauses of disease were chronic viral hepatitis and/or alcohol abuse.From five of the HCC samples paraffin-embedded tissue includingtumour surrounding fibrotic/cirrhotic liver was available for analysisby immuno-histochemistry.

2.4. RNA isolation

Total RNA was isolated using TRIzol reagent (Invitrogen, USA)according to the instructions of the manufacturer. RNA concentrationwas determined photometrically.

2.5. Quantitative real-time (QRT)-PCR

Two micrograms of total RNA was reverse transcribed withMMLV reverse transcriptase (Sigma) resulting in 100 ll cDNA. Twoto four microlitres cDNA was used as template for each PCR. Taqmanassays for rat and human activins bA, bE, fst, fstl-3, and b2-microglob-ulin (b2mg) were from Applied Biosystems (USA) and were performedfollowing the guidelines of the manufacturer on an ABI Prism 7000Sequence Detection System. Expression data were normalised tob2mg. Tumour-surrounding normal liver tissue from the same animalas well as two human normal liver samples were used as calibrators.All samples were analysed in duplicate.

2.6. Antibody preparation and Western blot

Antiserum against activin bE was prepared against a synthetic pep-tide corresponding to residues (C)HSAVFSLLKANNPWPASTS(C)of the protein. The peptide was coupled to keyhole limpet hemocyaninand used for immunisation of adult female New Zealand White rabbitsby a standard procedure [25]. The IgG fraction was affinity-purified onprotein A–Sepharose (Amersham, UK). The resulting antibody recog-nised recombinant human activin bE monomer produced in E. coli inWestern blots and showed no cross-reactivity with recombinant activ-ins A and C. Protein extracts were prepared and Western blots per-formed as described previously [14] using the anti-fst mAb clone85918 (R&D System, USA).

2.7. Immunostaining

Tissue samples were fixed in 4% buffered formalin or alternativelyfor rat activin E staining in Carnoy’s solution and embedded in paraf-fin. Two micrometers sections were deparaffinated, and antigen-re-trieval for formalin fixed tissue was done by heating the sections inthe microwave in 0.01 M citrate buffer. Sections were incubated withprimary antibodies in 0.1% BSA/PBS overnight at 4 �C. The followingprimary antibodies were used: mouse monoclonal antibody against

R5T4

R5T3

R5T2

R5T1

R4T3

R4T2

R4T1

R3T2

R3T1

R2T3

R2T2

R2T1

R1T2

R1T1

Fstl-3Fst

ßEßA

840

144

A

M. Grusch et al. / Journal of Hepatology 45 (2006) 673–680 675

activin A (Serotec) diluted 1:50, rabbit antiserum against activin Ediluted 1:500, mouse mAb clone 85918 to fst (R&D System, USA,human samples) diluted 1:20, mouse monoclonal antibody to fst 315(clone H10, rat samples) [26] diluted 1:100, goat polyclonal antibodyto fstl-3 (Santa Cruz, USA) diluted 1:50. Sections were washed inPBS with 0.5% Tween 20 and incubated with HRP-coupled secondaryantibodies (Dako, Denmark) at a dilution of 1:200. DAB (Dako) wasused as chromogen to detect peroxidase activity. Sections were coun-terstained with hemalaun and mounted in Kaiser’s glycerol gelatine(Merck, Germany).

2.8. Isolation of primary hepatocytes

Rat liver cells were isolated by collagenase perfusion at day 20 afterNNM treatment according to the method of Seglen with modificationsdescribed previously [27,28]. Hepatocytes were seeded at a density of30.000 viable cells/cm2 into collagen-coated 3.5 cm dishes in WE2medium with 10% FCS as previously described [28]. After one hourcells were washed and thereafter cultivated in WE2 medium withoutFCS. Treatment was started four hours later.

2.9. Determination of DNA replication

DNA replication in primary hepatocytes was assessed by incorpo-ration of [3H]thymidine (0.5 lCi [3H]thymidine/ml medium) aspublished [29]. H4IIE-C3 rat hepatoma cells were cultured in RPMI-1640 with 10% FCS. 5 · 104 cells per well were seeded in 24-well platesand after 24 h treated with 25 ng/ml follistatin, or 10 ng/ml activin A.BrdU was added 48 h later for 60 min. BrdU staining was conductedwith the cell proliferation kit from Amersham Biosciences (UK)according to the instructions of the manufacturer. One thousand cellsper well and three wells per treatment were analysed and dataexpressed as labelling index (LI) representing the percentage of labelledcells.

