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Apoptotic Response to TGF-β in Fetal Hepatocytes Depends upon Their State of Differentiation
Experimental Cell Research 252, 281–291 (1999) Article ID excr.1999.4624, available online at http://www.idealibrary.com on
Apoptotic Response to TGF-b in Fetal Hepatocytes Depends upon Their State of Differentiation ´ lvarez,† J. M. Lo´pez Pedrosa,‡ C. Roncero,* M. Benito,* and I. Fabregat* ,1 A. Sa´nchez,* A. M. A *Departamento de Bioquı´mica y Biologı´a Molecular II, Centro Mixto CSIC/UCM and †Centro de Citometrı´a de Flujo y Microscopı´a Confocal, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain; and ‡Departamento I1D, Abbot Laboratories, 18004 Granada, Spain
Transforming growth factor-b (TGF-b1) induces death of fetal hepatocytes by an apoptotic mechanism. However, even when very high concentrations and/or long-term exposure to the cytokine is used, 40 –50% of cells always survive. The process of cell survival is coincident with changes in morphology and phenotype, with cells showing a fibroblastic appearance and eliciting an epithelial–fibroblastic transition. Surviving cells continue responding to TGF-b in terms of growth control. Expression of liver-specific genes is very low in these cells; this effect is due to the decrease in their rate of transcription as soon as 2 h after the addition of the factor. Surviving cells present a decreased DNA binding activity for liver-enriched transcription factors, an increased DNA binding activity for AP-1, and a high expression of protooncogenes. These cells are immature hepatocytes since in the presence of the appropriate signal (i.e., epidermal growth factor), they can differentiate, organizing in cell clusters and increasing both liver-specific mRNA expression and liver-enriched transcription factor activity. In accord with these results, TGF-b, secreted at high concentrations during liver carcinogenesis, might induce death of normal cells while providing a selective advantage for the survival of cells that are “partially transformed” or “less differentiated” and unresponsive to the factor. © 1999 Academic Press Key Words: hepatocyte; TGF-b; apoptosis; differentiation; hepatocarcinogenesis.
INTRODUCTION
Apoptosis seems to be the predominant type of active cell death in the liver [1]. Endogenous factors, such as transforming growth factor-b (TGF-b1), activin A, Fas ligand, and tumor necrosis factor-a (TNF-a), may be involved in the induction of apoptosis [2]. The action of these death factors seems to be counteracted by growth factors, such as epidermal growth factor (EGF) [3], 1 To whom correspondence and reprint requests should be addressed. Fax: 34-91-3941779. E-mail: [email protected].
hepatocyte growth factor [4], or keratinocyte growth factor [5]. During the first stages of hepatocarcinogenesis in rat, apoptotic activity gradually increases from normal liver to putative preneoplastic foci [6]. Preneoplastic liver cells are even more susceptible than normal hepatocytes to stimulation of cell death, suggesting that tumor initiation, at the organ level, can be reversed by preferential elimination of initiated cells [6]. Tumor promoters and nongenotoxic carcinogens inhibit active cell death, thereby increasing the accumulation of preneoplastic cells and accelerating the development of cancer [2, 6]. The TGF-b family comprises a large number of structurally related polypeptide factors, each capable of regulating several cellular processes, including cell proliferation, lineage determination, differentiation, motility, adhesion, and death [7]. TGF-b has been proposed to play an important role in hepatocarcinogenesis, however, much controversy exits as to its exact contribution. Indeed, it has been proposed that TGF-b secreted during liver carcinogenesis may inhibit liver cell proliferation [8], suppress transformation [9, 10], and induce apoptosis [11]. Disruption of the TGF-b pathway at the prereceptor, receptor, or postreceptor levels occurs in hepatocellular carcinoma (HCC) and can cause dysregulation of apoptosis [12]. However, some findings also suggest an association of the activated TGF-b gene transcription with progression of hepatocarcinogenesis: (i) human HCC strongly expresses TGF-b mRNA and protein in vivo [13]; (ii) an elevated urinary TGF-b level, used as a tumor marker, is related to poor survival in cirrhotic HCC [14]; (iii) constitutive expression of mature TGF-b in the liver accelerates hepatocarcinogenesis in transgenic mice [15]; and (iv) TGF-b promotes spontaneous transformation of cultured rat liver epithelial cells [16]. Fetal rat hepatocytes in primary culture are immature cells capable of carrying out both proliferation and differentiation processes simultaneously. We have shown that TGF-b inhibited growth of fetal hepatocytes, arresting cells in G1 and down-regulating c-myc expression [17]. Furthermore, TGF-b acted synergisti-
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0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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cally with EGF, promoting morphological changes related to differentiation of these cells [18]. In addition to these functions and when used at higher concentrations, TGF-b induced fetal hepatocyte apoptosis [19] by a mechanism dependent on protein synthesis [20]. The dose and time dependence study of this cell death process showed that, regardless of the TGF-b concentration and the duration of exposure to the cytokine, 40 – 50% of cells always survived. In this work we have tried to analyze the morphological and molecular characteristics of these “TGF-b-resistant” cells. This population of cells responds to TGF-b in terms of growth inhibition but not in terms of cell death and appears to be composed of less-differentiated hepatocytes. The relevance of these findings in hepatocarcinogenesis will be discussed. MATERIALS AND METHODS Materials. Human recombinant TGF-b was from Calbiochem (La Jolla, CA) and human recombinant EGF was kindly provided by Serono Laboratories (Madrid, Spain). Fetal and neonatal calf serum and culture media were from Imperial Laboratories (Hampshire, UK). Radiochemicals were from ICN (Irvine, CA). The multiprimer DNA labeling system was from Amersham (Buckinghamshire, UK). The nick-translation labeling system was from Gibco BRL (Gaithersburg, MD). Other reagents were from Sigma Chemical Co. (St. Louis, MO) or Boe¨hringer Mannheim (Mannheim, Germany). FITCconjugated sheep anti-mouse immunoglobulins and monoclonal antivimentin (clone V9) were obtained from Boe¨hringer Mannheim. TRITC-conjugated goat anti-rabbit immunoglobulins were obtained from Dako Corp. (Santa Barbara, CA). Professor O. Bachs and Dr. R. Bastos (Barcelona, Spain) kindly provided polyclonal anti-cytokeratin 18 (CK18). Immunofluorescence mounting medium was from ICN Biomedicals Inc. (Costa Mesa, CA). Cell isolation and culture. Hepatocytes from 20-day-old fetal Wistar rats were isolated as previously described by collagenase disruption (2.5 3 10 6 cells/fetus) [21] and plated on plastic (noncoated) dishes in arginine-free medium 199, supplemented with ornithine (200 mM), fetal calf serum (10%), penicillin (120 mg/ml), and streptomycin (100 mg/ml). Cells were incubated in 5% CO 2, at 37°C for 4 h, allowing cell attachment to plates. Media were changed at that time and replaced by medium of the same composition except that 10% fetal calf serum was replaced by 2% newborn bovine serum. After 18 –20 h, the medium was again replaced with medium of identical composition but in the absence of serum. Two hours later, cells were ready for all the experiments described below. Light microscopy. After incubation of cells in the absence or in the presence of the different factors, they were washed twice with PBS and fixed in Bouin (71% picric acid/24% formaldehyde/5% acetic acid) solution at room temperature for 30 min. Fixative was then removed and cells were extensively rinsed with 70% ethanol and finally with PBS. Cells were visualized in a Nikon Eclipse TE 300 microscope and photographed using Kodak Elite chrome 100 films. TGF-b-mediated cytotoxicity assay. After cell incubation in the absence or in the presence of different factors, the medium was discarded and the remaining viable adherent cells were stained with crystal violet (0.2% in 2% ethanol) for 20 min, as previously described [17]. Remaining viable cells were calculated as the percentage absorbance with respect to control cells (incubated in the absence of growth factors). Analysis of DNA fragmentation. Rat fetal hepatocytes were washed twice with PBS, scraped, and pelleted at 4°C. Cells were
resuspended in 500 ml of buffer containing 10 mM EDTA, 0.25% Triton X-100, 2.5 mM Tris–HCl, pH 8.0, and stored at 4°C for 15 min. Intact nuclei were pelleted and eliminated by centrifugation at 500g for 10 min and the supernatant was centrifuged at 25,000g at 4°C for 30 min. DNA in the supernatant was precipitated at 280°C after addition of 2 vol of ethanol (70% final concentration), pelleted by microcentrifugation at 4°C for 15 min, dried, resuspended in 200 ml of 10 mM Tris–HCl, 1 mM EDTA, pH 8.0 (TE buffer), and incubated at 37°C for 30 min with 0.1 mg/ml RNase A and for 2–3 h with 0.25 mg/ml Proteinase K. DNA was purified by phenol– chloroform extraction and precipitated at 270°C after (1/10 vol) 3 M sodium acetate, pH 5.3, and (2 vol of) ethanol were added. Precipitated DNA was dissolved in TE buffer containing 30% glycerol, 1 mg/ml ethidium bromide and electrophoresed in a 1.5% agarose gel. Gel was visualized and photographed under transmitted UV light with a Polaroid camera. Confocal microscopy studies. For immunofluorescence detection of intermediate filaments (vimentin and cytokeratins), cells were washed twice with PBS, fixed in methanol (220°C) for 2 min, and processed for double immunofluorescence [18], by applying a mixture of mouse and rabbit primary antibodies, followed by extensive washing and incubation with a mixture of FITC-conjugated anti-mouse and TRITC-conjugated goat anti-rabbit. Primary antibodies were applied for 1 h at 37°C, followed by 43 5 min washes in PBS, a 45-min incubation with fluorescence-conjugated antibodies, and four final washes of 5 min each in PBS. For detection of actin filaments, cells were washed with PBS and fixed in 3% paraformaldehyde for 30 min at room temperature. Following this period, after the cells were washed with PBS, they were stained with TRITC-conjugated phalloidin that binds F-actin filaments. This probe was reconstituted in 60% methanol/40% DMSO at a concentration of 100 mg/ml and diluted 1/500 before use with PBS, 0.1% BSA, 0.01% azide, pH 7.2. It was applied for 1 h at 37°C in a humidified atmosphere in the dark, followed by extensive washing with PBS. For visualization, sections of the plates were coverslipped using immunofluorescence medium and examined in an MRC-1024 (Bio-Rad, Hempstead, and UK) confocal microscope adapted to an inverted Nikon Eclipse TE 300 microscope. Images were taken with 488-nm laser excitation for FITCconjugated antibodies and 514-nm laser excitation for TRITCconjugated antibodies. Fluorescence emissions were detected through a 513/24-nm bandpass filter for FITC and a 605/15-nm bandpass filter for TRITC. DNA synthesis assay. After incubation of the cells for 48 h in the absence or in the presence of the mitogen, DNA synthesis was evaluated by [ 3H]thymidine incorporation into TCA-precipitable material during the last 40 h, as we have previously described [22]. Analysis of cells’ DNA content by flow cytometry. The ploidy determination of hepatocytes was estimated by flow cytometry DNA analysis as previously described [19]. Cells were detached from dishes by addition of 0.25% trypsin– 0.02% EDTA and the DNA content per cell was evaluated in a FACScan flow cytometer (BectonDickinson, San Jose, CA), after cells were stained with propidium iodide, using Kinesis 50 (Bio-Rad). For the computer analysis, only signals from single cells were considered (10,000 cells/assay). RNA isolation and Northern blot analysis. Total RNA was isolated as described by Chomczynski and Sacchi [23]. For each assay, RNA was extracted from the pooled cells of two 92-mm-diameter dishes, denatured in 50% formamide, 2.2 M formaldehyde, 20 mM Mops, pH 7.0, 6% glycerol at 65°C for 15 min, separated by size on gels containing 0.9% agarose and 0.66 M formaldehyde, and blotted on GeneScreen membranes (NEN Research Products, Dupont, Boston, MA). cDNA probe origin and hybridization conditions were previously described [3, 17, 24]. a-Fetoprotein, albumin, and 18S ribosomal cDNAs were labeled with [a- 32P]dCTP by nick-translation, whereas c-fos, c-jun, c-myc, H-ras, and HNF-4 were labeled by random priming reaction. Serial hybridization with the different probes was performed successively.
