Cell Proliferation and Apoptosis Are Altered in Mice Deficient in the NF-{kappa}B p50 Subunit after Treatment with the Peroxisome Proliferator Ciprofibrate

Job C. Tharappel*,{dagger}, Aysegul Nalca{ddagger}, Aaron B. Owens§, Leila Ghabrial{ddagger}, Elizabeth C. Konz*, Howard P. Glauert*,{dagger},§ and Brett T. Spear*,{dagger},{ddagger},1

* Graduate Center for Nutritional Sciences, {dagger} Graduate Center for Toxicology, {ddagger} Department of Microbiology, Immunology, and Molecular Genetics, § Department of Nutrition and Food Science, and Department of Pathology and Laboratory Medicine, University of Kentucky, Lexington, Kentucky 40536

Received April 17, 2003; accepted July 14, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously showed that the peroxisome proliferator ciprofibrate increases hepatic NF-{kappa}B DNA binding activity in rats, mice, and hepatoma cell lines. Here, we analyzed the response to ciprofibrate in mice that lack the NF-{kappa}B p50 gene (p50-/-). Wild-type and p50-/- mice were fed a diet with or without 0.01% ciprofibrate for 10 days. NF-{kappa}B DNA binding activity was present and increased after ciprofibrate treatment in wild-type mice, but was not detected in p50-/- mice. The untreated p50-/- mice had a higher level of hepatic cell proliferation, as measured by BrdU labeling, than did untreated wild-type mice. However, the increase in proliferation was greater in ciprofibrate-fed wild-type mice than in ciprofibrate-fed p50-/- mice. The apoptotic index was low in wild-type mice in the presence or absence of ciprofibrate. Apoptosis was increased in untreated p50-/- mice compared to wild-type mice; apoptosis was reduced in p50-/- mice after ciprofibrate feeding. The c-Jun and JunB mRNA levels were higher in untreated p50-/- mice than in untreated control mice; c-Jun mRNA levels increased, whereas JunB mRNA levels decreased in both groups after ciprofibrate treatment. The c-Jun and JunB protein levels were the same in untreated wild-type and p50-/- mice and increased in both groups after ciprofibrate treatment. Several apoptosis-related mRNAs were higher in untreated p50-/- mice compared to untreated control mice; expression of these genes increased in both groups after ciprofibrate treatment. These data indicate that NF-{kappa}B contributes to the proliferative and apoptotic changes that occur in the liver in response to ciprofibrate.

Key Words: liver; NF-{kappa}B; proliferation; apoptosis; peroxisome proliferator; ciprofibrate; gene knock-out mice.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferators are a group of chemically distinct compounds capable of eliciting a persistent peroxisome proliferation in hepatocytes and promoting liver tumors in rats and mice (Cattley et al., 1998Go; Rao and Reddy, 1987Go). These chemicals activate peroxisome proliferator-activated receptors, which leads to an increase in the size and number of peroxisomes, as well as activating genes encoding several enzymes of the peroxisomal ß-oxidation pathway (Eacho and Feller, 1991Go; Schoonjans et al., 1996Go). The rate-limiting enzyme of this pathway, fatty acyl CoA oxidase (FAO), produces hydrogen peroxide (H2O2) as a by-product. The activity of this enzyme is increased 10- to 15-fold by peroxisome proliferators such as ciprofibrate (Rao and Reddy, 1987Go). In contrast, the activity of the H2O2-detoxifying enzyme catalase is only increased about 2-fold by peroxisome proliferators (Rao and Reddy, 1987Go). It has been proposed that this imbalance in FAO and catalase induction may result in the accumulation of H2O2, which could at least partially be responsible for the effects of peroxisome proliferators. Several studies have found that the administration of peroxisome proliferators leads to lipid peroxidation and oxidative DNA damage, but other studies have not (Antonenkov et al., 1989Go; Cattley and Glover, 1993Go; Conway et al., 1989Go; Elliott and Elcombe, 1987Go; Glauert et al., 1992Go; Goel et al., 1986Go; Hegi et al., 1990Go; Huang et al., 1994Go; Huber et al., 1991Go; Kasai et al., 1989Go; Lake et al., 1987Go; Reddy et al., 1982Go; Srinivasan et al., 1990Go; Takagi et al., 1990aGo,bGo). Peroxisome proliferators have been found to decrease the levels of several cellular antioxidants and antioxidant enzymes, including vitamins C and E, and glutathione peroxidase (Conway et al., 1989Go; Glauert et al., 1992Go; Goel et al., 1986Go; Lake et al., 1989Go).

In addition to these biochemical changes, peroxisome proliferators increase cell proliferation in the liver soon after they are administered (Reddy and Lalwani, 1983Go). Cell proliferation eventually returns to basal levels for many peroxisome proliferators, but remains elevated for others (Chen et al., 1994Go; Eacho et al., 1991Go; Marsman et al., 1988Go; Yeldandi et al., 1989Go). In addition to stimulating DNA synthesis, peroxisome proliferators have been shown to inhibit apoptosis in normal and preneoplastic hepatocytes (Bayly et al., 1993Go, 1994Go; Roberts et al., 1995Go; Schulte-Hermann et al., 1995Go). The withdrawal of peroxisome proliferators leads to rapid reduction in liver weight, presumably in part by apoptosis (Schulte-Hermann et al., 1995Go).

