Phenobarbital promotes liver growth in c-myc/TGF-{alpha} transgenic mice by inducing hypertrophy and inhibiting apoptosis

Sean Sanders and Snorri S. Thorgeirsson1

Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Phenobarbital (PB) is a non-genotoxic liver tumor promoter used extensively in initiation–promotion protocols. To determine the mode of PB action, double transgenic mice overexpressing both the c-myc and transforming growth factor (TGF)-{alpha} genes were treated with PB in the food for 10 weeks, from 3 weeks of age. After 3–4 weeks on PB a peak in liver mass was noted, which subsequently leveled off at a value ~30% above untreated animals. The mitotic index in mice given PB peaked at 1 week of treatment and was significantly elevated compared with untreated animals. No significant difference between treated and untreated animals was seen thereafter, although a trend of PB-associated mitotic suppression was noticeable. The apoptotic index also showed a trend of suppression compared with untreated animals, significant after prolonged PB administration. Dysplastic hepatocytes were more prominent in PB-treated mice than untreated animals, particularly pericentrally. Removal of PB from the diet at 4 weeks of treatment led to a dramatic increase in apoptosis. This accompanied a drop in the liver mass to the level of untreated controls by 10 days. Throughout the study, PB-treated animals showed markedly lower levels of TGF-ß1 ligand, coincident with an elevated level of the anti-apoptotic protein Bcl-2. On withdrawal of PB, the levels of all these proteins rapidly changed to mirror those seen in untreated mice. In all treatment groups, no change in the levels of epidermal growth factor receptor, TGF-ß receptors I and II or Bcl-xS/L were seen. We conclude from our data that PB stimulates liver growth in double transgenic c-myc/TGF-{alpha} mice by induction of liver hypertrophy and inhibition of apoptosis, brought about by both a decrease in signaling through the TGF-ß pathway and an increase in Bcl-2. The data support the hypothesis that PB promotes neoplastic development through a reduction in the incidence of cell death.

Abbreviations: DAB, 3,3'-diaminobenzidine; ECL, enhanced chemiluminescence; EGFR, epidermal growth factor receptor; H&E, hematoxylin and eosin; MT, metallothionein; NF-{kappa}B, nuclear factor {kappa}B; PB, phenobarbital; TGF, transforming growth factor; TGF-ß RI, transforming growth factor receptor I; TGF-ß RII, transforming growth factor receptor II.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Phenobarbital (PB) is a non-genotoxic barbiturate, used for many years as a liver tumor promoter in rodents (1,2) and an anti-epilepsy drug in humans (3). The biochemical effects of PB are exceedingly pleotropic, being both context- and strain-dependent. The main effects are pericentral hypertrophy (4) and transient hyperplasia (47), but also include increased (8) or decreased (7) sensitivity to transforming growth factor (TGF)-ß and other growth factors (9,10), decreased translocation of protein kinase C (8,11), a decrease in the number of epidermal growth factor receptors (EGFR) (4,6), increased levels of cytochrome P-450 (12,13), a decrease in gap junction communication (14), and changes in thyroid hormones (15,16).

Recent work has shown that PB can block apoptosis following chemical (2-acetyl-aminofluorene) or physical (UV radiation) insult in vitro in rat hepatocytes (17). In initiation–promotion studies in rats, PB withdrawal resulted in increased apoptosis (1820). Kaufmann et al. demonstrated that TGF-{alpha}, an autocrine growth factor, could replace PB in cultures which are dependent on a constant supply of PB for survival (21). Furthermore, colony formation ability using either TGF-{alpha} or PB was additive and yielded a saturated response when combined at optimal concentrations. More recently, TGF-{alpha} has been shown to be a survival factor in hepatocellular carcinogenesis (22).

It has been hypothesized that PB may act via the same autocrine growth pathway as TGF-{alpha} to promote hepatocarcinogenesis through inhibition of apoptosis (2124). To address this possibility, we made use of a double transgenic mouse model generated in our laboratory carrying both TGF-{alpha} and c-myc transgenes (25). We used this c-myc/TGF-{alpha} system as a means to examine the effect of a further PB-induced growth stimulus on hepatocytes already placed under pressure to proliferate. Co-expression of the transgenes has previously been shown to result in a dramatic acceleration of neoplasia in the c-myc/TGF-{alpha} mouse liver when compared with single transgenic mice (26) and this was thought to be due to the suppression of apoptosis by TGF-{alpha} (22).