TGF-b HEPATOCYTE DEATH AND DIFFERENTIATION Nuclear preparation and gel mobility shift assay. DNA-binding protein extracts from fetal hepatocytes were prepared as we previously described [18, 19], starting with 5 3 10 6 cells. The cell suspension was resuspended in 10 mM Hepes–KOH, pH 7.9, 1.5 mM MgCl 2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM PMSF (Buffer A), at 4°C, allowed to swell on ice for 10 min, and then vortexed for 10 s. Samples were centrifuged at 14,000g for 10 s and the supernatant was discarded. The pellet was resuspended in cold Buffer C (20 mM Hepes–KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl 2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF, and 0.75 mg each of leupeptin, antipain, and aprotinin per milliliter) and incubated on ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation at 14,000g for 2 min at 4°C and the supernatant fraction was stored at 280°C. Proteins were measured using the Bio-Rad protein reagent following the recommendations of the supplier. The gel mobility shift assay was performed essentially as described by Wen and Locker [25]. Synthetic oligonucleotides were prepared using an oligonucleotide synthesizer (Pharmacia Biotech Inc.) and with the sequences that we described previously [18, 19]. Labeling was performed by using Klenow polymerase and a- 32Plabeled deoxyadenosine triphosphate. The binding reaction mixture contained 0.5 ng of doubled-stranded oligonucleotide probe, 2 mg of poly (dI– dC), and 5 mg protein in Buffer C supplemented with 35 mM MgCl 2. After a 20-min incubation at 4°C, the mixture was applied to a 6% polyacrylamide gel. Gels were run at 0.8 V/cm 2 in 0.53 Tris– borate–EDTA running buffer for 2–3 h, at room temperature. The dried gel was then autoradiographed. Nuclear run-on transcription assays. Nuclei were isolated from the pooled cells of 10 –12 92-mm-diameter plates [26] and stored at 270°C in 50 mM Hepes, pH 7.9, 150 mM NaCl, 0.1 mM EDTA, 5 mM dithiothreitol, 0.125 mM phenylmethylsulfonyl fluoride and 50% glycerol. 32P-labeled RNA transcripts were purified by the method of Linial et al. [27] using NICK columns (Pharmacia LKB Biotechnology Inc.) and following the manufacturers instructions. In vitro elongation reactions, denaturation of DNA probes, application of DNA to GeneScreen membranes, hybridization of the 32P-labeled RNA transcripts to the membrane– bound DNA, posthybridization washes, and autoradiography were carried out as before [24]. We used the same cDNAs probes than in Northern blot experiments, except in the case of AFP, where a genomic probe (a gift of Dr. J. L. Danan, Paris) was used to increase the efficiency of hybridization.
RESULTS
Effect of TGF-b on Rat Fetal Hepatocyte Death When fetal hepatocytes in primary culture were incubated in the presence of 2 ng/ml TGF-b for 24 h, several changes occurred. First, between 12 and 24 h after the addition of the factor, around 50% of the cells died by apoptosis and detached from the plastic (Fig. 1). This process was coincident with the appearance of DNA cleavage that presented a peak around 8 –16 h (Fig. 1C). We never could find a DNA ladder at later times (Fig. 1C), even when new TGF-b was added (results not shown). Higher concentrations of TGF-b or exposure for longer times did not increase the percentage of cells that responded to the factor (Fig. 1B). Second, cells suffered changes in morphology in response to TGF-b. In controls (serum-free medium without any addition) small clusters of hepatocytes could be observed (Fig. 1A, top). In contrast, TGF-b-treated
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cells progressively acquired a fibroblast-like aspect; they were flat, were spindle-like, and migrated to the empty spaces. After 24 h of treatment, all the cells that were not sensitive to the apoptotic effect of TGF-b appeared to have a fibroblastic appearance (Fig. 1A, bottom). Changes in Expression of Cytokeratin and Vimentin Intermediate Filaments and Cytoskeleton Distribution Induced by TGF-b in Fetal Hepatocytes Hepatocytes are epithelial cells; thus, in differentiated states and under normal physiological conditions, they present only cytokeratin as the intermediate filament. In contrast to this, fibroblastic cells or transitional cells express vimentin [28, 29]. We decided to characterize the intermediate filament expression in TGF-b-treated cells. We present the results obtained by using double immunofluorescence with anti-cytokeratin and anti-vimentin on control (untreated) and TGF-b-treated (24 h) fetal hepatocytes (Figs. 2A and 2B). As can be observed, control cells organized in clusters were only cytokeratin positive (Fig. 2A). However, TGF-b treatment induced a progressive increase in the number of vimentin-positive cells. Coexpression of vimentin and cytokeratin filaments could be observed in most of the fibroblast-like cells (Fig. 2B). Vimentin expression was absent in the cells undergoing the death process. Actin assembly also changes during epithelial–mesenchyma transitions. For this, we decided to study the effect of apoptotic concentrations of TGF-b on actin distribution. Laser confocal microscopy observations allowed us to visualize the distribution of F-actin in cells stained with rhodamine–phalloidin (Figs. 2C and 2D). In controls (untreated cells), hepatocytes in the middle of the clusters presented a ring-like distribution, which is typical of epithelial cells that organize actin bundles in areas of cell to cell contact (Fig. 2C). In contrast, fibroblast-like cells that survive to TGF-binduced apoptosis presented abundant stress fibers. The ring-like cortical F-actin was not observed, except in those cells that were rounded and in the process of death (Fig. 2D). Proliferative and Apoptotic Response of TGF-bPretreated Hepatocytes To compare the growth properties of the two cell populations, i.e., control (untreated) and TGF-b-pretreated hepatocytes, we decided to analyze the proportion of cells in each phase of the cell cycle. After treatment for 24 h with 6TGF-b, cell DNA content was estimated by flow cytometry as described under Materials and Methods. As shown in Fig. 3A, most of the fetal hepatocytes are in the G1 phase of the cell cycle.
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FIG. 1. Effect of TGF-b on rat fetal hepatocyte death. Fetal rat hepatocytes were isolated, plated, and treated as described under Materials and Methods. (A) Light microscopy photographs of cells incubated for 24 h in the absence (control) or in the presence of 2 ng/ml TGF-b (TGF-b). (B) Cell viability at different times after TGF-b addition. Two different concentrations of the factor were used: (open circle) 2 ng/ml; (closed circle) 5 ng/ml. Results are expressed as a percentage with respect to control, untreated cells and are the mean 6 SEM of five different experiments with triplicate determinations. (C) DNA fragmentation: cytosolic DNA from cells incubated for 12 or 24 h in the absence (C) or in the presence of 2 ng/ml TGF-b (T) was analyzed in 1.5% agarose gels, as described under Materials and Methods. One representative experiment of five is shown.