In spite of numerous studies, the link between peroxisome proliferators and hepatocarcinogenesis on a molecular and cellular level is not fully understood. In addition, the link between active oxygen production by peroxisome proliferator-induced enzymes and carcinogenesis has not been demonstrated. Recently, we have shown that the peroxisome proliferator ciprofibrate increases the DNA binding activity of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) in rats and mice (Li et al., 1996Go; Nilakantan et al., 1998Go; Tharappel et al., 2001Go). NF-{kappa}B is normally found in the cytoplasm as an inactive complex consisting of two subunits (p50 and p65), which are bound to an inhibitory subunit, I{kappa}B; upon activation, NF-{kappa}B is released from I{kappa}B and translocates to the nucleus, where it increases the transcription of specific genes (Verma et al., 1995Go). NF-{kappa}B is important in the activation of genes that regulate cell proliferation and apoptosis in various cell types (Barkett and Gilmore, 1999Go; Beg et al., 1995Go; Demartin et al., 1999Go; Sha et al., 1995Go). Reactive oxygen species, including H2O2, are potent activators of NF-{kappa}B, while the addition of antioxidants such as vitamin E can block activation of NF-{kappa}B (Gabbita et al., 2000Go; Meyer et al., 1994Go). This has led us to hypothesize that peroxisome proliferators activate NF-{kappa}B through the induction of H2O2-generating enzymes such as FAO or through the down-regulation of antioxidants and antioxidant enzymes, such as vitamin E and glutathione peroxidase. We subsequently found that NF-{kappa}B can be activated by the overexpression of FAO in Cos cells, and that ciprofibrate-induced NF-{kappa}B activation can be inhibited by vitamin E or N-acetyl cysteine in rat hepatoma cells, by dietary vitamin E in rats, and by catalase overexpression in mice (Calfee et al., 1998Go; Li et al., 2000aGo,bGo; Nilakantan et al., 1998Go). Catalase overexpression also inhibited ciprofibrate-induced cell proliferation in hepatocytes (Nilakantan et al., 1998Go), thus raising the question of whether NF-{kappa}B activation is important in changes in cell proliferation induced by peroxisome proliferators.

In this study, we examined the hypothesis that the activation of NF-{kappa}B is necessary for peroxisome proliferator-induced changes in hepatocyte cell proliferation and apoptosis. We used a mouse model that is deficient in the p50 subunit of NF-{kappa}B (Sha et al., 1995Go). Wild-type and p50-/- mice were administered the peroxisome proliferator ciprofibrate for 10 days; cell proliferation, apoptosis, and the expression of genes related to cell proliferation and apoptosis were then quantified.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Ciprofibrate was a gift from Sanofi Research (Malvern, PA). Anti-bromodeoxyuridine (BrdU) antibody was purchased from Becton Dickinson (San Jose, CA); all other antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). The consensus NF-{kappa}B oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') was obtained from Promega (Madison, WI). The Vectastain kit for immunostaining was obtained from Vector Laboratories (Burlingame, CA). Alzet 1003D osmotic pumps for BrdU infusion were purchased from Durect (Cupertino, CA). The antigen retrieval solution Citra was purchased from BioGenex (San Ramon, CA). The super signal chemiluminescent substrate kit was purchased from Pierce (Rockford, IL). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Experimental design.
Eight 7–8-week-old male B6.129 mice homozygous for NF-{kappa}B (p50) mutation (p50-/-) and eight B6129SF2/J age-matched wild-type controls were obtained from the Jackson Laboratory (Bar Harbor, ME). Four mice from each group were fed either a diet containing 0.01% ciprofibrate or a control diet for 10 days; after eight days Alzet 1003D osmotic pumps containing 20 mg/ml BrdU solution were implanted sc. After 10 days, mice were euthanized and livers removed. A portion of the liver was fixed in formalin for histology, and the remainder was frozen in liquid nitrogen and then stored at -80°C.

Electrophoretic mobility shift assay.
Nuclear extracts were prepared from the frozen tissue by the method of Derykere and Gannon (1994)Go, as modified by Tharappel et al. (2001)Go. Five µg of nuclear extract was incubated with 0.5 µg poly (dI-dC) in a binding buffer (50 mM KCl, 10 mM Hepes-KOH, pH 7.9, 6.5 mM dithiothreitol, and 10% glycerol) for 5 min, followed by 20 min incubation at room temperature after adding 20,000 cpm of the {gamma}32P end-labeled NF-{kappa}B oligonucleotide probe. After incubation, samples were resolved on 7% nondenaturing polyacrylamide gels, which were then dried under vacuum and exposed overnight at -80°C to Kodak XOMAT-AR film.

BrdU immunostaining.
Tissue sections were fixed in formalin and embedded in paraffin blocks. Sections (5 µm thick) were prepared and stained with an anti-BrdU antibody using the Vecta Stain ABC kit according to the manufacturer’s protocol. Cells containing newly synthesized DNA were visualized by dark brown labeled nuclei. Roughly 3000 total nuclei were randomly counted from each slide and labeling indices were calculated.