In this paper we show that treatment with PB caused an elevated rate of increase in liver mass over untreated mice, with an early, transient peak in the mitotic index. Suppression of apoptosis was observed in animals on PB, coincident with an elevation in the level of Bcl-2 and concomitant depression of TGF-ß1. Withdrawal of PB resulted in a dramatic, but short-lived, increase in cell death, accompanied by a drop in liver mass. Although PB, in this system, clearly has an effect additional to that of TGF-{alpha}, the results are consistent with previous data demonstrating suppression of apoptosis by PB (1719) and provide an explanation for the promoting effects observed during murine hepatocarcinogenesis.


    Materials and methods
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 Materials and methods
 Results
 Discussion
 References
 
Animals and tissue
c-myc/TGF-{alpha} mice were derived as previously described (25) by making use of (C57BL/6JxCBA/J)c-myc and [(C57BL/6JxCBA/J)xCD1]TGF-{alpha} mouse strains. The TGF-{alpha} gene was under the control of the human metallothionein (MT) promoter (27), while the c-myc gene was controlled by an albumin promoter (25). To fully activate expression of the MT–TGF-{alpha} gene, 50 mM zinc chloride was included in the drinking water of all animals from the time of weaning (3 weeks of age). PB was administered to experimental animals in the food at a concentration of 0.05%. Mice were kept on a 12 h dark/light cycle (6 a.m. to 6 p.m.). Animal housing and care was carried out according to NIH guidelines.

Animals were weighed, killed by cervical dislocation and the liver immediately removed and weighed. All tissue was taken between 9 a.m. and 11 a.m. to minimize diurnal variations in physiological and biochemical variables. A portion of the liver was fixed overnight in 10% phosphate-buffered formalin and the remainder frozen for future biochemical analysis.

Tissues taken specifically for immunohistochemistry were washed and fixed by perfusion in situ through the inferior vena cava using first cold phosphate-buffered saline and then cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Samples were post-fixed in the above fixative for 3 h and embedded in paraffin.

Quantitation
At least 2000 nuclei were counted per animal on hematoxylin and eosin (H&E) stained slides and three to four animals per time point were used. Mitotic figures and apoptotic events were scored per 100 nuclei. Apoptotic cells, single apoptotic bodies and closely clustered apoptotic bodies were all scored as single events. Any cell which demonstrated markers of both apoptosis (circular bodies of condensed chromatin, eosinophilic cytoplasm, shrunken appearance) and necrosis (swollen appearance, localized inflammatory infiltration, pyknotic nuclei) was classified as a `single cell necrosis' or necrobiotic cell (28) and was included in the apoptotic index. These cells were seen very rarely except in those animals recently removed from PB treatment. They were isolated and showed no other signs of inflammation apart from very localized mononuclear infiltrate. Occasional areas of coagulative necrosis (i.e. patches made up of a large number of necrosing cells, with extensive infiltrate and inflammation) were not included in the quantitation. All statistical analysis was performed using a Mann–Whitney two-tailed test.

TUNEL staining
The in situ Cell Death Detection Kit, POD (Boehringer Mannheim, Indianapolis, IN) was used to detect the position of cells undergoing apoptosis and confirm the H&E results. Paraffin-embedded sections (5 µm) were deparaffinized and treated with proteinase K (20 µg/ml, 15 min), followed by a 30 min block in 0.3% H2O2 in methanol and permeabilization on ice for 2 min in 0.1% sodium citrate with 0.1% Triton X-100. The remainder of the procedure followed the protocol supplied by the manufacturer. 3,3'-Diaminobenzidine (DAB) staining was carried out for 5 min and slides were counterstained with Mayer's hematoxylin. The TUNEL stain generally stained both apoptotic cells and `single cell necroses' (see above).