Cells that survived the TGF-b apoptotic effect presented similar proportions of cells in each phase of the cell cycle. We wanted to know whether these “TGF-b resistant” cells continue to be sensitive to this factor in terms of growth inhibition. For this we pretreated fetal hepatocytes in the presence of 2 ng/ml TGF-b for 24 h. Then, dishes were vigorously washed with PBS, to remove all the dead cells, and the medium was replaced with another medium in the absence of the cytokine. After 4 – 8 h, cells were used for proliferation assays as we have previously described [22], in the absence or in the presence of EGF at 20 ng/ml and/or TGF-b at 0.5 ng/ml (this dose inhibits growth but it is insufficient to induce apoptosis in normal hepatocytes; see [17]). Non-pretreated fetal hepatocytes were used as control. As can be observed in Fig. 3B, the mitogen-independent DNA synthesis of TGF-b-pretreated hepatocytes is lower than that observed in control, untreated cells, but they
continued responding to a mitogen, such as EGF, duplicating [ 3H]thymidine incorporation into DNA. TGF-b completely blocked the increase in DNA synthesis induced by EGF in both untreated and TGF-bpretreated hepatocytes. Similar results were observed when we analyzed the cell cycle phases by flow cytometry (results not shown). Thus, cells that were resistant to the TGF-b apoptotic effect maintained sensitivity to this factor in terms of inhibition of mitogen-induced proliferation. In contrast to these results, when we analyzed the ability of TGF-b to induce apoptosis in these cells (Fig. 4), we found that after pretreatment with TGF-b for 24 h, the cells that have not died never respond to the cytokine in terms of cell death. We measured the percentage of cells with a DNA content lower than 2C (Fig. 4) or with a DNA ladder (results not shown) and we could not find any response to TGF-b in pretreated cells. In contrast, control (un-
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FIG. 2. Changes in expression of cytokeratin and vimentin intermediate filaments and cytoskeleton distribution induced by TGF-b in fetal hepatocytes. (A and B) Immunofluorescence detection of vimentin and cytokeratin 18 of fetal hepatocytes cultured in the absence (A) or in the presence of 2 ng/ml TGF-b for 24 h (B). Cells were double-immunostained with mouse anti-vimentin or rabbit anti-cytokeratin 18. Fluorescent secondary antibodies were TRITC-conjugated goat anti-rabbit immunoglobulins (in red, CK 18) or FITC-conjugated goat anti-mouse immunoglobulins (in green, vimentin). Areas where vimentin and CK18 colocalize appear yellow. Note that in TGF-pretreated hepatocytes almost all the cells are yellow. (C and D) Actin filament organization of fetal hepatocytes cultured in the absence (C) or in the presence of 2 ng/ml TGF-b for 24 h (D). Actin filaments were stained with rhodamine-conjugated phalloidin. In C, a typical epithelial actin distribution (arrow) is found. In D, stress fibers (star) can be observed in the fibroblastic-like cells and nonpolarized actin fibers (cross) are found in cells undergoing the death process. Bar is 50 mm in all cases.
treated) cells responded to the cytokine increasing the percentage of cells with a DNA content lower than 2C. EGF was a protective agent, as we have previously described [3]. Identical results were obtained when we analyzed cell viability after 24 h in the presence of the factor (results not shown). All
these results provide evidence that growth inhibitory and apoptotic pathways induced by TGF-b in hepatocytes are independent pathways, as we have previously proposed [19], and only the apoptotic signal transduction is blocked in those TGF-b-resistant cells.
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FIG. 3. Proliferative response of TGF-b-pretreated fetal hepatocytes. (A) Fetal hepatocytes were pretreated for 24 h in the absence (C) or in the presence (T) of 2 ng/ ml TGF-b. After this time, the percentage of cells in each phase of the cell cycle was analyzed by flow cytometry. (B) After a 24-h pretreatment of fetal hepatocytes with 2 ng/ml TGF-b, medium was replaced by another medium in the absence of the cytokine. 4 – 8 h later, cells were used for analysis of DNA synthesis, measured as [ 3H]thymidine incorporation (see Materials and Methods), in the absence (control, C) or in the presence of EGF (20 ng/ml) (E), and/or TGF-b (0.5 ng/ml) (E1T and T, respectively). Non-pretreated fetal hepatocytes were used as control to compare with the response of pretreated hepatocytes. Results are expressed as dpm/10 6cells and are the mean 6 SEM of three independent experiments with duplicate determinations.
Analysis of Gene Expression in TGF-b-Pretreated Hepatocytes Following these results, we wanted to know the molecular characteristics of these cells in terms of differentiation markers. First, we analyzed gene expression by Northern blot. For this, after treatment of fetal hepatocytes for 24 h in the presence of 2 ng/ml TGF-b, we washed the cells with PBS and then we extracted RNA from attached cells. Results are presented in Fig. 5A. Cells that survived TGF-b-induced apoptosis presented higher levels of protooncogenes (such as c-fos, c-jun, c-myc, or H-ras), whereas liver-specific gene expression was clearly decreased (albumin, a-fetoprotein, or hepatocyte nuclear factor-4), when compared with control, untreated, cells. We also obtained nuclear extracts and analyzed their DNA binding activity to specific oligonucleotides for hepatocyte nuclear factors
(HNF-1, HNF-3, and HNF-4), for AP-1 and NF-kB, in gel-shift experiments. Results can be observed in Fig. 5B. AP-1 binding activity clearly increased in response to the cytokine, as we previously found [19]. However, DNA binding activity of liver-enriched transcription factors was clearly decreased. These results suggest that TGF-b-resistant cells were immature or less differentiated hepatocytes. It has been previously suggested that TGF-b blocks the NF-kB binding to DNA and that increasing the activity of this factor suppresses the apoptotic effect [30]. We also observed that nuclear extracts from TGF-b-treated cells presented very low binding activity to a specific oligonucleotide for the NF-kB binding sequence (Fig. 5B). Nevertheless, these cells did not die. To determine whether TGF-b, by itself, could induce dedifferentiation of fetal hepatocytes we performed “in vitro” transcription (run-on) assays with nuclei isolated from cells after 2 h of treatment in the absence (C) or in the presence of 2 ng/ml TGF-b (T) (Fig. 5C). We could observe that gene transcription was diminished for most of the liver-specific genes (albumin, a-fetoprotein, HNF-4) in TGF-b-treated hepatocytes. As a positive control for this experiment, we analyzed fibronectin transcription. The expression of this gene, which codes for an extracellular matrix protein, increased in response to TGF-b, as has been described [31]. All these results could suggest that fetal hepatocytes dedifferentiate in response to TGF-b and this dedifferentiation might be related to their ability to survive in the presence of this factor. Redifferentiation of TGF-b-Pretreated Fetal Hepatocytes To confirm that cells surviving the apoptotic effect of TGF-b were dedifferentiated hepatocytes, we decided to induce their redifferentiation. We selected EGF, which in addition to being a potent mitogen for fetal hepatocytes [32], is a survival factor [3] and induces differentiation of these cells [18, 32]. Cells were pretreated with 2 ng/ml TGF-b for 24 h; then, medium was removed and replaced by another medium of identical composition but in the absence of TGF-b. After 4 – 8 h, EGF (20 ng/ml) was added to some dishes for studies of redifferentiation (EGF treated). Other dishes remained without any addition (untreated). As shown in Fig. 3, cells responded to EGF by entering the cell cycle. In addition to this, 24 h later, cells that were incubated with EGF appeared different in morphology (Fig. 6A, bottom). Cells organized forming clusters of parenchyma-like hepatocytes where biliar canaliculi could be observed. Immunocytochemical analysis of these colonies demonstrated that they were positive for cytokeratin 18 and negative for vimentin (results not shown). In serum-free medium without EGF or other
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FIG. 4. Apoptotic response of TGF-b-pretreated fetal hepatocytes. Fetal hepatocytes were pretreated for 24 h in the presence of 2 ng/ ml TGF-b. After being washed with PBS, medium was replaced in the absence of the cytokine. 4 – 8 h later, cells were incubated in the absence (C) or in the presence of EGF (20 ng/ml) (E) and/or TGF-b (2 ng/ml) (E1T and T, respectively). After 8 –12 h, cells were used for analysis of cell DNA content by flow cytometry. Non-pretreated fetal hepatocytes were used as control to compare with the response of pretreated hepatocytes. (Top) A representative experiment of three is shown, where the percentage of hypodiploid cells has been calculated. (Bottom) Mean 6 SEM of percentage of hypodiploid cells from three independent experiments.
mitogens (Fig. 6A, top), cells retained a fibroblastic morphology, even after 48 h. In both cases, untreated and EGF-treated cells, the culture could be maintained for a further 2–3 days. After this time, cells died. Adding 10% fetal calf serum could induce maintenance of the culture for 4 –5 days. Under these conditions, partial areas of differentiated hepatocytes could also be observed (results not shown). To corroborate this apparent redifferentiation of hepatocytes, we analyzed the relative levels of molecular markers in differentiated cells (EGF-treated) versus dedifferentiated cells (untreated). Cells that were pretreated with TGF-b and then incubated for 24 h in the presence of EGF presented higher mRNA levels of AFP or HNF-4 (Fig. 6B) and enhanced DNA binding activity
for the liver-enriched transcription factors, particularly, HNF-3 and HNF-4 (Fig. 6C), when compared with untreated, dedifferentiated cells. NF-kB activity also increased as a consequence of the incubation in the presence of the mitogen. Figure 6 also shows a lane with the molecular markers (mRNA levels and DNA binding activity for transcription factors) of fetal (control, untreated) hepatocytes after the same number of days in culture. As can be observed, after 24 h in the presence of EGF, TGF-b-pretreated hepatocytes presented a differentiation status similar to that of cultured fetal hepatocytes. All these results suggest that cells that survived the apoptosis induced by TGF-b were dedifferentiated hepatocytes, capable of redifferentiating in the presence of a suitable signal, such as EGF.
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FIG. 5. Analysis of gene expression in TGF-b-pretreated fetal hepatocytes. Fetal rat hepatocytes were isolated, plated, and treated as described under Materials and Methods. Then, cells were incubated in the absence (C) or in the presence of 2 ng/ml TGF-b (T). After 24 h, RNA isolated from cells was used for Northern blot experiments to visualize mRNA levels (A) and nuclear extracts from parallel dishes were used for gel-shift experiments (B). Also, nuclei isolated from cells incubated for only 2 h in the absence (C) or in the presence (T) of TGF-b were used for in vitro transcription experiments (C). For this, we used the same cDNAs probes as in A, except in the case of AFP, where a genomic probe (a gift from Dr. J. L. Danan, Paris) was used to increase the efficiency of hybridization. In each case a representative experiment of three is shown. AFP, a-fetoprotein; ALB, albumin; HNF, hepatocyte nuclear factor; FN, fibronectin; pGEM, background for run on experiments, using pGEM plasmid for hybridization with nuclear transcripts.
