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Carcinogenesis Advance Access originally published online on August 4, 2005
Carcinogenesis 2006 27(1):152-161; doi:10.1093/carcin/bgi202
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Carcinogenesis vol.27 no.1 © Oxford University Press 2005; all rights reserved.

Non-parenchymal liver cells support the growth advantage in the first stages of hepatocarcinogenesis

Claudia Drucker, Wolfram Parzefall, Olga Teufelhofer, Michael Grusch, Adolf Ellinger 1, Rolf Schulte-Hermann and Bettina Grasl-Kraupp *

Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria and 1 Department for Anatomy and Cell Biology, Division: Cell Biology and Ultrastructural Research, Medical University of Vienna, Schwarzspanierstrasse 13, A-1090 Vienna, Austria

* To whom correspondence should be addressed. Tel: +43 1 4277 65137; Fax: +43 1 4277 9651; Email: bettina.grasl-kraupp{at}meduniwien.ac.at


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Hepatocellular carcinoma almost always arises in chronically inflamed livers. We developed a culture model to study the role of non-parenchymal cells (NPCs) for inflammation-driven hepatocarcinogenesis. Rats were treated with the carcinogen N-nitrosomorpholine, which induced initiated hepatocytes expressing the marker placental glutathione-S-transferase (GSTp). After 21 days two preparations of hepatocytes were made: (i) conventional ones (Hep-conv) containing NPCs and (ii) hepatocytes purified of NPCs (Hep-pur). Initiated hepatocytes, being positive for GSTp (GSTp-pos) were present in both preparations and were cultured along with normal hepatocytes, being negative for GSTp (GSTp-neg). Under any culture condition DNA synthesis was ~4-fold higher in GSTp-pos than in GSTp-neg hepatocytes demonstrating the inherent growth advantage of the first stages of hepatocarcinogenesis. Hepatocytes showed ~3-fold lower rates of DNA synthesis in Hep-pur than in Hep-conv, which was elevated above Hep-conv levels by addition of NPC or NPC-supernatant. Pretreatment of NPCs with proinflammatory lipopolysaccharide (LPS) further increased DNA synthesis. Thus, NPCs release soluble growth stimulators. Next we investigated the effect of specific cytokines produced by NPCs. Tumour necrosis factor {alpha} and interleukin 6 barely altered DNA synthesis, whereas hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and the heparin-binding epidermal growth factor-like growth factor (HB-EGF) were potent inducers of DNA replication in both, GSTp-neg and GSTp-pos cells. In conclusion, DNA synthesis of hepatocytes is increased by factors released from NPCs, an effect augmented by LPS-stimulation. NPC-derived cytokines, such as KGF, HGF and HB-EGF, stimulate DNA synthesis preferentially in initiated hepatocytes, presumably resulting in tumour promotion. Similar mechanisms may contribute to carcinogenesis in human inflammatory liver diseases.

Abbreviations: EC, endothelial cell; GSTp-neg, negative for placental glutathione-S-transferase; GSTp-pos, positive for placental glutathione-S-transferase; HB-EGF, heparin-binding epidermal growth factor like growth factor; Hep-conv, conventional primary hepatocyte culture; Hep-pur, purified primary hepatocyte culture; HGF, hepatocyte growth factor; IL-6, interleukin 6; KC, Kupffer cell; KGF, keratinocyte growth factor; LI, labelling index; LPS, lipopolysaccharide; NNM, N-nitrosomorpholine; NPC, non-parenchymal liver cell; ROS, reactive oxygen species; TNF{alpha}, tumour necrosis factor {alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Hepatocellular carcinoma is one of the most common fatal cancers worldwide accounting for at least half a million deaths per year (1). Chronic viral infections, dietary exposure to aflatoxin-B1 or excessive ethanol intake have been identified as major risk factors (2). The resulting chronic hepatitis and cirrhosis predispose to the development of malignant liver cells. It is assumed that unresolved inflammation evokes cell turnover in an effort to restore tissue homeostasis (3–6). In this process, deregulated cytokine production and aberrant cytokine signalling may lead to altered cell growth, differentiation and apoptosis (3–10). The result may be the formation and selection of premalignant hepatocytes that increasingly lose normal growth control and that may give rise to cancer.