Immunohistochemistry
Staining for Bax and Bcl-2 was carried out as previously described (29). Briefly, following deparaffinization and blocking of endogenous peroxidase in 0.3% H2O2 in methanol, sections were microwaved for two rounds of 6 min in 10 mM sodium citrate and placed in preblock solution (2% bovine serum albumin, 1% mouse serum, 1.5% normal goat serum, 0.1% Triton X-100, in 100 mM Tris, 550 mM NaCl and 10 mM KCl). Incubation with primary antibody [1 µg/ml of Santa Cruz Biotechnology (Santa Cruz, CA) anti-bax P-19 or 1:1000 dilution of PharMingen (San Diego, CA) anti-bcl-2] was overnight at 4°C. Secondary antibody application and development were carried out using the Vectastain Elite Rabbit Kit (Vectastain, Burlingame, CA) according to the supplied protocol. Final development utilized either DAB (Bcl-2) or VIP (Bax; Vectastain) staining for 10 min and sections were counterstained with Mayer's hematoxylin.

TGF-ß1 immunohistochemistry was carried out as above, except that the microwave step was replaced by incubation with hyaluronidase (1 mg/ml bovine serum hyaluronidase in 0.1 mM sodium acetate) for 30 min at 37°C. Primary antibody was LC[1-30], a kind gift from Dr Kathy Flanders.

Western blotting
Protein was extracted from frozen mouse liver in lysis buffer (30 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Tergitol NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol and 5 mM EDTA). Inhibitors were added just before use (Complete, Mini; Boehringer Mannheim). Tissue was mechanically homogenized and sonicated, followed by centrifugation at 14 000 g for 15 min. The supernatant was collected as crude extract and the protein concentration determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). For each gel, 40 µg protein was loaded per lane, electrophoresed for 2–8 h at room temperature and blotted onto nitrocellulose. Membranes were checked for equal loading by Ponceau-S staining, followed by blocking and incubation overnight at 4°C in primary antibody [1:500 dilution of PharMingen anti-bcl-2 or 1–2 µg/ml for Santa Cruz antibodies against transforming growth factor receptor I (TGF-ß RI), transforming growth factor receptor II (TGF-ß RII), EGFR, Bax, Bcl-xS/L and phosphotyrosine], followed by secondary antibody (1:10 000) incubation and visualization using the enhanced chemiluminescence system (ECL; Amersham, Arlington Heights, IL). The positive control for Bcl-2 was MI mouse myeloma cell line (PharMingen).


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Gross and histological changes
Livers were noticeably larger after extended PB treatment and exhibited a softer and more spongy consistency. Nevertheless, the mice appeared generally to be in good health and their body mass was not otherwise dramatically changed. No tumors were found during the course of the study.

At the start of the experiment, the liver mass:body mass ratio was 5.11 ± 0.45% (mean ± SD; Figure 1Go). This increased rapidly in both treated and untreated animals, but more sharply in the former. A significant difference (P < 0.03) was noticeable after only 1 week from the start of the experiment. The liver mass in the PB-treated group peaked at between 3 and 4 weeks and then declined slightly to plateau at a level ~30% above that of untreated animals. After 10 weeks of treatment, the liver mass was significantly different in treated and untreated mice, at 13.1 ± 0.48 and 9.3 ± 0.53%, respectively (P < 0.03).



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Fig. 1. Effect of PB administration and withdrawal on liver mass. Solid lines show results of feeding double transgenic c-myc/TGF-{alpha} mice with normal chow (open triangles) or chow containing 0.05% PB (closed squares). Note that liver mass (expressed as a percentage of body mass) peaked at 3–4 weeks and reached a plateau at ~13% (30% higher than untreated). The dashed line with closed triangles shows the decrease in liver mass:body mass ratio (%) when mice were taken off PB after 4 weeks of treatment (error bars omitted for visual clarity and did not exceed 3% of the mean). Within 10 days these mice had achieved a ratio (%) identical to untreated animals. The difference in liver mass:body mass ratio between treated and untreated mice was statistically significant at all time points from 1 week of treatment (P < 0.03).

 
Microscopically, the most noticeable and obvious effect of PB was on liver morphology. In mice treated with PB there was evidence of extensive dysplasia, particularly prominent pericentrally, as well as aberrant mitotic figures and abnormal nuclei (Figure 2AGo). These features were evident even by 7 weeks of age (4 weeks of treatment) and were more pronounced in treated than untreated animals. By 10 weeks, PB-treated mice displayed grossly hypertrophic cells, especially in the region of the central vein (Figure 3EGo). These cells were often many-fold larger than those found periportally in treated animals or generally in untreated animals.