DISCUSSION
Escape from the inhibitory control of growth or the regulation of apoptosis is regarded as one of the key steps in cancer. Transforming growth factor b-1 has been proposed to play an important role in epithelial
carcinogenesis, since it is known to be a potent negative epithelial cell growth regulator, an apoptotic factor, and a modulator of cellular phenotype. During the past years, our group has been attempting to resolve the multiple, and sometimes contradictory, roles of TGF-b1 in liver development. For this purpose, we
FIG. 6. Redifferentiation of TGF-b-pretreated fetal hepatocytes. Fetal hepatocytes were pretreated for 24 h in the presence of 2 ng/ml TGF-b. After being washed with PBS, medium was replaced in the absence of the cytokine; 4 – 8 h later, cells were incubated in the absence or in the presence of EGF (20 ng/ml) for a further 24 h. (A) Light-microscopy photographs of the cells. (B) RNA isolated from cells was used for Northern blot experiments to visualize mRNA levels. An additional lane shows mRNA levels in fetal hepatocytes (control) after the same number of days in culture. (C) Nuclear extracts from parallel dishes were used for gel-shift experiments. A representative experiment of three is shown. Abbreviations are the same as those used in Fig. 5.
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have studied its effect on growth, differentiation, and death of fetal hepatocytes in primary culture [3, 17– 21]. In this work, we show that these cells respond to TGF-b inducing apoptosis (Fig. 1). However, some cells survive this apoptotic effect. Survival is coincident with changes in morphology (Fig. 1) and phenotype (Fig. 2). Thus, cells able to overcome the apoptotic signals present a fibroblastic appearance, an increased motility, and a replacement of the cytokeratin network by vimentin as the intermediate filament. During embryogenesis and organogenesis, epithelial cells can detach from the epithelium and adopt a mesenchymal phenotype with locomotory properties. When this happens, the acquisition of motility is correlated with dramatic changes in the program of cell differentiation. The migrating fibroblast-like cells no longer appear epithelial, but they adopt a mesenchymal phenotype, coincident with the loss of specific gene expression [33]. This kind of phenotype transition has been described to occur in vitro in neonatal hepatocytes [28] and it is also frequently observed in malignant carcinoma cells where it is associated with the acquisition of invasive and metastatic phenotypes [34]. Previous works have related TGF-b to transdifferentiation of epithelial cells [35], with the type I receptor being implicated in the response [36]. Furthermore, TGF-b elicited epithelial– mesenchymal transition appears to be related to its ability to enhance malignant progression in skin carcinogenesis [37, 38]. We show here that these “TGF-b-unresponsive” cells in terms of apoptosis (Fig. 4) continue responding to this factor in terms of inhibiting the proliferation induced by a mitogen, such as EGF (Fig. 3). This fact would indicate that type I/type II receptor signaling seems to be unaltered in these cells. Apoptosis and growth control elicited by TGF-b appear to be closely related but independent pathways in epithelial cells. In mammalian epithelial cells, Smads 2 and 3 mediate growth inhibition and transcriptional activation of TGF-b and activin reporter genes [7]. However, Smad 2 and Smad 3 appear to have quantitatively different capabilities regarding the induction of apoptosis [39]. In fetal hepatocytes, apoptosis induced by TGF-b implicates oxygen radical (ROS) production and oxidative stress [19]. Antioxidants [19] or caspase inhibitors [40] are able to completely block TGF-b-induced cell death without affecting its ability to inhibit growth [19] or down-regulate cyclin A [40]. Thus, some postreceptor apoptotic signaling pathways might be altered in these dedifferentiated fetal hepatocytes, allowing them to overcome the apoptotic signal elicited by TGF-b, without affecting its growth-control ability. Fetal hepatocytes that survive the apoptotic effect of TGF-b present a low-level expression of liver-specific genes (Fig. 5A), a high-level expression of protoconcogenes (Fig. 5A), and decreased DNA binding activity
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for liver-enriched transcription factors, such as HNF-1, HNF-3, and HNF-4 (Fig. 5B). TGF-b appears to have a direct action on dedifferentiation of fetal hepatocytes, since run-on assays showed that as soon as 2 h after addition of the factor, transcription of liver-enriched genes is clearly diminished (Fig. 5C). This effect is reversible, since we can induce the redifferentiation of these cells when TGF-b is removed and EGF is added (Fig. 6). EGF is a survival signal for the apoptosis induced by TGF-b in fetal hepatocytes [3] and by itself, or synergistically with TGF-b or hormones, induces changes related to differentiation of these cells [18, 32]. Furthermore, EGF contributes to the maintenance of an epithelial morphology in neonatal hepatocytes in primary culture [29]. The ability of these TGF-b-resistant cells to differentiate in response to EGF confirms the hypothesis that they are potentially less-differentiated parenchymal hepatocytes. Although further work will be necessary to elucidate the survival pathway that it is activated and/or the apoptotic signal that it is blocked in these cells, AP-1 might be inducing the expression of antioxidant genes that could protect the hepatocytes against the oxidative stress induced by TGF-b. Interestingly, c-fos could be also responsible for the epithelium–mesenchyme transition, as has been previously proposed [41]. In contrast to this, we exclude a possible role of NF-kB in these effects, since its DNA binding activity is very low in TGF-b-pretreated hepatocytes. Furthermore, considering the high level of H-ras and c-myc expression that appears in TGF-bpretreated hepatocytes, previous works have shown that overexpression of ras induces TGF-b resistance in liver epithelial cells, related to the up-regulation of c-myc and coincident with an increasing malignancy [42, 43]. Taking all these results together, we propose that TGF-b, in addition to its known role as growth inhibitor and apoptosis activator, could be promoting some phenotypic, morphological, and molecular changes in fetal hepatocytes that allow them to escape its apoptotic effect. Alternatively, two populations of fetal hepatocytes exist, one, more differentiated, that has the necessary signaling pathway for TGF-b-induced apoptosis and another one, less differentiated, that either does not have the intracellular signaling or suffers an adaptation and survives in the presence of TGF-b. This last population or both cell types might be predisposed to respond to TGF-b by a switch to a fibroblastic phenotype, as has been seen for other epithelial cells. Further work will be necessary to distinguish between the two possibilities. TGF-b has been found to be a potent negative regulator of proliferation for normal, regenerating, preneoplastic and neoplastic hepatocytes in culture [8]. In contrast, most of the hepatoma cell lines are resistant to TGF-b in terms of apoptosis [44]. Furthermore, cells
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overexpressing active TGF-b, but not latent TGF-b, show increased tumorigenicity in nude mice [45]. It has been proposed that increasing TGF-b synthesis confers several proliferation-independent phenotypic changes that may be of significance for the survival of the tumor cell inoculum and for tumor formation and development [45]. Substantial evidence now exists indicating that disruption of the TGF-b1-induced apoptosis is an important and integral part of the multistage process of hepatocellular carcinoma [12]. From the data presented in this paper, we could hypothesize that TGF-b, secreted at high concentrations during liver carcinogenesis [1], might inhibit proliferation and induce death of normal cells while providing a selective advantage for the survival of cells that are partially transformed or less differentiated and unresponsive to the factor. According to this, TGF-b would play a dual role in the progression of hepatocarcinogenesis, i.e., acting as a suppressor of early stages of tumor progression but also stimulating selection of undifferentiated, TGF-b-unresponsive cells. We thank Drs. J. L. Danan, J. E. Darnell, R. O. Hynes, E. Rozengurt, E. Santos, and M. Yaniv for providing plasmids and oligonucleotides for gene expression analysis; Drs. O. Basch and R. Bastos for their gift of polyclonal anti-CK-45, and A. Va´zquez for expert assistance with the flow cytometer. We are most grateful to Drs. A. Cano, M. Ferna´ndez, J. Gil, M. Quintanilla, and S. Vilaro´ for stimulating comments and helpful discussion of the results presented in this paper. This work was supported by grants from the Ministerio de Sanidad y Consumo (FIS 95/1530) and the Ministerio de Educacio´n y Cultura (PM97/0052). A. Sa´nchez was the recipient of a fellowship from the Comunidad de Madrid.
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Serra, R., Carbonetto, S., Lord, M., and Isom, H. C. (1994). Transforming growth factor b1 suppresses transformation in hepatocytes by regulating a1b1 integrin expression. Cell Growth Differ. 5, 509 –517.
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Oberhammer, F., Bursch, W., Parzefall, W., Breit, P., Erber, E., Stadler, M., and Schulte-Hermann (1991). Effect of transforming growth factor b on cell death of cultured rat hepatocytes. Cancer Res. 51, 2478 –2485.
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Thorgeirsson, S. S., Teramoto, T., and Factor, V. M. (1998). Dysregulation of apoptosis in hepatocellular carcinoma. Semin. Liver Dis. 18, 115–122.
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Ito, N., Kawata, S., Tamura, S., Takaishi, K., Shirai, Y., Kiso, S., Yabuuchi, I., Matsuda, Y., Nishioka, M., and Tarui, S. (1991). Elevated levels of transforming growth factor beta messenger RNA and its polypeptide in human hepatocellular carcinoma. Cancer Res. 51, 4080 – 4083.
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Tsai, J. F., Jeng, J. E., Chuang, L. Y., Yang, M. L., Ho, M. S., Chang, W. Y., Hsich, M. Y., Lin, Z. Y., and Tsai, J. H. (1997). Elevated urinary transforming growth factor-b1 level as a tumour marker and predictor of poor survival in cirrhotic hepatocellular carcinoma. Br. J. Cancer 76, 244 –250.