Interactions between tumour prestages and the microenvironment appear crucial for the development of liver cancer and are yet incompletely understood. The present work focuses on the role of non-parenchymal liver cells (NPCs) as a source of growth regulatory cytokines involved in inflammation-driven hepatocarcinogenesis. These cytokines may stem from different cell populations (3–7). Hepatocyte cords are flanked by sinusoidal structures consisting of endothelial cells (ECs), tissue macrophages [Kupffer cells (KCs)] and stellate cells. In case of cell damage KCs and ECs exhibit antigen-presenting function and attract lymphocytes. Both cell types are also activated when their toll-like receptors bind molecular components derived from microorganisms, such as lipopolysaccharide (LPS). As a result, proinflammatory mediators including cytokines, chemokines, prostaglandins, cytolytic proteases and reactive oxygen species (ROS) are released and may favour the development of cancer (7,9–12).

Rat liver provides valuable models to study the very first stages of carcinogenesis (13–17). Within a few days after treatment with genotoxic carcinogens single initiated hepatocytes appear, that selectively express placental glutathione-S-transferase (GSTp-pos) cells (14,15). A considerable fraction of these cells develops via premalignant GSTp-pos lesions to hepatocellular carcinoma. The rate of replication of GSTp-pos cells is higher than in normal hepatocytes from the two-cell stage onwards and is further increased by treatment with various tumour promoters or increased food intake (15–17). This indicates an enhanced sensitivity of the initiated cells towards different exogenous and endogeneous growth stimuli. The present study describes a unique cell culture system of unaltered and initiated GSTp-pos hepatocytes (18,19). It allows to simulate the single components of epithelial–mesenchymal interactions in the first stages of hepatocarcinogenesis and to study whether the growth advantage of initiated hepatocytes is affected by NPC-derived cytokines.

On the basis of our own and other data, we selected tumour necrosis factor {alpha} (TNF{alpha}), interleukin-6 (IL-6), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF) and heparin-binding epidermal growth factor like growth factor (HB-EGF) as potential key-players in inflammation-driven hepatocarcinogenesis. All of these factors are upregulated in human inflammatory liver diseases (7,20–24). TNF{alpha} and IL-6 are important and early mediators in the hepatic acute phase response (3–8). Upon proinflammatory stimuli they are immediately secreted by KCs and ECs and trigger the production of further cytokines and growth factors, such as KGF and HGF (24–26). When released by NPCs, KGF and HGF exert their growth stimulatory effect exclusively on epithelial liver cells which suggests a central role of these factors as paracrine regulators of mesenchymal–epithelial interactions in the inflamed liver (3–7,24). HB-EGF is expressed in NPCs as an immediate early gene upon stimulation by LPS or wounding indicating importance for liver tissue repair (27). For TNF{alpha}, KGF, HGF and HB-EGF an unequivocal stimulatory effect on the growth of hepatocytes has been reported so far (28–35). However, none of the cytokines has been studied for possible DNA synthesis induction in the first stages of tumour development.

We found that DNA synthesis of initiated hepatocytes increases in the presence of NPCs, which is augmented by LPS-stimulation of NPCs, and that KGF, HGF and HB-EGF may be candidate factors responsible for the observed growth-stimulatory effect of NPCs. Since these cytokines stimulated DNA synthesis preferentially in GSTp-pos hepatocytes it is suggested that cytokines released by NPCs promote the development of cancer in chronically inflamed livers.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Animals and treatment
Male SPF Wistar rats, ~4 weeks old, were obtained from and kept at the ‘Decentralized Biomedical Facilities of the Medical University of Vienna’ under standardized SPF-conditions (diet: ssniffR/M-H-10mm-extrudated, V1536-000, Soest, FGR). N-nitrosomorpholine (NNM; Sigma, St Louis, MO) was dissolved in phosphate-buffered saline (PBS, pH 7.4) immediately before treatment and was applied as one dose (250 mg/10 ml PBS/kg body wt) by gavage. All experiments were performed according to the Austrian guidelines for animal care and protection, and were approved by the ‘Committee for Animal Protection’ of the Medical University of Vienna.