Apoptosis assay.
The terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) apoptosis assay kit was purchased from Intergen (Purchase, NY). The assay was performed on paraffin sections following the manufacturer’s protocol. At least 3000 nuclei were randomly counted per slide and the apoptotic index was expressed as the percentage of number of labeled apoptotic bodies of the total number of nuclei counted.

Western blotting.
Lysis buffer (1% Nonidet P-40, 0.1% SDS, 0.1 mg/ml PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, and 1x PBS) was added to frozen liver tissue (1 part liver tissue to 4 parts lysis buffer) and homogenized using an Ultra-Turrax homogenizer (Tekmar Co. Cincinnati, OH). Homogenates were centrifuged at 10,000 x g for 20 min, and the supernatants were again centrifuged at 100,000 x g for one h. Supernatants were collected, aliquoted, and stored at -80°C. One aliquot was used for a protein assay using the BCA protocol (Pierce, Rockford, IL). The samples were denatured by boiling for 5 min with 2x gel loading buffer (17.3% glycerol, 1.25 M ß-mercaptoethanol, 5.2% SDS, 0.22 M Tris, pH 6.8, 1–2 mg bromophenol blue). Twenty-five µg of protein from each sample was electrophoresed (4% stacking gel and 8.5% separating gel at 175 volts for one h) and then electroblotted onto nitrocellulose membranes (Life Technologies, Carlsbad, California) at 100 volts for one h. Membranes were then incubated for one h in a blocking buffer (5% fat-free dry instant powdered milk, 1mM Tris-base, 15 mM NaCl, and 0.05% Tween-20) at room temperature with shaking. The primary and secondary antibodies were diluted in blocking buffer. They were then incubated with the membrane while shaking for one h, starting with the primary antibody and followed by the secondary antibody at room temperature. The membranes were then washed with 3 changes of wash buffer (1mM Tris-base, 15 mM NaCl, and 0.05% Tween-20) after the primary and secondary antibody incubations. A Super Signal chemiluminescent substrate kit from Pierce was used to detect antibodies bound to the membrane.

Ribonuclease protection assays.
Total RNA was prepared from frozen liver sections using the LiCl method. The RiboQuantTM RNase Protection Assay Kits were obtained from Becton Dickinson Pharmingen (San Diego, CA). RNase protection assays (RPAs) were performed with 30 µg of total RNA following the manufacturer’s protocol. The Fos/Jun (cat# 556194) and apoptosis (cat# 556192) multiprobe templates were used.

FAO assay.
FAO activity in liver tissue homogenates was determined using lauroyl CoA as the substrate as described by Poosch and Yamazaki (1986)Go.

Statistical analysis.
Results were first analyzed by two-way analysis of variance (ANOVA). If a significant interaction was observed, results were further analyzed using Bonferroni’s test. The results are reported as means ± SEM.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we tested whether NF-{kappa}B contributed to changes in cell proliferation and apoptosis in response to treatment with peroxisome proliferators. To accomplish this, we fed an unrefined diet containing 0.01% ciprofibrate (or a control unrefined diet) to mice that were deficient in the p50 subunit of NF-{kappa}B (p50-/-; [Sha et al., 1995Go]) or to wild-type control mice. After ten days of feeding, mice were euthanized; Alzet osmotic pumps containing bromodeoxyuridine were implanted two days before euthanasia. Tissue sections were prepared, and the remaining portion of the liver was frozen in liquid nitrogen and stored at -80°C. We examined whether wild-type or p50-/- mice were responding to ciprofibrate treatment by the increase in liver weights and the activity of hepatic FAO. The p50-/- mice weighed significantly less than wild-type mice at the beginning of the study, but there were no significant differences in the final body weights, although mice receiving ciprofibrate weighed slightly more than nontreated mice (p = 0.063), and p50-/- mice weighed slightly less than wild-type mice (p = 0.15) (Table 1Go). Liver-to-body weight ratios were significantly increased in both wild-type and p50-/- mice fed ciprofibrate compared to mice receiving a normal diet. As a further measure of the response to ciprofibrate treatment, FAO activity was examined in liver homogenates (Fig. 1Go). FAO is the rate-limiting enzyme in the peroxisomal ß-oxidation pathway and highly induced by peroxisome proliferators (Rao and Reddy, 1987Go). In mice receiving control diets, FAO levels were slightly higher (p = 0.3) in wild-type animals compared to p50-/- mice. A strong induction in FAO levels was seen in both groups after ciprofibrate treatment; over seven-fold and roughly 10-fold in wild-type and p50-/- mice, respectively. Thus, both the p50-/- and wild-type mice respond to ciprofibrate as measured by liver weight gain and FAO induction.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Influence of p50 Deletion and Ciprofibrate (CIP) on Liver and Body Weights
 


View larger version (28K):
[in this window]
[in a new window]
 
FIG. 1. Effect of ciprofibrate on fatty acyl CoA oxidase (FAO) activity in p50-/- and wild-type mice. Mice were fed a control diet or a diet containing 0.01% ciprofibrate for 10 days. The activity of FAO was quantified in liver homogenates. Data are means ± SE. Values with different superscripts are significantly different, by Bonferroni’s test (p < 0.05).