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Factor, V. M., Kao, C. Y., Santoni-Rugiu, E., Woitach, J. T., Jensen, M. R., and Thorgeirsson, S. S. (1997). Constitutive expression of mature transforming growth factor beta1 in liver accelerates hepatocarcinogenesis in transgenic mice. Cancer Res. 57, 2089 –2095.
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Zhang, X., Wang, T., Batist, G., and Tsao, M. S. (1994). Transforming growth factor beta 1 promotes spontaneous transformation of cultured rat liver epithelial cells. Cancer Res. 54, 6122– 6128. ´ lvarez, A. M., Benito, M., and Fabregat, I. (1995). Sa´nchez, A., A Transforming growth factor b modulates growth and differentiation of fetal hepatocytes in primary culture. J. Cell. Physiol. 165, 398 – 405. ´ lvarez, A. M., Roncero, C., Vilaro´, S., Sa´nchez, A., Pagan, R., A Benito, M., and Fabregat, I. (1998). Transforming growth factor-b (TGF-b) and EGF promote cord-like structures that indicate terminal differentiation of fetal hepatocytes in primary culture. Exp. Cell Res. 242, 27–37. ´ lvarez, A. M., Benito, M., and Fabregat, I. (1996) Sa´nchez, A., A Apoptosis induced by transforming growth factor–b in fetal hepatocyte primary cultures. J. Biol. Chem. 271, 7416 –7422. ´ lvarez, A. M., Benito, M., and Fabregat, I. (1997). Sa´nchez, A., A Cycloheximide prevents apoptosis, reactive oxygen species production and glutathione depletion induced by transforming growth factor b in fetal rat hepatocytes in primary culture. Hepatology 26, 935–943.
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Apoptotic Response to TGF-b in Fetal Hepatocytes Depends upon Their State of Differentiation ´ lvarez,† J. M. Lo´pez Pedrosa,‡ C. Roncero,* M. Benito,* and I. Fabregat* ,1 A. Sa´nchez,* A. M. A *Departamento de Bioquı´mica y Biologı´a Molecular II, Centro Mixto CSIC/UCM and †Centro de Citometrı´a de Flujo y Microscopı´a Confocal, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain; and ‡Departamento I1D, Abbot Laboratories, 18004 Granada, Spain
Transforming growth factor-b (TGF-b1) induces death of fetal hepatocytes by an apoptotic mechanism. However, even when very high concentrations and/or long-term exposure to the cytokine is used, 40 –50% of cells always survive. The process of cell survival is coincident with changes in morphology and phenotype, with cells showing a fibroblastic appearance and eliciting an epithelial–fibroblastic transition. Surviving cells continue responding to TGF-b in terms of growth control. Expression of liver-specific genes is very low in these cells; this effect is due to the decrease in their rate of transcription as soon as 2 h after the addition of the factor. Surviving cells present a decreased DNA binding activity for liver-enriched transcription factors, an increased DNA binding activity for AP-1, and a high expression of protooncogenes. These cells are immature hepatocytes since in the presence of the appropriate signal (i.e., epidermal growth factor), they can differentiate, organizing in cell clusters and increasing both liver-specific mRNA expression and liver-enriched transcription factor activity. In accord with these results, TGF-b, secreted at high concentrations during liver carcinogenesis, might induce death of normal cells while providing a selective advantage for the survival of cells that are “partially transformed” or “less differentiated” and unresponsive to the factor. © 1999 Academic Press Key Words: hepatocyte; TGF-b; apoptosis; differentiation; hepatocarcinogenesis.
INTRODUCTION
Apoptosis seems to be the predominant type of active cell death in the liver [1]. Endogenous factors, such as transforming growth factor-b (TGF-b1), activin A, Fas ligand, and tumor necrosis factor-a (TNF-a), may be involved in the induction of apoptosis [2]. The action of these death factors seems to be counteracted by growth factors, such as epidermal growth factor (EGF) [3], 1 To whom correspondence and reprint requests should be addressed. Fax: 34-91-3941779. E-mail: [email protected].
hepatocyte growth factor [4], or keratinocyte growth factor [5]. During the first stages of hepatocarcinogenesis in rat, apoptotic activity gradually increases from normal liver to putative preneoplastic foci [6]. Preneoplastic liver cells are even more susceptible than normal hepatocytes to stimulation of cell death, suggesting that tumor initiation, at the organ level, can be reversed by preferential elimination of initiated cells [6]. Tumor promoters and nongenotoxic carcinogens inhibit active cell death, thereby increasing the accumulation of preneoplastic cells and accelerating the development of cancer [2, 6]. The TGF-b family comprises a large number of structurally related polypeptide factors, each capable of regulating several cellular processes, including cell proliferation, lineage determination, differentiation, motility, adhesion, and death [7]. TGF-b has been proposed to play an important role in hepatocarcinogenesis, however, much controversy exits as to its exact contribution. Indeed, it has been proposed that TGF-b secreted during liver carcinogenesis may inhibit liver cell proliferation [8], suppress transformation [9, 10], and induce apoptosis [11]. Disruption of the TGF-b pathway at the prereceptor, receptor, or postreceptor levels occurs in hepatocellular carcinoma (HCC) and can cause dysregulation of apoptosis [12]. However, some findings also suggest an association of the activated TGF-b gene transcription with progression of hepatocarcinogenesis: (i) human HCC strongly expresses TGF-b mRNA and protein in vivo [13]; (ii) an elevated urinary TGF-b level, used as a tumor marker, is related to poor survival in cirrhotic HCC [14]; (iii) constitutive expression of mature TGF-b in the liver accelerates hepatocarcinogenesis in transgenic mice [15]; and (iv) TGF-b promotes spontaneous transformation of cultured rat liver epithelial cells [16]. Fetal rat hepatocytes in primary culture are immature cells capable of carrying out both proliferation and differentiation processes simultaneously. We have shown that TGF-b inhibited growth of fetal hepatocytes, arresting cells in G1 and down-regulating c-myc expression [17]. Furthermore, TGF-b acted synergisti-
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cally with EGF, promoting morphological changes related to differentiation of these cells [18]. In addition to these functions and when used at higher concentrations, TGF-b induced fetal hepatocyte apoptosis [19] by a mechanism dependent on protein synthesis [20]. The dose and time dependence study of this cell death process showed that, regardless of the TGF-b concentration and the duration of exposure to the cytokine, 40 – 50% of cells always survived. In this work we have tried to analyze the morphological and molecular characteristics of these “TGF-b-resistant” cells. This population of cells responds to TGF-b in terms of growth inhibition but not in terms of cell death and appears to be composed of less-differentiated hepatocytes. The relevance of these findings in hepatocarcinogenesis will be discussed. MATERIALS AND METHODS Materials. Human recombinant TGF-b was from Calbiochem (La Jolla, CA) and human recombinant EGF was kindly provided by Serono Laboratories (Madrid, Spain). Fetal and neonatal calf serum and culture media were from Imperial Laboratories (Hampshire, UK). Radiochemicals were from ICN (Irvine, CA). The multiprimer DNA labeling system was from Amersham (Buckinghamshire, UK). The nick-translation labeling system was from Gibco BRL (Gaithersburg, MD). Other reagents were from Sigma Chemical Co. (St. Louis, MO) or Boe¨hringer Mannheim (Mannheim, Germany). FITCconjugated sheep anti-mouse immunoglobulins and monoclonal antivimentin (clone V9) were obtained from Boe¨hringer Mannheim. TRITC-conjugated goat anti-rabbit immunoglobulins were obtained from Dako Corp. (Santa Barbara, CA). Professor O. Bachs and Dr. R. Bastos (Barcelona, Spain) kindly provided polyclonal anti-cytokeratin 18 (CK18). Immunofluorescence mounting medium was from ICN Biomedicals Inc. (Costa Mesa, CA). Cell isolation and culture. Hepatocytes from 20-day-old fetal Wistar rats were isolated as previously described by collagenase disruption (2.5 3 10 6 cells/fetus) [21] and plated on plastic (noncoated) dishes in arginine-free medium 199, supplemented with ornithine (200 mM), fetal calf serum (10%), penicillin (120 mg/ml), and streptomycin (100 mg/ml). Cells were incubated in 5% CO 2, at 37°C for 4 h, allowing cell attachment to plates. Media were changed at that time and replaced by medium of the same composition except that 10% fetal calf serum was replaced by 2% newborn bovine serum. After 18 –20 h, the medium was again replaced with medium of identical composition but in the absence of serum. Two hours later, cells were ready for all the experiments described below. Light microscopy. After incubation of cells in the absence or in the presence of the different factors, they were washed twice with PBS and fixed in Bouin (71% picric acid/24% formaldehyde/5% acetic acid) solution at room temperature for 30 min. Fixative was then removed and cells were extensively rinsed with 70% ethanol and finally with PBS. Cells were visualized in a Nikon Eclipse TE 300 microscope and photographed using Kodak Elite chrome 100 films. TGF-b-mediated cytotoxicity assay. After cell incubation in the absence or in the presence of different factors, the medium was discarded and the remaining viable adherent cells were stained with crystal violet (0.2% in 2% ethanol) for 20 min, as previously described [17]. Remaining viable cells were calculated as the percentage absorbance with respect to control cells (incubated in the absence of growth factors). Analysis of DNA fragmentation. Rat fetal hepatocytes were washed twice with PBS, scraped, and pelleted at 4°C. Cells were