Primary cultures
Twenty-one days after NNM-treatment livers were perfused with collagenase (36). The cell suspension obtained was used to separate NPCs from parenchymal cells by low-speed centrifugation (70 g, 3 min, 4°C). Then the supernatant was centrifuged at 650 g for 7 min at 4°C. The cell pellet was resuspended in HBSS and centrifuged on a density cushion of Percoll (25% and 50%) at 1800 g for 15 min at 4°C. The NPC fraction located between 20% and 50% Percoll was collected, centrifuged at 650 g for 7 min at 4°C and resuspended in WE2 containing 10% FCS. The pellet obtained by the initial centrifugation served to prepare conventional hepatocytes (Hep-conv) via four low-speed sedimentation steps (15 g, 5 min, 4°C) and purified hepatocytes (Hep-pur) via one additional centrifugation (50 g, 10 min, 4°C) in 49% Percoll. Cells were seeded on collagen-coated Petri dishes, which allows attachment of hepatocytes and the different types of NPCs. For further details on cell separation and culture conditions, see refs (18,19).

Treatment of cultures
Stock solutions were prepared for IL-6 (100 µg/ml aqua bidest; Strathmann, Hamburg, Germany), TNF{alpha} [50 µg/ml PBS containing 0.1% bovine serum albumin (BSA); Biodesign, Carmel, NY], KGF (10 µg/ml PBS/0.1% BSA; Sigma), HGF (20 µg/ml PBS/0.1% BSA; Sigma), and HB-EGF (100 µg/ml PBS/0.1% BSA; Sigma). Stock solutions were added to the medium for final concentrations, as indicated below.

Immunostaining of culture plates
Primary antibodies and dilutions: rabbit anti-rat Yp-subunit of GSTp (1:5000; Biotrin-International, Dublin, Eire); rabbit anti-human albumin (1:500; DakoCytomation, Glostrup, Denmark); rabbit anti-human van Willebrand factor (1:70; Sigma); mouse anti-human {alpha}-smooth muscle actin (1:150; Dakocytomation), mouse anti-rat ED2 (1:4000; Serotec, Oxford, UK).

Cells in culture were fixed for 90 min at room temperature with 4% buffered formalin according to Lillie and were then kept in distilled water at 4°C until immunostaining. The staining procedure was as follows: hydrogen peroxide to block endogenous peroxidases (3%, 20 min, room temperature); primary antibodies were diluted in 2.5% BSA in TBS (0.05 M Tris, 0.3 M NaCl, pH 7.6) and were applied overnight at 4°C; rinsing with TBS; secondary antibodies were diluted in 2.5% BSA–TBS (alkaline-phosphatase-labelled goat-anti-rabbit IgG or alkaline-phosphatase-labelled goat-anti-mouse IgG, both: 1:100, DakoCytomation) and were applied for 90 min at room temperature; rinsing with TBS was followed by incubation with 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium chloride (Boehringer, Mannheim, Germany) for colour development. Omission of the primary antibodies served as control.

Histochemical detection of unspecific esterase in KCs followed the method of Horwitz et al. (37), except that {alpha}-naphthylbutyrate was used instead of {alpha}-naphthylacetate. Cells were counterstained with methylgreen.

Determination of vitality, DNA content and DNA replication
Vitality and DNA content of cultured cells was assessed by methods described by Labarca and Paiger (38) and Grusch et al. (39), respectively. For autoradiography newly synthesized DNA was labelled with [3H]thymidine (60–80 Ci/mmol; ARC, St Louis, MO, USA) which was added at 0.5 µCi/ml medium 24 h before harvesting. For further processing see ref. (18). In each individual experiment 2000 nuclei of GSTp-neg cells and 600 nuclei of GSTp-pos cells were evaluated. The labelling index (LI) was calculated as percentage of labelled hepatocyte nuclei per total number of hepatocyte nuclei counted. For biochemical determination of DNA replication, [3H]thymidine was added at 2 µCi/ml for 2 h. The reaction was stopped by placing the cultures on ice and aspiration of the medium. Cells were harvested and the incorporated radioactivity was assessed by scintillation counting.

Electron microscopy
Isolated cell fractions were pelleted, fixed in 2% glutaraldehyde, osmificated and embedded in Epon. Ultrathin sections were stained and processed as described by Bursch et al. (40).