 
We previously showed that treatment of rats and mice with ciprofibrate for 10 days led to increased hepatic NF-{kappa}B DNA binding activity. To insure that the mice in this study were responsive to ciprofibrate as measured by NF-{kappa}B induction, and to determine whether the lack of the p50 gene affected this induction, we performed EMSAs using nuclear extracts prepared from the different groups of mice. Consistent with previous studies, we saw an increase in NF-{kappa}B DNA binding activity in wild-type mice treated with ciprofibrate compared to untreated control mice (Fig. 2Go). In contrast, levels of NF{kappa}B DNA binding activity in p50-/- mice were dramatically lower than in wild-type mice. In addition, we observed no change in NF-{kappa}B activity after 10 days of ciprofibrate treatment in the p50-/- mice. Thus, the lack of p50 resulted in substantially lower levels of NF-{kappa}B in both untreated and treated animals.



View larger version (76K):
[in this window]
[in a new window]
 
FIG. 2. NF-{kappa}B activity in wild-type and p p50-/- mice fed a control diet or 0.01% ciprofibrate for 10 days. Nuclear extracts were prepared from mouse livers and used in EMSAs with a radiolabeled oligonucleotide containing a NF-{kappa}B binding site. After electrophoresis, gels were dried and subjected to autoradiography. The radioactive probe was incubated with no extract (lane 1, Free Probe, F.P.), control Hela cell extract (lane 14), or 5 µg of nuclear extract from wild-type mice (lanes 2–7) or p50-/- mice (lanes 8–13) that were fed a control diet (lanes 2–4, 8–10) or ciprofibrate diet (lanes 5–7, 11–13). For lanes 2–13, each lane represents a single mouse.

 
Peroxisome proliferators in rodents lead to hepatomegaly, in part, by increasing cell proliferation (Chen et al., 1994Go; Eacho et al., 1991Go; Marsman et al., 1988Go; Reddy and Lalwani, 1983Go; Yeldandi et al., 1989Go). To examine the role of p50 in DNA synthesis, we quantified BrdU uptake, which serves as a measure of proliferation, by staining liver sections with anti-BrdU antibodies (Fig. 3Go). These data indicated that the BrdU labeling index was higher in untreated p50-/- mice than in untreated wild-type mice, although this was not statistically significant. The BrdU labeling index increased over 23-fold in wild-type mice after ciprofibrate treatment. In contrast, BrdU labeling increased only 2.2-fold in p50-/- mice after 10 days of ciprofibrate feeding. Thus, the NF-{kappa}B p50 subunit is required for normal cell proliferation after treatment with peroxisome proliferators.



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 3. Effect of ciprofibrate on hepatocyte proliferation in p50-/- and wild-type mice. Mice were fed a control diet or a diet containing 0.01% ciprofibrate for 10 days. Two days before euthanasia, Alzet osmotic pumps containing bromodeoxyuridine (BrdU) were implanted sc. Histological sections for the liver were immunohistochemically stained for BrdU, and labeling indexes were determined in hepatocytes to determine the rate of DNA synthesis. Data are means ± SE. Values with different superscripts are significantly different, using Bonferonni’s test (p < 0.05).

 
Increased cell proliferation in the liver is associated with increased AP-1 activity. AP-1 is a heterodimer composed of c-Fos and c-Jun, both of which are members, respectively, of the Fos and Jun families of transcription factors. To monitor changes in the expression of genes in these families, we performed RPAs using the Fos/Jun RPA kit (Fig. 4Go, Table 2Go). Levels of c-Jun, JunD, and JunB were significantly higher in untreated p50-/- mice than in untreated control mice; no significant differences were apparent in the levels of Fra2. These increases in c-Jun, JunD, and JunB mRNA are consistent with the BrdU data (Fig. 3Go), which showed increased proliferation in untreated p50-/- mice. Levels of c-Jun mRNA increased after ciprofibrate treatment in both wild-type and p50-/- mice, consistent with the increased cell proliferation seen in these animals. In contrast, JunB and JunD levels were reduced in both groups after ciprofibrate treatment. To confirm that the changes in mRNA levels resulted in increased protein, we performed western blots using antibodies specific for members of the Jun family (Fig. 5Go). Levels of c-Jun and JunB appeared to be roughly the same in untreated wild-type and p50-/- mice, which is in contrast to the change in mRNA levels between these two groups of mice. However, levels of c-Jun and JunB were increased in both groups after ciprofibrate treatment, consistent with the increased cell proliferation seen in these two groups.