resuspended in 500 ml of buffer containing 10 mM EDTA, 0.25% Triton X-100, 2.5 mM Tris–HCl, pH 8.0, and stored at 4°C for 15 min. Intact nuclei were pelleted and eliminated by centrifugation at 500g for 10 min and the supernatant was centrifuged at 25,000g at 4°C for 30 min. DNA in the supernatant was precipitated at 280°C after addition of 2 vol of ethanol (70% final concentration), pelleted by microcentrifugation at 4°C for 15 min, dried, resuspended in 200 ml of 10 mM Tris–HCl, 1 mM EDTA, pH 8.0 (TE buffer), and incubated at 37°C for 30 min with 0.1 mg/ml RNase A and for 2–3 h with 0.25 mg/ml Proteinase K. DNA was purified by phenol– chloroform extraction and precipitated at 270°C after (1/10 vol) 3 M sodium acetate, pH 5.3, and (2 vol of) ethanol were added. Precipitated DNA was dissolved in TE buffer containing 30% glycerol, 1 mg/ml ethidium bromide and electrophoresed in a 1.5% agarose gel. Gel was visualized and photographed under transmitted UV light with a Polaroid camera. Confocal microscopy studies. For immunofluorescence detection of intermediate filaments (vimentin and cytokeratins), cells were washed twice with PBS, fixed in methanol (220°C) for 2 min, and processed for double immunofluorescence [18], by applying a mixture of mouse and rabbit primary antibodies, followed by extensive washing and incubation with a mixture of FITC-conjugated anti-mouse and TRITC-conjugated goat anti-rabbit. Primary antibodies were applied for 1 h at 37°C, followed by 43 5 min washes in PBS, a 45-min incubation with fluorescence-conjugated antibodies, and four final washes of 5 min each in PBS. For detection of actin filaments, cells were washed with PBS and fixed in 3% paraformaldehyde for 30 min at room temperature. Following this period, after the cells were washed with PBS, they were stained with TRITC-conjugated phalloidin that binds F-actin filaments. This probe was reconstituted in 60% methanol/40% DMSO at a concentration of 100 mg/ml and diluted 1/500 before use with PBS, 0.1% BSA, 0.01% azide, pH 7.2. It was applied for 1 h at 37°C in a humidified atmosphere in the dark, followed by extensive washing with PBS. For visualization, sections of the plates were coverslipped using immunofluorescence medium and examined in an MRC-1024 (Bio-Rad, Hempstead, and UK) confocal microscope adapted to an inverted Nikon Eclipse TE 300 microscope. Images were taken with 488-nm laser excitation for FITCconjugated antibodies and 514-nm laser excitation for TRITCconjugated antibodies. Fluorescence emissions were detected through a 513/24-nm bandpass filter for FITC and a 605/15-nm bandpass filter for TRITC. DNA synthesis assay. After incubation of the cells for 48 h in the absence or in the presence of the mitogen, DNA synthesis was evaluated by [ 3H]thymidine incorporation into TCA-precipitable material during the last 40 h, as we have previously described [22]. Analysis of cells’ DNA content by flow cytometry. The ploidy determination of hepatocytes was estimated by flow cytometry DNA analysis as previously described [19]. Cells were detached from dishes by addition of 0.25% trypsin– 0.02% EDTA and the DNA content per cell was evaluated in a FACScan flow cytometer (BectonDickinson, San Jose, CA), after cells were stained with propidium iodide, using Kinesis 50 (Bio-Rad). For the computer analysis, only signals from single cells were considered (10,000 cells/assay). RNA isolation and Northern blot analysis. Total RNA was isolated as described by Chomczynski and Sacchi [23]. For each assay, RNA was extracted from the pooled cells of two 92-mm-diameter dishes, denatured in 50% formamide, 2.2 M formaldehyde, 20 mM Mops, pH 7.0, 6% glycerol at 65°C for 15 min, separated by size on gels containing 0.9% agarose and 0.66 M formaldehyde, and blotted on GeneScreen membranes (NEN Research Products, Dupont, Boston, MA). cDNA probe origin and hybridization conditions were previously described [3, 17, 24]. a-Fetoprotein, albumin, and 18S ribosomal cDNAs were labeled with [a- 32P]dCTP by nick-translation, whereas c-fos, c-jun, c-myc, H-ras, and HNF-4 were labeled by random priming reaction. Serial hybridization with the different probes was performed successively.
TGF-b HEPATOCYTE DEATH AND DIFFERENTIATION Nuclear preparation and gel mobility shift assay. DNA-binding protein extracts from fetal hepatocytes were prepared as we previously described [18, 19], starting with 5 3 10 6 cells. The cell suspension was resuspended in 10 mM Hepes–KOH, pH 7.9, 1.5 mM MgCl 2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM PMSF (Buffer A), at 4°C, allowed to swell on ice for 10 min, and then vortexed for 10 s. Samples were centrifuged at 14,000g for 10 s and the supernatant was discarded. The pellet was resuspended in cold Buffer C (20 mM Hepes–KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl 2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF, and 0.75 mg each of leupeptin, antipain, and aprotinin per milliliter) and incubated on ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation at 14,000g for 2 min at 4°C and the supernatant fraction was stored at 280°C. Proteins were measured using the Bio-Rad protein reagent following the recommendations of the supplier. The gel mobility shift assay was performed essentially as described by Wen and Locker [25]. Synthetic oligonucleotides were prepared using an oligonucleotide synthesizer (Pharmacia Biotech Inc.) and with the sequences that we described previously [18, 19]. Labeling was performed by using Klenow polymerase and a- 32Plabeled deoxyadenosine triphosphate. The binding reaction mixture contained 0.5 ng of doubled-stranded oligonucleotide probe, 2 mg of poly (dI– dC), and 5 mg protein in Buffer C supplemented with 35 mM MgCl 2. After a 20-min incubation at 4°C, the mixture was applied to a 6% polyacrylamide gel. Gels were run at 0.8 V/cm 2 in 0.53 Tris– borate–EDTA running buffer for 2–3 h, at room temperature. The dried gel was then autoradiographed. Nuclear run-on transcription assays. Nuclei were isolated from the pooled cells of 10 –12 92-mm-diameter plates [26] and stored at 270°C in 50 mM Hepes, pH 7.9, 150 mM NaCl, 0.1 mM EDTA, 5 mM dithiothreitol, 0.125 mM phenylmethylsulfonyl fluoride and 50% glycerol. 32P-labeled RNA transcripts were purified by the method of Linial et al. [27] using NICK columns (Pharmacia LKB Biotechnology Inc.) and following the manufacturers instructions. In vitro elongation reactions, denaturation of DNA probes, application of DNA to GeneScreen membranes, hybridization of the 32P-labeled RNA transcripts to the membrane– bound DNA, posthybridization washes, and autoradiography were carried out as before [24]. We used the same cDNAs probes than in Northern blot experiments, except in the case of AFP, where a genomic probe (a gift of Dr. J. L. Danan, Paris) was used to increase the efficiency of hybridization.
RESULTS
Effect of TGF-b on Rat Fetal Hepatocyte Death When fetal hepatocytes in primary culture were incubated in the presence of 2 ng/ml TGF-b for 24 h, several changes occurred. First, between 12 and 24 h after the addition of the factor, around 50% of the cells died by apoptosis and detached from the plastic (Fig. 1). This process was coincident with the appearance of DNA cleavage that presented a peak around 8 –16 h (Fig. 1C). We never could find a DNA ladder at later times (Fig. 1C), even when new TGF-b was added (results not shown). Higher concentrations of TGF-b or exposure for longer times did not increase the percentage of cells that responded to the factor (Fig. 1B). Second, cells suffered changes in morphology in response to TGF-b. In controls (serum-free medium without any addition) small clusters of hepatocytes could be observed (Fig. 1A, top). In contrast, TGF-b-treated
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cells progressively acquired a fibroblast-like aspect; they were flat, were spindle-like, and migrated to the empty spaces. After 24 h of treatment, all the cells that were not sensitive to the apoptotic effect of TGF-b appeared to have a fibroblastic appearance (Fig. 1A, bottom). Changes in Expression of Cytokeratin and Vimentin Intermediate Filaments and Cytoskeleton Distribution Induced by TGF-b in Fetal Hepatocytes Hepatocytes are epithelial cells; thus, in differentiated states and under normal physiological conditions, they present only cytokeratin as the intermediate filament. In contrast to this, fibroblastic cells or transitional cells express vimentin [28, 29]. We decided to characterize the intermediate filament expression in TGF-b-treated cells. We present the results obtained by using double immunofluorescence with anti-cytokeratin and anti-vimentin on control (untreated) and TGF-b-treated (24 h) fetal hepatocytes (Figs. 2A and 2B). As can be observed, control cells organized in clusters were only cytokeratin positive (Fig. 2A). However, TGF-b treatment induced a progressive increase in the number of vimentin-positive cells. Coexpression of vimentin and cytokeratin filaments could be observed in most of the fibroblast-like cells (Fig. 2B). Vimentin expression was absent in the cells undergoing the death process. Actin assembly also changes during epithelial–mesenchyma transitions. For this, we decided to study the effect of apoptotic concentrations of TGF-b on actin distribution. Laser confocal microscopy observations allowed us to visualize the distribution of F-actin in cells stained with rhodamine–phalloidin (Figs. 2C and 2D). In controls (untreated cells), hepatocytes in the middle of the clusters presented a ring-like distribution, which is typical of epithelial cells that organize actin bundles in areas of cell to cell contact (Fig. 2C). In contrast, fibroblast-like cells that survive to TGF-binduced apoptosis presented abundant stress fibers. The ring-like cortical F-actin was not observed, except in those cells that were rounded and in the process of death (Fig. 2D). Proliferative and Apoptotic Response of TGF-bPretreated Hepatocytes To compare the growth properties of the two cell populations, i.e., control (untreated) and TGF-b-pretreated hepatocytes, we decided to analyze the proportion of cells in each phase of the cell cycle. After treatment for 24 h with 6TGF-b, cell DNA content was estimated by flow cytometry as described under Materials and Methods. As shown in Fig. 3A, most of the fetal hepatocytes are in the G1 phase of the cell cycle.