Reverse transcription–polymerase chain reaction (RT–PCR)
Total RNA was extracted using Trizol-Reagent (Life-Technologies, Rockville, MD) and was transcribed applying random primer [p(dN)6; Roche, Mannheim, Germany] and MMLV reverse transcriptase (Sigma). For conventional PCR conditions, forward and reverse primers were as follows: rat KGF (forward: 5'-AGC-TAC-AGT-AGA-GGC-TCA-AG-3'; reverse: 5'-GGA-TCC-GTG-TCA-GTA-TCC-AT-3'; annealing temperature 57°C), rat HGF (forward: 5'-GTG-TGC-CAA-CAG-GTG-CAT-CA-3'; reverse: 5'-ACC-GTT-GCA-GGT-CAT-GCA-TT-3' annealing temperature 58°C), rat HB-EGF (forward: 5'-ATG-AAG-CTG-CTG-CCG-TCG-GTG-GT-3'; reverse: 5'-ACC-GCC-ATC-TCA-GAA-GTA-GCC-3'; annealing temperature 65°C). PCR products were electrophoresed in 1.2% agarose gels, stained with ethidium bromide and visualized by UV-light.

Quantitative RT–PCR
TaqMan-based ‘Assays-on-demand’ were obtained from Applied Biosystems (Foster City, CA) for rat ß2-microglobulin and rat HB-EGF. The ABI-Prism PCR standard protocol was applied on an ABI-Prism/7000 Sequence Detection System. All data were analysed in duplicates. Computer-assisted quantification of mRNA levels was carried out with ABI-Prism/7000 SDS-software; mRNA levels were normalized with respect to ß2-microglobulin as an internal standard.

Assay of superoxide release
NPCs (0.15 x 106/well) were seeded in 96-well microplates and 10 ng/ml LPS (Sigma) or 16 µM Phorbol-12-myristate-13-acetate (PMA; Sigma) were added. Superoxide was measured using the assay based on reduction of cytochrome c by superoxide, as described in detail elsewhere (41).

ELISA of TNF{alpha}
TNF{alpha} levels in cell culture supernatants were measured by the rat TNF{alpha} Module Set (Bender MedSystems, Vienna, Austria) according to the manufacturer's instructions. Further materials not included in the module set are tetramethylbenzidine-peroxidase-substrate solution for colour development (KPL, Gaithersburg, MD) and microtiter plates (No 3590; Costar, Corning, NY).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Primary cultures of liver cells
Twenty-one days after NNM-treatment, cells were isolated from the livers by collagenase perfusion and three different preparations of cells were made: Hep-conv still containing NPCs, Hep-pur and purified NPCs.

The different cell types within the preparations were identified by staining for albumin (hepatocyte marker), {alpha}-smooth muscle actin and Van Willebrand factor (EC markers), and for ED-2 and unspecific esterase (KC markers) (Figure 1). The immunostains served to determine relative frequencies of the different cell types. At 48 h KCs and ECs together constitute the majority of the NPCs in primary culture with KCs being ~5-times more frequent than ECs. Stellate cells were seen occasionally (Figure 2). The incidence of contaminating NPCs in Hep-pur was only one-tenth (0.5 ± 0.5%; n = 4) when compared with Hep-conv (4.7 ± 3.8%; n = 6). The NPC fraction was ~98% pure as assessed by electron microscopy, albumin-immunostains and by using the hepatocyte-specific cleavage of resorufin-O-glucoside to resorufin (Figures 1 and 2) (19).



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  		Fig. 1. Parenchymal and non-parenchymal liver cells isolated from an NNM-treated rat liver. In (A) and (B) cultured GSTp-pos hepatocyte (blue) 72 h after isolation showing incorporated [3H]thymidine (black spots). In (C–F) non-parenchymal cells were isolated and cultured for 24 h: KCs stained for ED2 (blue) in (C) and unspecific esterase (red) in (D); ECs stained for {alpha}-smooth muscle actin (blue) in (E) and for van-Willebrand factor (blue) in (F); Magnifications: 1:150 (A, C–F). 1:40 (B). See online Supplementary material for a colour version of this figure.