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Fig. 2. (A) H&E staining of animal on PB for 4 weeks. Note many mitotic figures (arrowed), a number of which are aberrant. Magnification 200x. (B) TUNEL staining 3 days after removal of PB. Numerous stained apoptotic cells can be seen. Magnification 200x. (C) Immunocytochemistry for Bcl-2 in animal treated with PB for 10 weeks. Staining comparable to 4 weeks on PB. Note strong signal, especially around central vein (CV). Magnification 200x. (D) As for (C), but in animal remaining untreated for 10 weeks. Arrow indicates apoptotic body. (Inset) 20 days after PB removal. Magnification 200x. (E) TGF-ß1 immunocytochemistry, showing very little staining in PB-treated mouse after 4 weeks. (Inset) 3 days after removal of PB staining is noticeably increased. Magnification 100x. (F) As for (E) in untreated mouse of same age. More extensive and stronger staining is obvious. Magnification 100x.

 


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Fig. 3. Bax immunohistochemistry. (A) Mouse on PB for 4 weeks. Strong and heterogeneous staining, particularly perivascularly. Magnification 100x. (B) High magnification of same animal as in (A), showing heterogeneity in staining between cells. Magnification 800x. (C) As for (A) in age-matched, untreated mouse. Staining is homogeneous and less intense. (Inset) Control using 10-fold excess of Bax peptide supplied with antibody. Magnification 100x. (D) Higher magnification of liver in (C). Note absence of nuclear stain. Magnification 800x. (E) PB-treated mouse after 10 weeks. Staining has become more homogeneous and is now identical to the untreated animal. Magnification 150x. (F) Untreated mouse, age-matched with that from (E). Magnification 150x. (G) Animal 3 days after withdrawal of PB. Note that staining is still somewhat heterogeneous (compare A). Magnification 150x. (H) Liver of mouse 20 days after withdrawal of PB. Bax staining is now homogeneous and indistinguishable from untreated animals (compare with C and F). Magnification 150x. In all panels, sections were counterstained with hematoxylin. CV, central vein; PV, portal vein.

 
Effect on apoptosis and mitosis
After a short period of PB treatment (1 week), a significant elevation in the mitotic index was seen (Figure 4AGo), while continued treatment elicited a possible suppression of cell proliferation. At times after 4 weeks in PB-treated mice, the apoptotic index appeared to be suppressed, averaging ~0.26%, while the number of apoptoses in untreated animals was maintained at a constant level of ~0.51% for the duration of the experiment (Figure 4BGo). The difference in apoptotic indices between treated and untreated mice was statistically significant (P < 0.05) only at 10 weeks.



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Fig. 4. Percentage (A) mitosis and (B) apoptosis in PB-treated and untreated mice over the full course of the study. PB treatment induced no significant change in the number of mitoses, apart from a transient increase at 1 week (A). After 4 weeks on PB, a trend of suppression of apoptoses could be seen in PB-treated animals relative to untreated mice (B). *Significantly different from PB-treated (P < 0.05). Error bars, ±SEM.

 
In order to further examine the effect of PB on cell death suppression, mice were treated with PB for 4 weeks and then returned to a PB-free diet. A dramatic and rapid increase in the apoptotic index (and in TUNEL stained cell death), which included a rise in the number of `single cell necroses', was seen after only 3 days off treatment, peaking at a level >4-fold that of mice remaining on PB (Figures 2B and 5GoGo). Liver mass fell in concert with this increase in cell death. By 10 days after PB withdrawal, the apoptotic index had returned to a value comparable with those in mice undergoing continued treatment with PB.



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Fig. 5. Effect of withdrawal of PB. Solid thin line (open diamonds) shows the liver mass:body mass ratio (%) taken from Figure 1Go. The apoptotic index (solid line, closed squares) showed a peak at 3–7 days off PB, with values returning to normal by 10 days. This peak coincided with a rapid drop in liver mass:body mass ratio (%). Untreated animals (dotted line, closed triangles) showed unchanged levels of apoptosis. Error bars, ±SEM.