R6T2

R6T1

0.01 0.1 1 10 1000.25 0.5 2.5 5 25 50

0.01 0.1 1 10 1000.25 0.5 2.5 5 25 50

R4T2

R4T1

R3T2

R3T1

R2T1

R1T1ßAßE

FstFstl-3

1400

9300

B

Fig. 1. Expression of Follistatin (fst), follistatin like-3 (fstl-3), activin

bA, and activin bE mRNAs in NNM/PB-induced (A) and nafenopin-

induced (B) liver tumours relative to surrounding liver from the same

animal. Expression in surrounding liver was arbitrarily set as 1.

Individual animals are indicated by R and individual tumours by T

numbers. Values > 100-fold are given as numbers adjacent to the

respective bars.

3. Results

3.1. Transcript Expression of Fst, Fstl-3, activin bA, and

bE in rat liver tumours

mRNA expression levels of the activin-antagonisingproteins fst and fstl-3 as well as of the activin subunitsbA, and bE were measured in a set of 16 liver tumoursfrom six rats treated with NNM/PB. Transcripts wereconsidered overexpressed or down-regulated when theirexpression level in the tumour was >2 x or <0.5 x that ofthe corresponding normal liver.

Fst was overexpressed in 10/16 (63%) tumours, andfstl-3 in 14/16 tumours (88%, Fig. 1A). Only in onetumour (6%, R4T1) neither fst nor fstl-3 was increased,whereas 9 tumours (56%) showed overexpression ofboth factors. While fstl-3 was up-regulated in a higherfraction of tumours, fst reached very high expressionlevels in some cases. For example, two tumours showeda more than 100-fold increase of fst. A completely differ-ent picture was obtained with the two activin subunits.The bA transcript was down-regulated in 13/16 (81%),and the bE transcript in 12/16 tumours (75%, Fig. 1A).Downregulation of bA tended to be stronger than thatof bE. All tumours investigated showed either down-reg-ulation of activin subunits, or up-regulation of activinantagonists, or a combination of both effects. Median

Table 1

Activin/follistatin mRNA expression

Follistatin Fstl-3 Activin bA Activin bE

NNM/PB-induced tumours 3.8 3.2 0.2 0.4Nafenopin-induced tumours 4.4 16.7 0.3 1.2Human liver tumours 5.5 0.2 0.6 0.1H4IIE-C3 cells 0.2 430 0.02 0.9

Transcript expression levels of the investigated genes in NNM/PB-induced and nafenopin-induced rat tumours (medians) relative to surroundinglivers (arbitrarily set as 1), as well as in human liver tumours (medians) and in the rat hepatoma cell line H4IIE-C3 relative to normal human and ratlivers, respectively.


expression levels of the investigated genes relative to sur-rounding livers are given in Table 1. Fst and bA tran-scripts were also analysed by RNAse protection assay.The results confirmed those of the QRT-PCR analysis(data not shown).

Pearson correlation coefficients (r) between the inves-tigated mRNA values showed a significant positive cor-relation between activin bA and bE subunit expression(r = 0.755, p = 0.007), whereas trends towards correla-tion between fst and fstl-3 (r = 0.401) and towardsinverse correlations between bA and fst (r = �0.283),bA and fstl-3 (r = �0.105), and bE and fst (r = �0.199)were not statistically significant.

When tumours were subgrouped into adenomas, car-cinoma stage 1, and carcinoma stage >1 according toestablished criteria [30], no obvious differences in expres-sion between the different subgroups were noticed exceptfor fstl-3, which was upregulated more than threefold in4/4 (100%) stage >1 carcinomas, 3/7 (43%) stage 1 car-cinomas, but only 1/5 (20%) adenomas.

To determine whether the observed changes in acti-vin/follistatin also occur in a second tumour model,we analysed six nafenopin-induced liver tumours from4 different rats (Fig. 1B). Fst was over-expressed in 4/6(67%), and fstl-3 in 6/6 (100%) tumours. Activin bA

was downregulated in 5/6 (83%) tumours. In contrastto the NNM/PB model, activin bE was not down-regu-lated in any of the nafenopin-induced tumours, butup-regulated in 2/6 (33%) tumours.

In the rat hepatoma cell line H4IIE-C3 fst expressionwas reduced compared to normal rat liver, but fstl-3expression was increased more than 400-fold (Table 1).H4IIE-C3 cells showed a strongly reduced activin bA level,but had retained significant expression of bE transcripts.

3.2. Protein expression in NNM/PB-induced liver

tumours

Immunostaining for both fst and fstl-3 showed aweak cytoplasmic staining of hepatocytes in tumour-sur-rounding liver sections and a much stronger staining inthe majority of tumours (Fig. 2A) which was in case offst also confirmed by Western blot analysis (Fig. 2B).There was a good agreement between mRNA expressionlevel and staining intensity.