View larger version (83K):
[in this window]
[in a new window]
 
FIG. 4. RPA analysis of Fos/Jun mRNAs in untreated and ciprofibrate treated wild-type and p50-/- mice. Thirty µg of total RNA was incubated with the Ambion Fos/Jun RPA template kit. RNAs were resolved on denaturing polyacrylamide gels which were subsequently dried and subjected to autoradiography. Each lane represents RNA from a single mouse. Lanes 1–4, untreated p50-/- mice; lanes 5–8, ciprofibrate-treated p50-/- mice; lanes 9–12, untreated wild-type mice; lanes 13–16, ciprofibrate-treated wild-type mice. Identities of the transcripts are noted on the right; transcripts for c-Fos, FosB, and Fra1 could not be detected.

 

View this table:
[in this window]
[in a new window]
 
TABLE 2 Quantitation of RPA Analysis from Figure 4Go
 


View larger version (69K):
[in this window]
[in a new window]
 
FIG. 5. Western analysis of c-jun and junB in untreated and ciprofibrate-fed wild-type and p50-/- mice. Protein samples were resolved by SDS-PAGE and electroblotted to nitrocellulose. Membranes were incubated with antibodies against c-Jun or JunB, and reprobed with ß-actin, as indicated. Each lane represents a protein sample from an individual mice; mouse genotype (wild-type or p50-/-) and treatment [control diet (control) or ciprofibrate (+Cip)] are designated.

 
Previous studies have shown that peroxisome proliferator treatment leads to a decrease in apoptosis in hepatocytes (Bayly et al., 1993Go, 1994Go; Roberts et al., 1995Go; Schulte-Hermann et al., 1995Go). Apoptosis was evaluated in this study with the TUNEL assay (Fig. 6Go). A low apoptotic index was observed in untreated wild-type mice; the level of apoptosis did not change after ciprofibrate feeding. Our inability to detect a ciprofibrate-mediated reduction in apoptosis may be due to the fact that the levels of TUNEL staining were simply too low in untreated mice. The level of apoptosis in untreated p50-/- mice was roughly 4-fold higher than was observed in untreated wild-type mice. This increase in apoptosis may be a consequence of impaired NF-{kappa}B activity in the absence of p50. Ciprofibrate treatment resulted in a significant decrease in TUNEL staining in p50-/- mice.



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 6. Effect of ciprofibrate on apoptosis in p50-/- and wild-type mice. Mice were fed a control diet or a diet containing 0.01% ciprofibrate for 10 days. Histological sections for the liver were used for TUNEL staining, and apoptotic indexes were determined in hepatocytes to determine the rate of apoptosis. Data are means ± SE. Values with different superscripts are significantly different, using Bonferroni’s test (p < 0.05).

 
The TUNEL assays demonstrated significant differences in apoptosis in the different groups of mice. To investigate these differences further, we performed RPAs to monitor the expression of several genes that are involved in apoptosis (Fig. 7Go, Table 3Go). The levels of mRNAs encoded by several of these genes, including FLICE, FADD, and FAS, were significantly higher in untreated p50-/- mice compared to untreated control mice. This correlates with the increased apoptosis seen in these animals. The expression of these genes as well as that of FAP significantly increases in both groups of mice after ciprofibrate treatment, despite the fact that no change in apoptosis was observed in treated wild-type mice, and in fact, a significant decrease in apoptosis was observed in p50-/- mice. The expression of RIP, however, was significantly decreased after ciprofibrate treatment. This suggests that anti-apoptotic signals after ciprofibrate treatment may counter any pro-apoptotic signals.



View larger version (82K):
[in this window]
[in a new window]
 
FIG. 7. RPA analysis of mRNAs of genes involved with apoptosis in untreated and ciprofibrate treated wild-type and p50-/- mice. Thirty µg of total RNA was incubated with the Ambion apoptosis RPA template kit. RNAs were resolved on denaturing polyacrylamide gels, which were subsequently dried and subjected to autoradiography. Each lane represents RNA from a single mouse. Lanes 1–4, untreated p50-/- mice; lanes 5–8, ciprofibrate-treated p50-/- mice; lanes 9–12, untreated wild-type mice; lanes 13–16, ciprofibrate-treated wild-type mice. Identities of the transcripts are noted on the right; transcripts for FasL, FAF, and TRAIL could not be detected.

 

View this table:
[in this window]
[in a new window]
 
TABLE 3 Quantitation of RPA Analysis from Figure 7Go
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The data presented here indicate that NF-{kappa}B plays an important role in the response to peroxisome proliferators in the liver. In wild-type mice, a large increase was seen in cell proliferation after the administration of ciprofibrate, as has been observed previously, but in the p50-/- mice, the increase in cell proliferation was much lower. Apoptosis was also affected: p50-/- mice had a much higher apoptotic index than wild-type mice, and the administration of ciprofibrate inhibited this increase.

These findings provide direct evidence that NF-{kappa}B is involved in peroxisome proliferator-induced changes in hepatic cell proliferation. Earlier studies have provided indirect evidence that NF-{kappa}B is important in the induction of cell proliferation by peroxisome proliferators. Nilakantan et al. (1998)Go found that catalase overexpression decreased both the activation of NF-{kappa}B and the induction of cell proliferation by ciprofibrate in mice. In studies comparing species that are responsive (rats) or nonresponsive (hamsters) to peroxisome proliferator-induced carcinogenesis, NF-{kappa}B was found to be activated by peroxisome proliferators in rats but not in hamsters, which correlated with the induction of cell proliferation in these two species (Durnford et al., 1998Go; Tharappel et al., 2001Go). While these previous studies only show correlations between peroxisome proliferator exposure, NF-{kappa}B activation, and the induction of cell proliferation, the present study clearly demonstrates that NF-{kappa}B activity is necessary for the maximum induction of cell proliferation by the peroxisome proliferator ciprofibrate.