 


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  		Fig. 2. Electron microscopy of NPC fraction. Numbers indicate KCs (1), stellate cells (2), ECs (3) and neutrophil granulocytes (4). Magnification, 1:2000.

 
DNA content, vitality and functions of NPCs in primary culture
Unlike Hep-conv and Hep-pur, DNA content and vitality of NPCs decreased between 28 and 48 h of culture (Figure 3). We checked whether isolated NPCs remain functionally active after isolation. Upon stimulation by LPS or PMA, NPCs were capable of enhancing the production of superoxide and TNF{alpha} (Figures 4 and 5). Basal generation of superoxide was negligible, whereas spontaneous TNF{alpha} release in control and NNM-pretreated NPCs was considerable. Following NNM-treatment the capacity to release TNF{alpha} was reduced, to release superoxide was enhanced.



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  		Fig. 3. DNA content and vitality of conventional and purified cultures of primary hepatocytes and of the NPC fraction. In (A) the data of the 6 h time point of investigation were arbitrarily set 1.00 and served to normalize the data of the subsequent time points. In (B) vital cells were identified as described by Grusch et al. (39) and their incidences (%) were determined by counting 2000 cells per plate. In (A) and (B) means ± SD from five (DNA content of NPCs), four (vitality of NPCs and Hep-pur) or two (DNA content of Hep-pur and Hep-conv) separate liver cell preparations are given. Statistics over time-course by Kruskal–Wallis test: aP < 0.05.

 


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  		Fig. 4. Release of superoxide by NPCs from control or NNM-treated livers. Isolated and purified NPCs remained untreated or were exposed to 10 ng LPS/ml medium or 16 µM PMA. The absorbance was read after 20 min of incubation. Means ± SD from four separate NPC-preparations are given. Statistics by Wilcoxon's test for control versus NNM-pretreatment: aP < 0.05.

 


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  		Fig. 5. Release of TNF{alpha} by NPCs from control or NNM-treated livers. Isolated and purified NPCs remained untreated or were exposed to 10 ng of LPS/ml medium. The release of TNF{alpha} to the medium was determined by ELISA (for details see Materials and methods). Symbols: NPCs from untreated rats (open circles) or from NNM-treated rats (closed circles). Means ± SD from four separate NPC-preparations are given. Statistics by Kruskal–Wallis test over time-course for control versus NNM-pretreatment: aP < 0.05.

 
NPCs stimulate DNA synthesis of GSTp-neg and GSTp-pos hepatocytes
After treatment with the genotoxic hepatocarcinogen NNM, single GSTp-pos cells and small preneoplastic GSTp-pos cell foci are formed in the liver (15). On day 21 cells were isolated and cultivated. GSTp-pos hepatocytes showed a ~4-fold higher rate of DNA replication as GSTp-neg cells. This indicates an inherent growth advantage of the first stages of hepatocarcinogenesis (Figure 6), as shown earlier (18).



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  		Fig. 6. DNA synthesis [LI (%)] in GSTp-neg and GSTp-pos hepatocytes prepared conventionally or in purified mode. Cells were cultivated for 72 h; [3H]thymidine was added 24 h before harvesting. DNA synthesis was determined by autoradiography. Symbols: GSTp-neg cells (open squares); GSTp-pos cells (closed squares). Means ± SD from 12 separate liver cell preparations are given. Statistics by Kruskal–Wallis test for GSTp-neg versus GST-pos cells over time-course: aP < 0.01; statistics by Wilcoxon's test for GSTp-neg cells, GSTp-pos cells and each time point (Hep-conv versus Hep-pur): bP < 0.05.

 
To reduce the contamination by NPCs, isolated hepatocytes were purified by density gradients (19). In the resulting culture (Hep-pur) the incidence of replicating GSTp-neg and GSTp-pos cells was decreased to ~30% when compared with cells in conventional cultures (Figure 6). Then, NPCs were added to purified hepatocytes at an equal ratio which approaches the relative frequencies in the intact liver. This elevated DNA synthesis in both GSTp-neg and GSTp-pos hepatocytes (Figure 7). When supernatant of unstimulated or LPS-stimulated NPCs was added to Hep-pur, DNA replication of primary hepatocytes was raised dramatically (Figure 7). These results provide strong evidence that the factors released from NPCs are soluble and overall are stimulators rather than inhibitors of hepatocyte growth. Furthermore, they suggest that activation of NPCs by proinflammatory events, such as exposure to LPS, favours growth of the first stages of hepatocarcinogenesis. Similar mechanisms may underlie the tumour-enhancing effect of inflammatory liver diseases.