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FIG. 1. Effect of TGF-b on rat fetal hepatocyte death. Fetal rat hepatocytes were isolated, plated, and treated as described under Materials and Methods. (A) Light microscopy photographs of cells incubated for 24 h in the absence (control) or in the presence of 2 ng/ml TGF-b (TGF-b). (B) Cell viability at different times after TGF-b addition. Two different concentrations of the factor were used: (open circle) 2 ng/ml; (closed circle) 5 ng/ml. Results are expressed as a percentage with respect to control, untreated cells and are the mean 6 SEM of five different experiments with triplicate determinations. (C) DNA fragmentation: cytosolic DNA from cells incubated for 12 or 24 h in the absence (C) or in the presence of 2 ng/ml TGF-b (T) was analyzed in 1.5% agarose gels, as described under Materials and Methods. One representative experiment of five is shown.
Cells that survived the TGF-b apoptotic effect presented similar proportions of cells in each phase of the cell cycle. We wanted to know whether these “TGF-b resistant” cells continue to be sensitive to this factor in terms of growth inhibition. For this we pretreated fetal hepatocytes in the presence of 2 ng/ml TGF-b for 24 h. Then, dishes were vigorously washed with PBS, to remove all the dead cells, and the medium was replaced with another medium in the absence of the cytokine. After 4 – 8 h, cells were used for proliferation assays as we have previously described [22], in the absence or in the presence of EGF at 20 ng/ml and/or TGF-b at 0.5 ng/ml (this dose inhibits growth but it is insufficient to induce apoptosis in normal hepatocytes; see [17]). Non-pretreated fetal hepatocytes were used as control. As can be observed in Fig. 3B, the mitogen-independent DNA synthesis of TGF-b-pretreated hepatocytes is lower than that observed in control, untreated cells, but they
continued responding to a mitogen, such as EGF, duplicating [ 3H]thymidine incorporation into DNA. TGF-b completely blocked the increase in DNA synthesis induced by EGF in both untreated and TGF-bpretreated hepatocytes. Similar results were observed when we analyzed the cell cycle phases by flow cytometry (results not shown). Thus, cells that were resistant to the TGF-b apoptotic effect maintained sensitivity to this factor in terms of inhibition of mitogen-induced proliferation. In contrast to these results, when we analyzed the ability of TGF-b to induce apoptosis in these cells (Fig. 4), we found that after pretreatment with TGF-b for 24 h, the cells that have not died never respond to the cytokine in terms of cell death. We measured the percentage of cells with a DNA content lower than 2C (Fig. 4) or with a DNA ladder (results not shown) and we could not find any response to TGF-b in pretreated cells. In contrast, control (un-
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FIG. 2. Changes in expression of cytokeratin and vimentin intermediate filaments and cytoskeleton distribution induced by TGF-b in fetal hepatocytes. (A and B) Immunofluorescence detection of vimentin and cytokeratin 18 of fetal hepatocytes cultured in the absence (A) or in the presence of 2 ng/ml TGF-b for 24 h (B). Cells were double-immunostained with mouse anti-vimentin or rabbit anti-cytokeratin 18. Fluorescent secondary antibodies were TRITC-conjugated goat anti-rabbit immunoglobulins (in red, CK 18) or FITC-conjugated goat anti-mouse immunoglobulins (in green, vimentin). Areas where vimentin and CK18 colocalize appear yellow. Note that in TGF-pretreated hepatocytes almost all the cells are yellow. (C and D) Actin filament organization of fetal hepatocytes cultured in the absence (C) or in the presence of 2 ng/ml TGF-b for 24 h (D). Actin filaments were stained with rhodamine-conjugated phalloidin. In C, a typical epithelial actin distribution (arrow) is found. In D, stress fibers (star) can be observed in the fibroblastic-like cells and nonpolarized actin fibers (cross) are found in cells undergoing the death process. Bar is 50 mm in all cases.
treated) cells responded to the cytokine increasing the percentage of cells with a DNA content lower than 2C. EGF was a protective agent, as we have previously described [3]. Identical results were obtained when we analyzed cell viability after 24 h in the presence of the factor (results not shown). All
these results provide evidence that growth inhibitory and apoptotic pathways induced by TGF-b in hepatocytes are independent pathways, as we have previously proposed [19], and only the apoptotic signal transduction is blocked in those TGF-b-resistant cells.
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FIG. 3. Proliferative response of TGF-b-pretreated fetal hepatocytes. (A) Fetal hepatocytes were pretreated for 24 h in the absence (C) or in the presence (T) of 2 ng/ ml TGF-b. After this time, the percentage of cells in each phase of the cell cycle was analyzed by flow cytometry. (B) After a 24-h pretreatment of fetal hepatocytes with 2 ng/ml TGF-b, medium was replaced by another medium in the absence of the cytokine. 4 – 8 h later, cells were used for analysis of DNA synthesis, measured as [ 3H]thymidine incorporation (see Materials and Methods), in the absence (control, C) or in the presence of EGF (20 ng/ml) (E), and/or TGF-b (0.5 ng/ml) (E1T and T, respectively). Non-pretreated fetal hepatocytes were used as control to compare with the response of pretreated hepatocytes. Results are expressed as dpm/10 6cells and are the mean 6 SEM of three independent experiments with duplicate determinations.
Analysis of Gene Expression in TGF-b-Pretreated Hepatocytes Following these results, we wanted to know the molecular characteristics of these cells in terms of differentiation markers. First, we analyzed gene expression by Northern blot. For this, after treatment of fetal hepatocytes for 24 h in the presence of 2 ng/ml TGF-b, we washed the cells with PBS and then we extracted RNA from attached cells. Results are presented in Fig. 5A. Cells that survived TGF-b-induced apoptosis presented higher levels of protooncogenes (such as c-fos, c-jun, c-myc, or H-ras), whereas liver-specific gene expression was clearly decreased (albumin, a-fetoprotein, or hepatocyte nuclear factor-4), when compared with control, untreated, cells. We also obtained nuclear extracts and analyzed their DNA binding activity to specific oligonucleotides for hepatocyte nuclear factors
(HNF-1, HNF-3, and HNF-4), for AP-1 and NF-kB, in gel-shift experiments. Results can be observed in Fig. 5B. AP-1 binding activity clearly increased in response to the cytokine, as we previously found [19]. However, DNA binding activity of liver-enriched transcription factors was clearly decreased. These results suggest that TGF-b-resistant cells were immature or less differentiated hepatocytes. It has been previously suggested that TGF-b blocks the NF-kB binding to DNA and that increasing the activity of this factor suppresses the apoptotic effect [30]. We also observed that nuclear extracts from TGF-b-treated cells presented very low binding activity to a specific oligonucleotide for the NF-kB binding sequence (Fig. 5B). Nevertheless, these cells did not die. To determine whether TGF-b, by itself, could induce dedifferentiation of fetal hepatocytes we performed “in vitro” transcription (run-on) assays with nuclei isolated from cells after 2 h of treatment in the absence (C) or in the presence of 2 ng/ml TGF-b (T) (Fig. 5C). We could observe that gene transcription was diminished for most of the liver-specific genes (albumin, a-fetoprotein, HNF-4) in TGF-b-treated hepatocytes. As a positive control for this experiment, we analyzed fibronectin transcription. The expression of this gene, which codes for an extracellular matrix protein, increased in response to TGF-b, as has been described [31]. All these results could suggest that fetal hepatocytes dedifferentiate in response to TGF-b and this dedifferentiation might be related to their ability to survive in the presence of this factor. Redifferentiation of TGF-b-Pretreated Fetal Hepatocytes To confirm that cells surviving the apoptotic effect of TGF-b were dedifferentiated hepatocytes, we decided to induce their redifferentiation. We selected EGF, which in addition to being a potent mitogen for fetal hepatocytes [32], is a survival factor [3] and induces differentiation of these cells [18, 32]. Cells were pretreated with 2 ng/ml TGF-b for 24 h; then, medium was removed and replaced by another medium of identical composition but in the absence of TGF-b. After 4 – 8 h, EGF (20 ng/ml) was added to some dishes for studies of redifferentiation (EGF treated). Other dishes remained without any addition (untreated). As shown in Fig. 3, cells responded to EGF by entering the cell cycle. In addition to this, 24 h later, cells that were incubated with EGF appeared different in morphology (Fig. 6A, bottom). Cells organized forming clusters of parenchyma-like hepatocytes where biliar canaliculi could be observed. Immunocytochemical analysis of these colonies demonstrated that they were positive for cytokeratin 18 and negative for vimentin (results not shown). In serum-free medium without EGF or other
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FIG. 4. Apoptotic response of TGF-b-pretreated fetal hepatocytes. Fetal hepatocytes were pretreated for 24 h in the presence of 2 ng/ ml TGF-b. After being washed with PBS, medium was replaced in the absence of the cytokine. 4 – 8 h later, cells were incubated in the absence (C) or in the presence of EGF (20 ng/ml) (E) and/or TGF-b (2 ng/ml) (E1T and T, respectively). After 8 –12 h, cells were used for analysis of cell DNA content by flow cytometry. Non-pretreated fetal hepatocytes were used as control to compare with the response of pretreated hepatocytes. (Top) A representative experiment of three is shown, where the percentage of hypodiploid cells has been calculated. (Bottom) Mean 6 SEM of percentage of hypodiploid cells from three independent experiments.
mitogens (Fig. 6A, top), cells retained a fibroblastic morphology, even after 48 h. In both cases, untreated and EGF-treated cells, the culture could be maintained for a further 2–3 days. After this time, cells died. Adding 10% fetal calf serum could induce maintenance of the culture for 4 –5 days. Under these conditions, partial areas of differentiated hepatocytes could also be observed (results not shown). To corroborate this apparent redifferentiation of hepatocytes, we analyzed the relative levels of molecular markers in differentiated cells (EGF-treated) versus dedifferentiated cells (untreated). Cells that were pretreated with TGF-b and then incubated for 24 h in the presence of EGF presented higher mRNA levels of AFP or HNF-4 (Fig. 6B) and enhanced DNA binding activity
for the liver-enriched transcription factors, particularly, HNF-3 and HNF-4 (Fig. 6C), when compared with untreated, dedifferentiated cells. NF-kB activity also increased as a consequence of the incubation in the presence of the mitogen. Figure 6 also shows a lane with the molecular markers (mRNA levels and DNA binding activity for transcription factors) of fetal (control, untreated) hepatocytes after the same number of days in culture. As can be observed, after 24 h in the presence of EGF, TGF-b-pretreated hepatocytes presented a differentiation status similar to that of cultured fetal hepatocytes. All these results suggest that cells that survived the apoptosis induced by TGF-b were dedifferentiated hepatocytes, capable of redifferentiating in the presence of a suitable signal, such as EGF.