In this study, we observed that c-Jun mRNA levels were increased and that JunB mRNA levels were decreased after ciprofibrate administration, whereas the protein levels of c-Jun and JunB were increased. This suggests that post-transcriptional mechanisms must account for differences in the levels of the Jun proteins after ciprofibrate treatment. In previous studies, peroxisome proliferators have been shown to increase the expression of c-Fos, c-Jun, JunB, FosB, and/or JunD in the BNL-CL.2 or ML-457 mouse liver cell lines and in primary rat hepatocytes (Ledwith et al., 1993Go, 1996Go; Pauley et al., 2002Go). The administration of peroxisome proliferators to rats, however, was found to have no effect on Fos expression (Bardot et al., 1995Go; Goldsworthy et al., 1994Go), or to slightly increase the expression of c-Fos and c-Jun (Hasmall et al., 1997Go). In a study that examined hepatic expression of the Fos/Jun family in p50-/- mice, DeAngelis et al.(2001)Go observed that c-Fos expression was the same in p50-/- and wild-type mice, both in untreated mice and in mice subjected to partial hepatectomy. The reasons for the lack of agreement among these studies and the present study are not clear.

Peroxisome proliferators have previously been shown to inhibit apoptosis in hepatocytes (Schulte-Hermann et al., 1995Go). NF-{kappa}B has been found to have anti-apoptotic activity in several cell types, including hepatic cell lines, by several agents, including TNF-{alpha} and TGF-ß (Barkett and Gilmore, 1999Go). In the present study, apoptosis was increased in untreated p50-/- mice compared to untreated wild-type mice, consistent with an anti-apoptotic role for NF-{kappa}B in the liver. Apoptosis was inhibited by ciprofibrate administration. One explanation for this inhibition is that ciprofibrate increases the DNA binding activity of other NF-{kappa}B family members, e.g., p65 homodimers or p65/p52 heterodimers, which have anti-apoptotic activity. Alternatively, ciprofibrate could be altering apoptosis through other signal transduction pathways. In this study, ciprofibrate administration to wild-type mice did not affect apoptosis; however, the apoptotic index was so low in wild-type mice that it would have been difficult to observe a further decrease induced by ciprofibrate.

The mRNA levels of several apoptosis-related genes were higher in untreated p50-/- mice compared to untreated wild-type mice, which correlates with the TUNEL data. However, levels of these mRNAs increased in both groups of mice after ciprofibrate treatment, despite the fact that apoptosis decreased or remained unchanged in p50-/- and wild-type mice, respectively. This suggests that additional anti-apoptotic mechanisms must also be occurring in the ciprofibrate-treated mice. Using specific inhibitors, Mounho and Thrall (1999)Go demonstrated that the extracellular signal-regulated kinase (ERK) pathway is important in the suppression of apoptosis by peroxisome proliferators in primary mouse hepatocytes. The peroxisome proliferator nafenopin reduced the expression of the negative growth regulator TGF-ß in primary rat hepatocytes (Strange and Roberts, 1996Go). Using proteomic analysis, Chevalier et al. (2000)Go observed the accumulation of three actin fragments, which are substrates of a pro-apoptotic enzyme, in primary rat hepatocytes after nafenopin treatment. No changes in apoptotic enzymes were observed, however, in proteomic analysis of diethylhexylphthalate-treated mice (Macdonald et al., 2001Go). Mice treated with Wy-14,643 showed increased expression of cell-death-inducing DNA-fragmentation factor-alpha in a microarray analysis (Cherkaoui-Malki et al., 2001Go). Clearly, more research is needed to understand the mechanisms by which peroxisome proliferators inhibit hepatocyte apoptosis, and the role of NF-{kappa}B in this process.

Several studies have used genetically modified mice to examine the role of NF-{kappa}B subunits on cell proliferation and apoptosis in the liver and other tissues. A clear role for NF-{kappa}B in inhibiting apoptosis by TNF-{alpha} or other apoptosis inducers in several cell types, including hepatocytes, has been demonstrated in studies in which NF-{kappa}B activity has been inhibited by the deletion of one of its subunits, the inhibition of its translocation, or the expression of a dominant negative form of I{kappa}B (Beg and Baltimore, 1996Go; Schoemaker et al., 2002Go; Vanantwerp et al., 1996Go; Wang et al., 1996Go; Xu et al., 1998bGo). In contrast to the dramatic affect that p50 has in the response to ciprofibrate, DNA synthesis and liver regeneration following partial hepatectomy or carbon tetrachloride treatment were not affected by the absence of the p50 subunit. In this latter study, increased levels of the p65 subunit may have compensated for the lack of p50 (DeAngelis et al., 2001Go). Similarly, the hepatic-specific expression of a truncated I{kappa}B{alpha} super-repressor did not affect DNA synthesis, apoptosis, or liver regeneration following partial hepatectomy, but led to increased apoptosis after treatment with TNF-{alpha} (Chaisson et al., 2002Go). Also, the hepatic inflammatory response after ischemia/reperfusion was not altered in p50-/- mice (Kato et al., 2002Go). In the RALA 255–10G rat hepatocyte cell line, expression of an I{kappa}B super-repressor inhibited cell proliferation but not apoptosis by TNF-{alpha} (Xu et al., 1998aGo). In addition, B cells lacking p50, RelB, or c-Rel (but not p52 or p65) have decreased proliferation in response to LPS (Horwitz et al., 1999Go; Kontgen et al., 1995Go; Sha et al., 1995Go; Snapper et al., 1996aGo,bGo). Overall, whether specific NF-{kappa}B subunits are essential for cell proliferation depends on the tissue and the stimulus for DNA synthesis.