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  		Fig. 7. Effect of coculture with NPCs or NPC-supernatant on DNA synthesis [LI (%)] of hepatocytes. In coculture experiments, NPCs were plated at a density of 300 000 viable cells/dish. After 1 h, 300 000 viable purified hepatocytes were added to the plates. In experiments with NPC supernatant, 3 000 000 viable NPCs/dish were allowed to attach for 1 h with 10% FCS and were subsequently exposed to 10 µg of LPS/ml medium (Sigma) for 30 min. Cells were washed and kept serum-free. After 4 h the NPC-supernatant was taken to replace the medium of the primary hepatocyte cultures. Under any condition cells were cultivated for 72 h; [3H]thymidine was added 24 h before harvesting. DNA synthesis was determined by autoradiography. Abbreviation: Sup, supernatant. Means ± SD from four separate liver cell preparations are given. Statistics by Wilcoxon's test: Hep-conv versus Hep-pur aP < 0.05; Hep-pur versus NPCs, supernatant, or LPS-supernatant: bP < 0.05; cP < 0.01.

 
IL-6 and TNF{alpha} exert moderate effects on DNA synthesis of GSTp-neg and GSTp-pos hepatocytes
We investigated the effects of IL-6 and TNF{alpha}, proinflammatory cytokines released by NPCs. At the highest concentration of IL-6 we found a 2-fold, barely significant increase in DNA synthesis (Figure 8). When discriminating the replication rate of GSTp-neg and GSTp-pos hepatocytes no stimulatory effect of IL-6 was evident.



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  		Fig. 8. Effect of IL-6 on DNA synthesis in GSTp-neg and GSTp-pos hepatocytes. Treatment of cultures commenced 4 h after plating (time point 0) and was renewed with a medium change at 48 h. Cells were cultivated for 72 h; [3H]thymidine was added 2 h in (A) and 24 h in (B) before harvesting. DNA synthesis was determined by scintillation counting (A) or separately in GSTp-neg and GSTp-pos cells by autoradiography (B). Symbols: GSTp-neg cells (open bars), GSTp-pos cells (closed bars). Means ± SD from four separate liver cell preparations are given. Statistics of dose-dependent effects by Kruskal–Wallis test: aP < 0.05.

 
TNF{alpha} induced a ~2-fold increase of DNA synthesis in both Hep-conv (x2.46 ± 0.9; n = 6, Wilcoxon's-test: P < 0.05) and Hep-pur (x2.27 ± 1.05; n = 6; P < 0.05) at 50 ng/ml medium. In a dose–reponse study TNF{alpha} stimulated DNA replication rather in the GSTp-pos cells than in the unaltered ones, which reached significance in Hep-pur only (Figure 9).



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  		Fig. 9. TNF{alpha}, KGF, HGF and HB-EGF induce DNA synthesis in GSTp-neg and GSTp-pos hepatocytes. Treatment of cultures with the factors commenced 4 h after plating (time point 0) and was renewed with a medium change at 48 h. Cells were cultivated for 72 h; [3H]thymidine was added 24 h before harvesting. DNA synthesis was determined by autoradiography. Symbols: GSTp-neg cells (open squares), GSTp-pos cells (closed squares). Means ± SD from four to six separate liver cell preparations are given. Statistics of dose-dependent effects by Kruskal–Wallis test: aP < 0.05; bP < 0.01.

 
HGF, KGF and HB-EGF are potent paracrine inducers of DNA synthesis of GSTp-neg and GSTp-pos hepatocytes
The mRNAs of HGF, KGF and HB-EGF were detectable in the NPC-fraction, but not in purified hepatocytes (Figure 10). This confirms that the factors are produced by the NPC-compartment.



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  		Fig. 10. Expression analyses of mRNAs of KGF, HGF and HB-EGF in Hep-pur and in unstimulated and LPS-stimulated NPCs.