 
Cellular changes induced by PB
Since we were interested in elucidating the mechanism by which PB might suppress apoptosis, we examined some of the genes involved in known apoptotic pathways. Western blotting showed a large increase in Bcl-2 protein (~26 kDa) levels in mice on PB (Figure 6Go). No Bcl-2 was present at 3 weeks of age (0 week time point, Figure 6Go) and only very small amounts were detectable at other times in untreated animals. At 4 weeks on PB, Bcl-2 was markedly elevated compared with untreated controls and remained so in PB-treated animals for the duration of the study. When animals were taken off the PB diet, the Bcl-2 levels dropped sharply within 3 days and by 20 days had returned to the same level as in mice not treated with PB. The top, non-specific band is present at all time points except 0 weeks and shows no change whether animals are given PB or not. No change in Bcl-xS/L was seen by western blotting (data not shown).



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Fig. 6. Determination of Bcl-2 levels by western blot. While no Bcl-2 was present at the start of treatment (0 week time point, 3 weeks of age), PB treatment induced noticeable Bcl-2 protein production at all other times. A noticeable drop in Bcl-2 was seen after 3 days off PB (3d off), but by 20 days, no Bcl-2 was detected. Arrow indicates Bcl-2 (~26 kDa). C, Bcl-2 positive control (MI mouse myeloma cell line lysate).

 
By immunohistochemical staining it could also be seen that the overall level of Bcl-2 was increased in mice on PB (Figure 2CGo) and was slightly stronger in pericentral hepatocytes. In comparison, untreated animals showed weak staining (Figure 2DGo). In agreement with the western blotting data, removal of PB resulted in decreased Bcl-2 staining after 3 days, with near complete loss by 20 days (Figure 2DGo, inset).

When we examined the levels of TGF-ß1 by immunohistochemistry using the LC[1-30] antibody against mature TGF-ß1 (30), we found that administration of PB caused a clear decrease in TGF-ß1 staining (Figure 2EGo), compared with untreated animals (Figure 2FGo). TGF-ß1 returned to higher levels when PB was removed from the diet (Figure 2EGo, inset). The staining pattern in both treated and untreated animals was heterogeneous, with a predominantly perivascular distribution. By western blot, both TGF-ß RI and TGF-ß RII were present at identical levels in both the treated and untreated groups and immunohistochemistry also showed no difference in TGF-ß RII levels or distribution when mice were treated with PB (data not shown).

We made further use of western blotting to look at the pro-apoptotic protein Bax. No overall change in the concentration of Bax was seen when mice were treated with PB or removed from treatment (data not shown). By immunohistochemistry, the distribution of Bax changed dramatically after 4 weeks of PB treatment, with highly heterogeneous staining seen in animals given PB (Figure 3A and BGo), but homogeneous cytoplasmic staining seen in untreated animals throughout the experiment (Figure 3C and DGo). In the former group, approximately equal numbers of very strongly and very weakly stained cells were observed, with the strongest staining generally seen pericentrally. It should be noted that staining at 4 weeks in treated animals is both nuclear and cytoplasmic (Figure 3BGo), while without PB it is almost exclusively cytoplasmic (Figure 3DGo). Apoptotic and necrotic cells staining strongly for Bax were seen infrequently. To some extent, Bax staining co-localized with that of Bcl-2 in the pericentral area. However, the distribution of Bcl-2 was neither as defined nor as heterogeneous as that of Bax at 4 weeks of treatment (compare Figures 2C and 3AGoGo). Continued PB treatment saw less of a difference between treated and untreated groups, such that by 10 weeks almost no difference between the two could be seen: both groups showed predominantly homogeneous staining (compare Figure 3E and FGo). Removal of PB from the diet after 4 weeks of treatment resulted in a reassertion of the homogeneous Bax distribution seen in untreated animals; 3 days after PB removal the staining was mostly homogeneous (Figure 3GGo), with some residual, stronger staining perivascularly which had disappeared completely by 20 days (Figure 3HGo).

No changes were seen in general tyrosine phosphorylation, as determined by western blot using an anti-phosphotyrosine antibody (data not shown). In addition, contrary to reports in rats treated for extended periods with PB in initiation–promotion studies (6,8), no down-regulation of the EGFR was observed in those mice on the PB diet (data not shown).