Interestingly, the liver-to-body-weight ratios were higher in the p50-/- mice as well as in the ciprofibrate-treated mice. Higher liver weights have been observed many times previously in peroxisome proliferator-treated rats and mice, and the higher liver weights seen here correlate well with the induction of cell proliferation and inhibition of apoptosis by ciprofibrate. In the p50-/- mice, the increases in the liver to body weight ratio were much lower than those seen in mice administered ciprofibrate. The increases also did not correlate with changes in cell proliferation or apoptosis: in the untreated mice, both cell proliferation and apoptosis were increased by the deletion of p50, and in the ciprofibrate-treated mice, apoptosis was increased and cell proliferation was decreased. It is possible that the ratio was more affected by changes in body weight or that liver weights were even higher in p50-/- at the beginning of the study, a time at which this ratio could not be measured.

This study clearly shows that NF-{kappa}B is important in the proliferative and apoptotic changes induced by ciprofibrate. The molecular mechanisms of these changes, however, are not clear at this time. We found that ciprofibrate altered the expression of genes involved in cell proliferation and apoptosis, but the changes observed did not necessarily correlate with the effects on cell proliferation and apoptosis. Future studies will be needed to determine which NF-{kappa}B-regulated genes are responsible for alterations in cell proliferation and apoptosis induced by peroxisome proliferators.


    ACKNOWLEDGMENTS
 
This study was supported by National Cancer Institute grant CA74147 and by the Kentucky Agricultural Experiment Station. J. C. Tharappel was supported by a training grant from the National Institute of Environmental Health Sciences (ES07266). E. C. Konz was supported by a training grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK07778).


    NOTES
 
1 To whom correspondence should be addressed at Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536. Fax: (859) 257-8994. E-mail: bspear{at}uky.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antonenkov, V. D., Pirozhkov, S. V., Popova, S. V., and Panchenko, L. F. (1989). Effect of chronic ethanol, catalase inhibitor 3-amino-1,2,4-triazole and clofibrate treatment on lipid peroxidation in rat myocardium. Int. J. Biochem. 21, 1313–1318.[CrossRef][ISI][Medline]

Bardot, O., Clemencet, M. C., Malki, M. C., and Latruffe, N. (1995). Delayed effects of ciprofibrate on rat liver peroxisomal properties and proto-oncogene expression. Biochem. Pharmacol. 50, 1001–1006.[CrossRef][ISI][Medline]

Barkett, M., and Gilmore, T. D. (1999). Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene 18, 6910–6924.[CrossRef][ISI][Medline]

Bayly, A. C., French, N. J., Dive, C., and Roberts, R. A. (1993). Non-genotoxic hepatocarcinogenesis invitro—the fao hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis. J. Cell. Sci. 104, 307–315.[Abstract/Free Full Text]

Bayly, A. C., Roberts, R. A., and Dive, C. (1994). Suppression of liver cell apoptosis invitro by the non-genotoxic hepatocarcinogen and peroxisome proliferator nafenopin. J. Cell Biol. 125, 197–203.[Abstract]

Beg, A. A., and Baltimore, D. (1996). An essential role for NF-kappa B in preventing TNF-alpha-induced cell death. Science 274, 782–784.[Abstract/Free Full Text]

Beg, A. A., Sha, W. C., Bronson, R. T., Ghosh, S., and Baltimore, D. (1995). Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 376, 167–170.[CrossRef][ISI][Medline]

Calfee, K. G., Spear, B. T., and Glauert, H. P. (1998). Increased dietary vitamin E leads to decreased activation of hepatic NF-kB by the peroxisome proliferator ciprofibrate. FASEB J. 12, A563.