Activin E staining was strong in periportal andweaker in centrilobular hepatocytes in non-tumorousliver tissue, reminiscent of the expression patterndescribed previously for activin bE mRNA by in situhybridisation [17]. Tumour tissue had reduced immu-noreactivity for activin E (Fig. 2). In contrast to fst,fstl-3, and activin E, we did not observe a change inactivin A immunostaining corresponding to thedecreased transcript levels. Staining was weak in sec-tions from tumour surrounding liver, and furtherdownregulation of immunoreactivity in tumour tissuewas not detectable. In all cases omission of primaryantibodies resulted in complete loss of staining (notshown).

3.3. Expression of Fst, Fstl-3, activin bA, and bE in humanliver tumours

Changes in the activin/follistatin expression were alsoinvestigated in 11 human liver tumours. Similar to therat model, real-time PCR demonstrated a strong upreg-ulation of fst mRNA expression in 8/11 (73%) tumourtissues (Fig. 3 and Table 1). Immunostaining of humanfst protein demonstrated abundant expression already intumour-surrounding liver tissues which was, however,further enhanced in the malignant cells (Fig. 2). Inremarkable contrast to the rodent model, fstl-3 mRNAwas abundantly expressed in human normal tissues butconsistently downregulated in 9/11 (82%) tumours(Fig. 3). Like in rats, downregulation of bA (5/11,45%) and bE mRNA (10/11, 91%) was also observedin human carcinomas with a stronger and more consis-tent effect for bE (Fig. 3).

3.4. Effect of fst and activin A on DNA synthesis

Finally, it was investigated whether fst and activin Aaffect the growth of preneoplastic hepatocytes and hep-atoma cells. For this we utilised a well-characterised cellmodel for preneoplastic rat hepatocytes prepared fromrats three weeks after a single dose of NNM [29,31]and the rat hepatoma cell line H4IIE-C3. Preneoplasticrat hepatocytes, but not normal liver cells, express pla-cental type glutathione S-transferase (GSTp). Thisallows detection of preneoplastic (GSTp+) hepatocytes

-ns

-fts

LS 1 T A1 T B1 LS 2 T A2 T B2 LS 3 T3

LS

T A1 T B1 T A2 T B2 T3

0

2

3

BA

Fig. 2. Immunostaining (A) of fst, fstl-3, activin bA, and activin bE in rat and fst in human liver tumours (T) and surrounding liver tissue (SL). Between 5

and 15 rat liver tumours and 5 human liver tumours as well as in each case surrounding liver tissues were analysed and representative examples are shown.

Western blot analysis (B) of fst expression in NNM/PB-induced rat liver tumours (T) and surrounding livers (SL). For densitometry analysis fst band

intensities of the tumour samples were normalised to a non-specific band (ns) at 70 kD and expressed as fold control of the respective surrounding livers.


in primary culture and to assess their DNA synthesisrates compared to non-initiated (GSTp�) normal hepa-tocytes. In agreement with previous data [29], about 2%of the cultured cells showed positive staining with GSTpand had higher DNA synthesis rates (12.5%) thanGSTp� cells (2.5%, Fig. 4).

When cell cultures were treated with 25 ng/ml recom-binant fst, the labelling index of GSTp+ cells was further

increased to 22%. On GSTp� cells, on the other hand,fst treatment had only a marginal effect (Fig. 4). In con-trast, treatment of the cultures with 10 ng/ml recombi-nant human activin A reduced the labelling index inGSTp+ cells from 12.5% to the level of GSTp� cells(2.5%).

In the hepatoma cell line H4IIE-C3 treatment withactivin A decreased the labelling index from 43% in

NL

fst fstl-3 ßA ßE0.01

0.1

10

100

NL

noisserpxe dl of

Fig. 3. Expressions of follistatin (fst), follistatin like-3 (fstl-3), activin

bA, and activin bE transcripts in human liver tumours. QRT-PCR data

are given relatively to the mean of two normal liver samples (NL)

arbitrarily set as 1. Medians are shown as horizontal bars.

Gst-

Co Activin A Follistatin0

5

10

15

20

25

30

Gst+

10

20

30

p<0.0001

p=0.0257

LI %

LI %


untreated controls to 29%. Addition of recombinant fstdid not further induce the already high basal DNA syn-thesis rate (Fig. 4).