    Discussion
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 Abstract
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 Materials and methods
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 References
 
We have previously utilized a double transgenic mouse, carrying transgenes for both c-myc and TGF-{alpha}, as a model system in which to study hepatocellular carcinogenesis (22,25,26,3133). This transgenic model provides an experimental system in which the effects of both transgenes, either singly or combined, upon cellular responses such as proliferation and apoptosis can be analyzed in vivo. In this present study we have used this system in which cells are continually being pushed to grow and proliferate under the combined influence of both the c-myc and TGF-{alpha} transgenes. This has provided a means to ascertain the mechanism of action of PB in the context of TGF-{alpha}. We found a PB-induced suppression of cell death, which was released when the drug was withdrawn. Administration of PB resulted in an increase in Bcl-2 levels, but a decrease in TGF-ß1, consistent with a reduction in apoptosis. Bax showed a shift from a homogeneous, predominantly cytoplasmic localization to a heterogeneous, more punctate and nuclear distribution, but was not linked with any increase in apoptosis. Our results demonstrated a PB-dependent rise in liver mass in c-myc/TGF-{alpha} mice due both to hypertrophy and a reduction in cell death.

It has been shown previously that PB can suppress apoptosis (1719,34,35). This provides a potential mechanism by which it can act as a promoter in carcinogenesis: if initiated cells normally destined to die are protected, then these cells can continue to proliferate, accumulate DNA lesions and go on to form preneoplastic foci and eventually tumors. However, the means by which PB protects against cell death has not yet been determined.

Recent work has raised the question of whether PB could inhibit apoptosis by acting as a survival factor in a similar manner to TGF-{alpha}, or indeed via the same pathway (2224,36). Using TGF-{alpha} transgenic mice, two groups have shown that this mitogen can decrease the incidence of cell death in mammary tissue during involution (23,24). Similar experiments in our laboratory indicated that TGF-{alpha} may function as a survival factor in hepatocytes (22). Further work in vitro showed the ability of TGF-{alpha} to replace PB in PB-dependent rat liver cell culture (21). Here it was found that the effects of PB and TGF-{alpha} on the formation of colonies were additive at sub-optimal concentrations and saturating at optimal concentrations. This work was strongly suggestive of a common pathway by which PB and TGF-{alpha} promote cell survival. A final intriguing piece of evidence from in vivo work showed that in CD-1 mice, PB treatment produced an increase in liver mass only when the TGF-{alpha} transgene was expressed (37). This implies that for liver hypertrophy to occur in these mice, an additional stimulus is required, in this case in the form of TGF-{alpha}. Alternatively, it is possible that the TGF-{alpha} signaling pathway needs to be active for PB to exert its full effect.

We set out to determine whether PB indeed suppressed apoptosis in our model system and to investigate possible pathways, including that of TGF-{alpha}, by which it could exert these effects. Morphological changes were evident within 2–3 weeks after PB administration began (Figure 2AGo) and coincided with a rapid increase in liver mass (Figure 1Go), which was subsequently maintained at a constant level. This significant elevation was due initially to the transient spike in mitoses in the first week (Figure 4AGo), but was maintained predominantly through extensive cellular hypertrophy (Figure 2AGo). The dysplasia was particularly noticeable pericentrally and far more prominent in PB-treated mice, as was the occurrence of aberrant mitoses and abnormal nuclei. On removal of the drug, cells were released from the PB-induced block of cell death and an increase in apoptosis resulted (Figure 5Go). Unexpectedly, elevated levels of `single cell necrosis' were also observed. However, this could be explained by new in vitro evidence which suggests common pathways for both apoptotic and necrotic forms of cell death (38,39) and shows that the exact manner of death may be dependent not only on gene expression, but also on ATP levels (39). In our in vivo system, many other factors may play a role, including the two transgenes. In addition, others have found induction of necrosis on PB withdrawal, pointing to a suppression of both this form of cell death, as well as apoptosis, by PB treatment (28,40,41). This evidence would support the theory that apoptosis and necrosis represent parallel pathways of cell death which may have common or interacting pathways (4143). A further complication is raised by reports that it is not always easy or possible to simply distinguish between apoptoses and necroses and the end point of apoptotic cell death can appear `necrotic' (42,44). We therefore believe that the `necrotic' cell death seen is a true manifestation of a cellular reaction to the release from PB treatment and is related to the role of PB as a survival factor in this model.