 
HGF, KGF and HB-EGF all potently stimulated DNA replication of hepatocytes (Figure 9). This effect tended to be more pronounced in Hep-pur than in Hep-conv, probably because of the lower basal levels in DNA replication of Hep-pur. At the maximal concentration tested, the strongest induction of DNA synthesis in GSTp-pos hepatocytes, prepared conventionally or in purified mode, was found with HB-EGF, followed by HGF and KGF. Sixty percent of GSTp-pos hepatocytes in Hep-conv were stimulated by HB-EGF to replicate DNA, which is remarkably high for hepatocytes in primary culture. At the lowest concentrations of KGF, HGF and HB-EGF, the induction of DNA replication in Hep-pur was ~1.5-fold for GSTp-neg cells and ~4-fold for GSTp-pos hepatocytes. This indicates an almost selective response of the initiated cells to the growth factors at concentrations in the physiological range. At supraphysiological concentrations of the factors the replication rate of GSTp-neg cells came close to that of the initiated cells. In any case absolute increases in the pool of replicating cells were highest for the GSTp-pos hepatocytes. In conclusion, in this model system the mesenchymal factors KGF, HGF and HB-EGF induce a strong growth response preferentially in initiated hepatocytes, whereas IL-6 and TNF{alpha} exerted marginal effects.

LPS elevates HB-EGF expression in NPCs
LPS stimulation increased the mRNA of HB-EGF in NPCs x2.7 ± 1.2, as determined by conventional and quantitative RT–PCR (n = 3, Wilcoxon's test: P < 0.05). The mRNAs of KGF and HGF remained unaltered (Figure 10). This is consistent with the observation that these two factors are released in cases of demand by proteolytic activation of their precursors (5,24).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
The present paper describes the importance of NPCs and NPC-derived (inflammatory) cytokines for growth stimulation of the very first stages of hepatocarcinogenesis. There are numerous candidate factors possibly involved in this process. TNF{alpha} is one of the main NPC-derived mediators of the acute phase response. The maximum effect of TNF{alpha} on DNA synthesis was not more than 2-fold, as also shown by previous studies (28,30,31). Hasmall et al. (42) reported that removal of NPCs from Hep-conv prevented the TNF{alpha}-induced increase in DNA synthesis and the authors considered the NPC fraction as an important source of a cofactor required by TNF{alpha}. However, in the present study there was even a tendency towards higher induction of DNA replication by TNF{alpha} in Hep-pur than in Hep-conv. It appears possible that NNM-pretreatment altered the liver cells, e.g. NPCs derived from control livers produced considerably more TNF{alpha} and less superoxide than NPCs isolated from NNM-pretreated livers (Figures 4 and 5). Possibly hepatocytes adapted to the altered functions of the NNM-pretreated NPCs and showed reactions deviating from those of control hepatocytes.

The release of TNF{alpha} from KCs is associated with the enhanced formation of ROS by superoxide-generating NADPH oxidase (4,7,12). The resulting ROS are involved in host defence against pathogens. They may also cause DNA damage and thus are considered to be potential mutagens (11,43). Accordingly, deletion of the superoxide production in KCs of P47-PHOX–/– mice, harbouring a defective NADPH oxidase, reduces damage of liver DNA and cytotoxicity by the hepatocarcinogen diethylnitrosamine (12). The impact of an impaired superoxide production on hepatocarcinogenesis is currently under investigation.

Under the present experimental conditions the effect of IL-6 on DNA replication of hepatocytes was insignificant which agrees with previous data (5,7). Moreover, our study did not reveal an effect of this cytokine on initiated liver cells. It was suggested that the presence of IL-6 is essential for full hepatocyte replicative response towards other, yet unknown factors, e.g. in IL-6 –/– mice the regenerative response of the liver was impaired severely, an effect prevented by the application of IL-6 before partial hepatectomy (44). Thus IL-6 may exert different effects depending on absence/presence of essential cofactors in the medium. However, under our conditions the presence of NPCs provides multiple growth factors/cytokines in primary hepatocyte cultures, but did not reveal any (co)mitogenic activity of IL-6 on unaltered and initiated hepatocytes (Figure 8).