H4IIE-C3


25

50

75

p=0.0689LI %

Fig. 4. Impact of follistatin and activin A on DNA synthesis of primary

hepatocytes and hepatoma cells. Labelling indices (LI) of normal,

glutathione S-transferase-negative (Gst�) and preneoplastic, glutathione

S-transferase-positive (Gst+) hepatocytes in primary culture as well as

H4IIE-C3 rat hepatoma cells are shown. Cultures were treated with

solvent (Co), recombinant human follistatin 288 (25 ng/ml), or recombi-

nant human activin A (10 ng/ml) for 48 h and labelling indices were

determined as described [29]. Results are means ± SD of three indepen-

dent experiments. p values were calculated by unpaired t tests.

4. Discussion

Alterations in pathways promoting or suppressingcell growth have been described in diverse human can-cers and are believed to represent essential events intumour formation [32–34]. From a therapeutic perspec-tive these alterations offer the possibility to specificallytarget tumour cells on a rational basis. Polypeptidegrowth factors stimulating hepatocyte growth, likeIGF-II, TGFa, HB-EGF, as well as some members ofthe FGF family have been found overexpressed in livercirrhosis and hepatocellular carcinoma and are believedto contribute to the increased proliferation rates charac-teristic of (pre)neoplastic hepatocytes [35–41]. Theescape of (pre)neoplastic cells from growth-limitingand apoptosis-inducing signals represents an additionalmechanism leading to increased growth of tumoursand tumour prestages.

Several studies have indicated that the activin/follist-atin system critically contributes to homeostasis of cellgrowth in normal liver [12,20,42,21]. In the presentreport, we demonstrate that the balanced expression ofactivins and follistatins becomes shifted towards follist-atins during hepatocarcinogenesis. Previously, we havereported enhanced fst mRNA levels in rodent livertumours [43] and Mashima et al. [44] described releaseof fst from three human hepatoma cell lines. According-ly, Stove et al. [45] found fst overexpression in the Bowesmelanoma cell line. Here, we extend these investigationsto protein analysis in rat livers, to human liver tumours,and to fstl-3, a protein with similar properties as fst. Fstwas consistently overexpressed in human and rat livertumours. In contrast, fstl-3 expression was demonstrat-

ed to be low in normal liver but upregulated duringrat hepatocarcinogenesis while it was relatively high innormal human liver but consistently down-regulated intumours. This suggests a different expression regulationof fstl-3 in human and rat liver. Moreover, an impact ofthe tumor-initiating factors might be anticipated, i.e.chemical carcinogenesis in the rat model and persistentinflammation due to hepatitis and/or alcohol abuse incase of human liver cancers.

In contrast to activin inhibitors, mRNAs of both acti-vin subunits analysed were consistently reduced in a


high proportion of rat and human liver tumours. Down-regulation of activin signal transduction componentshas also been described in high-grade breast cancer[46]. Moreover, the activin type II receptor gene is fre-quently mutated in microsatellite-unstable colon andother gastrointestinal cancers [47,48].

Several of our observations suggest that deregulationof the activin/follistatin system plays an essential role inhepatocarcinogenesis. First, addition of recombinanthuman fst to primary cultures of hepatocytes fromNNM treated rats induced DNA synthesis preferentiallyin GSTp+ cells indicating that activin A is a growth-lim-iting factor for preneoplastic hepatocytes. Secondly,H4IIE-C3 hepatoma cells express high levels of the acti-vin inhibitor fstl-3 but almost undetectable amounts ofbA. Consequently and in agreement with a previousstudy [49], fst treatment did not increase DNA synthesis,suggesting that endogenously produced activin is nolonger growth-limiting for these cells. Thirdly, treatmentof GSTp+ hepatocytes with activin A reduced theirDNA synthesis rates to the level of GSTp�cells, indicat-ing that preneoplastic hepatocytes are very sensitive toactivin A, and that their growth advantage as comparedto normal hepatocytes could be eliminated by increasinghepatic activin concentrations. Sensitivity to activin Awas retained even in H4IIE-C3 cells, despite overexpres-sion of fstl-3, and has also been demonstrated for fst-ex-pressing human hepatoma cells [50,14].

In conclusion, our results suggest that deregulation ofthe activin/follistatin system may be a key event duringhepatocarcinogenesis and that restoration of the balancebetween activins and their respective inhibitors may be auseful strategy for prevention and therapy of livertumours.

Acknowledgements

The excellent technical assistance of K. Bukowska, H.Koudelka, and B. Mir-Karner is gratefully acknowl-edged. The work was supported by grants from the ‘‘Her-zfelder’sche Familienstiftung’’ to M.G. and to B.G.K.

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deregulation of the activin/follistatin system in hepatocarcinogenesis

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