We found that PB could still have a dramatic effect on liver size (Figure 1Go), even in mice already overexpressing the c-myc and TGF-{alpha} transgenes and in which the TGF-{alpha} pathway was most likely already maximally stimulated. Western blotting showed that the levels of EGFR and of phosphorylated proteins were identical in both treated and untreated groups, indicating that the TGF-{alpha} membrane signaling components were present and not affected by PB. We did not see down-regulation of the EGFR, as has been reported in rats (6,8), although it should be noted that the latter experiments utilized an initiation–promotion protocol and the PB was administered by a different route (in drinking water). This led us to conclude that the effect of PB was over and above that of TGF-{alpha}. Although not dismissing some potential influence on the TGF-{alpha} system or the possibility that PB is acting on one or more downstream components of the TGF-{alpha} signaling pathway, it seemed more likely that the PB effect was occurring through a separate pathway.

Our attention therefore turned to examining the TGF-ß pathway as a possible point of influence of PB. Western blotting showed no change in TGF-ß RI or RII levels, indicating that these proteins were present and unaffected by PB. This is in agreement with work done in rats in which no change in type I or II receptor levels was observed after long-term PB treatment (45,46). We did not determine whether the TGF-ß signaling cascade was intact, but corroborating evidence has shown that this pathway is functional in this system (E.Santoni-Rugiu and S.S.Thorgeirsson, in preparation). Histochemical staining for TGF-ß1 ligand showed a definite decrease in mature TGF-ß1 in the livers of mice treated with PB (Figure 2EGo). Similar work done by others with rats demonstrated a decrease in staining especially in the pericentral region, while periportal staining was increased (4,8). However, we saw a more general reduction in TGF-ß1 staining due to PB. The observed decrease in actual TGF-ß1 concentration is believed to be an important mechanism by which PB suppresses apoptosis.

Santoni-Rugui et al. showed that the pericentral, dysplastic hepatocytes in c-myc/TGF-{alpha} animals were more prone to apoptosis than the periportal cells and hypothesized that an elevation in TGF-ß1 specifically in these cells was the cause of apoptosis (26). No such distribution of apoptosis was seen over the time course of this experiment, possibly as a result of the continually low levels of TGF-ß1 (and therefore general suppression of apoptosis) observed in this system due to the administration of PB. On withdrawal of PB, there was a large and rapid increase in TGF-ß1 (Figure 2EGo, inset) along with the sharp rise in cell death (Figure 5Go), which fits well with a role for TGF-ß1 in induction of cell death. However, once again, no clear zonal distribution of apoptotic cells was observed.

Further evidence of the influence of PB on apoptosis came from western blot analysis to detect the anti-apoptotic protein Bcl-2. Results clearly showed an increase in Bcl-2 in mice treated with PB when compared with untreated controls at all times during the study. A decrease in Bcl-2 was observed when PB was removed from the diet (Figure 6Go), coincident with a dramatic increase in cell death (Figure 5Go). Identical results were found by immunostaining: overall levels of Bcl-2 rose considerably during PB administration, more noticeable pericentrally, compared with untreated controls (Figure 2C and DGo) and decreased on PB withdrawal (Figure 2DGo, inset). Amounts of Bcl-xS/L protein did not vary between treated and untreated groups (data not shown). PB therefore appears to be both suppressing TGF-ß1 levels and up-regulating Bcl-2: two mechanisms, not necessarily mutually exclusive, which could be responsible for PB-induced inhibition of apoptosis.

A number of groups have shown that phosphorylation and dephosphorylation of Bcl-2 can take place in vitro. This can activate (47,48) or inactivate (49) Bcl-2, respectively. The upper band seen in the Bcl-2 western blot (Figure 6Go) may be interpreted as being a phosphorylated form of Bcl-2 and PB simply induced dephosphorylation. However, we do not believe this to be the case, as this would imply that Bcl-2 is constantly in a phosphorylated state, a scenario inconsistent with previous studies (4752). In addition, immunohistochemical evidence shows a large increase in Bcl-2 in PB-treated animals (Figure 2CGo), in agreement with the indicated band on the western blot (Figure 6Go).