TNF{alpha} and IL-6 may rather prime for than directly induce the growth response in the liver, e.g. TNF{alpha} induces IL-6 expression in the liver, which in turn elevates the production of KGF and HGF (3–7,24,26). KGF appears to be one of the actual growth signals; it induced DNA replication of hepatocytes in vitro and in the intact rat (32,33). Our study indicates that initiated hepatocytes show a preferential response towards the growth stimulatory effect of this cytokine, which may also occur in inflammatory liver disease. KGF also protects hepatocytes in vivo and in vitro from apoptosis induced by the combined action of TNF{alpha} and inhibitors of transcription (24,45). This suggests that KGF is induced by proinflammatory cytokines as a kind of counteracting ‘anti-inflammatory shield’ resulting in accelerated reepithelialization and reduced cell death. Thus, a significant protective role of KGF for epithelial cells in an inflamed liver appears likely. Escape from apoptosis, together with the profound growth stimulatory effects, may promote outgrowth of initiated hepatocytes to (pre)malignant cell clones.

HGF is a further paracrine growth factor that is found here to act preferentially on initiated hepatocytes. Whereas 16% of the unaltered hepatocytes were induced to undergo DNA replication, 33% of the initiated hepatocytes were recruited to the pool of cycling cells. It has been suggested that the effects of HGF on DNA synthesis are mediated by prostaglandins (46). KCs and ECs are rich sources for prostaglandins, which are released upon stimulation with LPS or other proinflammatory stimuli (7). However, PGE2 and PGF2{alpha} induced DNA replication almost exclusively in the GSTp-neg cell population, whereas GSTp-pos cells remain largely unaffected (47). However, prostaglandins A2 and J2 depressed DNA synthesis preferentially in premaligant, GSTp-pos hepatocytes (47). It, therefore, appears unlikely that the strong growth stimulating effects of HGF on the initiated hepatocytes are mediated by prostaglandins. The enhanced sensitivity of the initiated cells towards HGF may be because of an elevated level of the HGF-receptor c-MET. At least in hepatocellular carcinoma of rats and humans this receptor is often upregulated (48).

Among the factors studied in the present work, HB-EGF was the most potent paracrine growth stimulator of initiated hepatocytes. In subsequent experiments, we found that equimolar concentration of EGF were similarly effective as HB-EGF on DNA replication of GSTp-pos hepatocytes (C.Drucker, manuscript in preparation). Thus, HB-EGF is a further member of the EGF-family, that may act as a potent endogenous tumour promoter, if similarly effective in vivo. In rodents and humans intrahepatic mRNA and plasma levels of HB-EGF rise considerably during regenerative liver growth (23,27,49). The main sources of HB-EGF appear to be ECs and KCs, which produce the factor upon stimulation by TNF{alpha}, LPS, or lysophosphatidylcholine, all of which are present at sites of inflammation (27). We observed that in LPS-stimulated NPCs HB-EGF mRNA induction occurred within half an hour, as is characteristic for immediate early genes. HB-EGF in turn induces the expression of HGF and VEGFA and appears to be among the key players in the cytokine/growth factor network in the diseased liver (49,50). The importance of HB-EGF for hepatocarcinogenesis is also emphasized by the fact, that it turns from a paracrine to an autocrine growth factor, preferentially produced by (pre)malignant hepatocytes. Accordingly, HB-EGF is upregulated in hepatoma cells and hepatocellular carcinoma of various mammalian species including humans (27,51).

In conclusion, the present work shows that a comprehensive understanding of the cytokine-cascades in the hepatocyte microenvironment may provide valuable tools to control inflammatory events and the concomitant development of liver cancer.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary material is available online at: http://www.carcin.oxfordjournals.org.


   Acknowledgments
 
The excellent technical assistance of B.Peter-Vörösmarty, K.Bukowska, H.Koudelka and B.Mir-Karner is gratefully acknowledged. This study was supported by ‘Herzfeldersche Familienstiftung’, Bürgermeisterfonds der Stadl Wien (study No 2338) and by the Austrian ‘Gen-Au Programme’ (study No GZ 200.058/6-VI/2/2002).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received March 22, 2005; revised July 27, 2005; accepted July 28, 2005.


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