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FIG. 5. Analysis of gene expression in TGF-b-pretreated fetal hepatocytes. Fetal rat hepatocytes were isolated, plated, and treated as described under Materials and Methods. Then, cells were incubated in the absence (C) or in the presence of 2 ng/ml TGF-b (T). After 24 h, RNA isolated from cells was used for Northern blot experiments to visualize mRNA levels (A) and nuclear extracts from parallel dishes were used for gel-shift experiments (B). Also, nuclei isolated from cells incubated for only 2 h in the absence (C) or in the presence (T) of TGF-b were used for in vitro transcription experiments (C). For this, we used the same cDNAs probes as in A, except in the case of AFP, where a genomic probe (a gift from Dr. J. L. Danan, Paris) was used to increase the efficiency of hybridization. In each case a representative experiment of three is shown. AFP, a-fetoprotein; ALB, albumin; HNF, hepatocyte nuclear factor; FN, fibronectin; pGEM, background for run on experiments, using pGEM plasmid for hybridization with nuclear transcripts.
DISCUSSION
Escape from the inhibitory control of growth or the regulation of apoptosis is regarded as one of the key steps in cancer. Transforming growth factor b-1 has been proposed to play an important role in epithelial
carcinogenesis, since it is known to be a potent negative epithelial cell growth regulator, an apoptotic factor, and a modulator of cellular phenotype. During the past years, our group has been attempting to resolve the multiple, and sometimes contradictory, roles of TGF-b1 in liver development. For this purpose, we
FIG. 6. Redifferentiation of TGF-b-pretreated fetal hepatocytes. Fetal hepatocytes were pretreated for 24 h in the presence of 2 ng/ml TGF-b. After being washed with PBS, medium was replaced in the absence of the cytokine; 4 – 8 h later, cells were incubated in the absence or in the presence of EGF (20 ng/ml) for a further 24 h. (A) Light-microscopy photographs of the cells. (B) RNA isolated from cells was used for Northern blot experiments to visualize mRNA levels. An additional lane shows mRNA levels in fetal hepatocytes (control) after the same number of days in culture. (C) Nuclear extracts from parallel dishes were used for gel-shift experiments. A representative experiment of three is shown. Abbreviations are the same as those used in Fig. 5.
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have studied its effect on growth, differentiation, and death of fetal hepatocytes in primary culture [3, 17– 21]. In this work, we show that these cells respond to TGF-b inducing apoptosis (Fig. 1). However, some cells survive this apoptotic effect. Survival is coincident with changes in morphology (Fig. 1) and phenotype (Fig. 2). Thus, cells able to overcome the apoptotic signals present a fibroblastic appearance, an increased motility, and a replacement of the cytokeratin network by vimentin as the intermediate filament. During embryogenesis and organogenesis, epithelial cells can detach from the epithelium and adopt a mesenchymal phenotype with locomotory properties. When this happens, the acquisition of motility is correlated with dramatic changes in the program of cell differentiation. The migrating fibroblast-like cells no longer appear epithelial, but they adopt a mesenchymal phenotype, coincident with the loss of specific gene expression [33]. This kind of phenotype transition has been described to occur in vitro in neonatal hepatocytes [28] and it is also frequently observed in malignant carcinoma cells where it is associated with the acquisition of invasive and metastatic phenotypes [34]. Previous works have related TGF-b to transdifferentiation of epithelial cells [35], with the type I receptor being implicated in the response [36]. Furthermore, TGF-b elicited epithelial– mesenchymal transition appears to be related to its ability to enhance malignant progression in skin carcinogenesis [37, 38]. We show here that these “TGF-b-unresponsive” cells in terms of apoptosis (Fig. 4) continue responding to this factor in terms of inhibiting the proliferation induced by a mitogen, such as EGF (Fig. 3). This fact would indicate that type I/type II receptor signaling seems to be unaltered in these cells. Apoptosis and growth control elicited by TGF-b appear to be closely related but independent pathways in epithelial cells. In mammalian epithelial cells, Smads 2 and 3 mediate growth inhibition and transcriptional activation of TGF-b and activin reporter genes [7]. However, Smad 2 and Smad 3 appear to have quantitatively different capabilities regarding the induction of apoptosis [39]. In fetal hepatocytes, apoptosis induced by TGF-b implicates oxygen radical (ROS) production and oxidative stress [19]. Antioxidants [19] or caspase inhibitors [40] are able to completely block TGF-b-induced cell death without affecting its ability to inhibit growth [19] or down-regulate cyclin A [40]. Thus, some postreceptor apoptotic signaling pathways might be altered in these dedifferentiated fetal hepatocytes, allowing them to overcome the apoptotic signal elicited by TGF-b, without affecting its growth-control ability. Fetal hepatocytes that survive the apoptotic effect of TGF-b present a low-level expression of liver-specific genes (Fig. 5A), a high-level expression of protoconcogenes (Fig. 5A), and decreased DNA binding activity
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for liver-enriched transcription factors, such as HNF-1, HNF-3, and HNF-4 (Fig. 5B). TGF-b appears to have a direct action on dedifferentiation of fetal hepatocytes, since run-on assays showed that as soon as 2 h after addition of the factor, transcription of liver-enriched genes is clearly diminished (Fig. 5C). This effect is reversible, since we can induce the redifferentiation of these cells when TGF-b is removed and EGF is added (Fig. 6). EGF is a survival signal for the apoptosis induced by TGF-b in fetal hepatocytes [3] and by itself, or synergistically with TGF-b or hormones, induces changes related to differentiation of these cells [18, 32]. Furthermore, EGF contributes to the maintenance of an epithelial morphology in neonatal hepatocytes in primary culture [29]. The ability of these TGF-b-resistant cells to differentiate in response to EGF confirms the hypothesis that they are potentially less-differentiated parenchymal hepatocytes. Although further work will be necessary to elucidate the survival pathway that it is activated and/or the apoptotic signal that it is blocked in these cells, AP-1 might be inducing the expression of antioxidant genes that could protect the hepatocytes against the oxidative stress induced by TGF-b. Interestingly, c-fos could be also responsible for the epithelium–mesenchyme transition, as has been previously proposed [41]. In contrast to this, we exclude a possible role of NF-kB in these effects, since its DNA binding activity is very low in TGF-b-pretreated hepatocytes. Furthermore, considering the high level of H-ras and c-myc expression that appears in TGF-bpretreated hepatocytes, previous works have shown that overexpression of ras induces TGF-b resistance in liver epithelial cells, related to the up-regulation of c-myc and coincident with an increasing malignancy [42, 43]. Taking all these results together, we propose that TGF-b, in addition to its known role as growth inhibitor and apoptosis activator, could be promoting some phenotypic, morphological, and molecular changes in fetal hepatocytes that allow them to escape its apoptotic effect. Alternatively, two populations of fetal hepatocytes exist, one, more differentiated, that has the necessary signaling pathway for TGF-b-induced apoptosis and another one, less differentiated, that either does not have the intracellular signaling or suffers an adaptation and survives in the presence of TGF-b. This last population or both cell types might be predisposed to respond to TGF-b by a switch to a fibroblastic phenotype, as has been seen for other epithelial cells. Further work will be necessary to distinguish between the two possibilities. TGF-b has been found to be a potent negative regulator of proliferation for normal, regenerating, preneoplastic and neoplastic hepatocytes in culture [8]. In contrast, most of the hepatoma cell lines are resistant to TGF-b in terms of apoptosis [44]. Furthermore, cells
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overexpressing active TGF-b, but not latent TGF-b, show increased tumorigenicity in nude mice [45]. It has been proposed that increasing TGF-b synthesis confers several proliferation-independent phenotypic changes that may be of significance for the survival of the tumor cell inoculum and for tumor formation and development [45]. Substantial evidence now exists indicating that disruption of the TGF-b1-induced apoptosis is an important and integral part of the multistage process of hepatocellular carcinoma [12]. From the data presented in this paper, we could hypothesize that TGF-b, secreted at high concentrations during liver carcinogenesis [1], might inhibit proliferation and induce death of normal cells while providing a selective advantage for the survival of cells that are partially transformed or less differentiated and unresponsive to the factor. According to this, TGF-b would play a dual role in the progression of hepatocarcinogenesis, i.e., acting as a suppressor of early stages of tumor progression but also stimulating selection of undifferentiated, TGF-b-unresponsive cells. We thank Drs. J. L. Danan, J. E. Darnell, R. O. Hynes, E. Rozengurt, E. Santos, and M. Yaniv for providing plasmids and oligonucleotides for gene expression analysis; Drs. O. Basch and R. Bastos for their gift of polyclonal anti-CK-45, and A. Va´zquez for expert assistance with the flow cytometer. We are most grateful to Drs. A. Cano, M. Ferna´ndez, J. Gil, M. Quintanilla, and S. Vilaro´ for stimulating comments and helpful discussion of the results presented in this paper. This work was supported by grants from the Ministerio de Sanidad y Consumo (FIS 95/1530) and the Ministerio de Educacio´n y Cultura (PM97/0052). A. Sa´nchez was the recipient of a fellowship from the Comunidad de Madrid.
8.
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