The concentration of Bax was unchanged by western blot, but the distribution of this pro-apoptotic protein changed dramatically in mice on PB as shown by immunohistochemistry (Figure 3A–DGo). After administration of PB for 4 weeks, Bax staining was stronger in ~50% of all cells, particularly in the pericentral region (Figure 3A and BGo), and nuclear staining was more prevalent. In contrast, Bax staining was more homogeneous with little nuclear staining in untreated animals (Figure 3C and DGo) and in PB-treated mice after 7–10 weeks (Figure 3EGo). This would suggest that the pericentral, more dysplastic cells in treated mice after 4 weeks should be more sensitive and show higher levels of apoptosis. This was not observed, likely due to protection afforded by constant and markedly higher Bcl-2 and lower TGF-ß1 levels (Figure 2C and EGo). The later, more homogeneous appearance of Bax staining seen after 7 and 10 weeks of exposure to PB may demonstrate an adaptive response to prolonged treatment (Figure 3EGo), while the heterogeneous staining of Bax at 4 weeks can be thought of as a reaction of the different populations of hepatocytes (dysplastic versus normal) in the transgenic liver to chemical insult from PB.

It was noted that the distribution of Bax in hepatocytes from mice treated with PB for 4 weeks (Figure 3BGo) was noticeably more nuclear and punctate than that in untreated animals (Figure 3DGo), which displayed little or no nuclear staining. This cytosol-to-membrane (mitochondrial and nuclear) redistribution has been argued to be an early event, or even a trigger, for apoptosis (53,54). However, as mentioned above, the much higher levels of Bcl-2 observed during PB administration would be predicted to protect against Bax-induced apoptosis in a similar manner to the ectopic overexpression of Bcl-2 in in vitro experimental systems (38,5559). This situation could explain the failure to observe a clear zonal distribution of apoptosis in PB-treated animals.

Removal of PB after 4 weeks of treatment allowed the re-establishment of homogeneous Bax staining, noticeable after only 3 days (Figure 3GGo) and almost complete by 20 days (Figure 3HGo). This provides evidence that the shift in Bax localization was a direct result of PB administration.

Previous studies using untreated c-myc/TGF-{alpha} mice demonstrated the presence of preneoplastic foci in 20% of animals at 8 weeks of age and adenomas in more than one third by 4 months (26). Although PB works as a tumor promoter, in the time frame of this study it did not appear to significantly accelerate the appearance of foci or tumors. A more extensive study is currently underway to determine any long-term effects on neoplastic development in c-myc/TGF-{alpha} mice exposed to PB.

Another facet of PB which is worthy of further characterization is its possible effect on nuclear factor {kappa}B (NF-{kappa}B). Some evidence exists that significant activation of NF-{kappa}B occurs in rats treated with PB for between 3 and 10 days (60) and it appears from knockout studies that NF-{kappa}B is crucial for hepatocyte survival, at least during embryogenesis (61). In addition, it has been shown that Bcl-2 can have an effect on the transactivation function of NF-{kappa}B (62). A clear function for NF-{kappa}B in apoptosis has, however, yet to be elucidated and our system may provide a means to further shed light on this question.

We have collected strong evidence, using a double transgenic mouse system, that administration of PB suppresses apoptosis via two inter-related pathways. Firstly, mature TGF-ß1 levels are depressed in an environment where the signaling pathway for TGF-ß-induced cell death is apparently intact. This would be predicted to result in a decrease in apoptosis (in the absence of elevated death signals through other pathways, for example, the Fas receptor or tumor necrosis factor-{alpha} receptor), as was observed. Secondly, the concentration of Bcl-2 was higher in PB-treated than in untreated mice, providing an additional level of protection. Bax, on the other hand, demonstrates only a change in distribution at a time when the liver mass:body mass ratio reaches steady-state with prolonged PB treatment. PB-induced stimulation of mitosis was transient, while its effect on hepatocellular hypertrophy was prolonged and extensive and evidently responsible for the maintenance of an elevated liver mass. These data, besides demonstrating that Bcl-2 does in fact play a part in liver physiology, have provided evidence for a possible mechanism by which PB can exert a tumor-promoting effect.


    Notes
 
1 To whom correspondence should be addressed Email: snorri_thorgeirsson{at}nih.gov Back


    Acknowledgments
 
We would sincerely like to thank Dr Valentina Factor for her invaluable assistance with the immunohistochemistry and manuscript preparation and Nancy Sanderson for her expert help with animal breeding.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 19, 1998; revised September 16, 1998; accepted October 2, 1998.