Cattley, R. C., DeLuca, J., Elcombe, C., Fenner-Crisp, P., Lake, B. G., Marsman, D. S., Pastoor, T. A., Popp, J. A., Robinson, D. E., Schwetz, B., et al. (1998). Do peroxisome proliferating compounds pose a hepatocarcinogenic hazard to humans? Regul. Toxicol. Pharmacol. 27, 47–60.[CrossRef][ISI]

Cattley, R. C., and Glover, S. E. (1993). Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: Relationship to carcinogenesis and nuclear localization. Carcinogenesis 14, 2495–2499.[Abstract]

Chaisson, M. L., Brooling, J. T., Ladiges, W., Tsai, S., and Fausto, N. (2002). Hepatocyte-specific inhibition of NF-kappa B leads to apoptosis after TNF treatment, but not after partial hepatectomy. J. Clin. Invest. 110, 193–202.[Abstract/Free Full Text]

Chen, H., Huang, C. Y., Wilson, M. W., Lay, L. T., Robertson, L. W., Chow, C. K., and Glauert, H. P. (1994). Effect of the peroxisome proliferators ciprofibrate and perfluorodecanoic acid on hepatic cell proliferation and toxicity in Sprague-Dawley rats. Carcinogenesis 15, 2847–2850.[Abstract]

Cherkaoui-Malki, M., Meyer, K., Cao, W. Q., Latruffe, N., Yeldandi, A. V., Rao, M. S., Bradfield, C. A., and Reddy, J. K. (2001). Identification of novel peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in mouse liver using cDNA microarray analysis. Gene Expr. 9, 291–304.[ISI][Medline]

Chevalier, S., Macdonald, N., Tonge, R., Rayner, S., Rowlinson, R., Shaw, J., Young, J., Davison, M., and Roberts, R. A. (2000). Proteomic analysis of differential protein expression in primary hepatocytes induced by EGF, tumour necrosis factor alpha or the peroxisome proliferator nafenopin. Eur. J. Biochem. 267, 4624–4634.[Abstract/Free Full Text]

Conway, J. G., Tomaszewski, K. E., Olson, M. J., Cattley, R. C., Marsman, D. S., and Popp, J. A. (1989). Relationship of oxidative damage to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and Wy-14,643. Carcinogenesis 10, 513–519.[Abstract]

DeAngelis, R. A., Kovalovich, K., Cressman, D. E., and Taub, R. (2001). Normal liver regeneration in p50/nuclear factor kappa B1 knockout mice. Hepatology 33, 915–924.[CrossRef][ISI][Medline]

Demartin, R., Schmid, J. A., and Hoferwarbinek, R. (1999). The NF-kappa B/Rel family of transcription factors in oncogenic transformation and apoptosis. Mutat. Res. 437, 231–243.[CrossRef][ISI][Medline]

Deryckere, F., and Gannon, F. (1994). A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 16, 405.[ISI][Medline]

Durnford, J. M., Hejtmancik, M. R., Kurtz, P. J., Renne, R. A., Gideon, K. M., Marsman, D. S., Vallant, M., Chhabra, R., and Cunningham, M. L. (1998). Peroxisomal enzyme activity and cell proliferation in rats, mice and hamsters exposed for 13-weeks to Wy-14,643 and gemfibrozil. Toxicol. Sci. 42, 11.[CrossRef]

Eacho, P. I., and Feller, D. R. (1991). Hepatic peroxisome proliferation induced by hypolipidemic drugs and other chemicals. In Antilipidemic Drugs, Medicinal, Chemical and Biochemical Aspects (D. T. Witiak, H. A. I. Newman, and D. R. Feller, Eds.), pp. 375–426. Elsevier Science, Amsterdam.

Eacho, P. I., Lanier, T. L., and Brodhecker, C. A. (1991). Hepatocellular DNA synthesis in rats given peroxisome proliferating agents: Comparison of WY-14,643 to clofibric acid, nafenopin and LY171883. Carcinogenesis 12, 1557–1561.[Abstract]

Elliott, B. M., and Elcombe, C. R. (1987). Lack of DNA damage or lipid peroxidation measured in vivo in the rat liver following treatment with peroxisomal proliferators. Carcinogenesis 8, 1213–1218.[Abstract]

Gabbita, S. P., Robinson, K. A., Stewart, C. A., Floyd, R. A., and Hensley, K. (2000). Redox regulatory mechanisms of cellular signal transduction. Arch. Biochem. Biophys. 376, 1–13.[CrossRef][ISI][Medline]

Glauert, H. P., Srinivasan, S., Tatum, V. L., Chen, L. C., Saxon, D. M., Lay, L. T., Borges, T., Baker, M., Chen, L. H., Robertson, L. W., et al. (1992). Effects of the peroxisome proliferators ciprofibrate and perfluorodecanoic acid on hepatic cellular antioxidants and lipid peroxidation in rats. Biochem. Pharmacol. 43, 1353–1359.[CrossRef][ISI][Medline]

Goel, S. K., Lalwani, N. D., and Reddy, J. K. (1986). Peroxisome proliferation and lipid peroxidation in rat liver. Cancer Res. 46, 1324–1330.[Abstract]

Goldsworthy, T. L., Goldsworthy, S. M., Sprankle, C. S., and Butterworth, B. E. (1994). Expression of myc, fos and Ha-ras associated with chemically induced cell proliferation in the rat liver. Cell Prolif. 27, 269–278.[ISI][Medline]

Hasmall, S. C., Pyrah, I. T. G., Soames, A. R., and Roberts, R. A. (1997). Expression of the immediate-early genes, c-fos, c-jun, and c-myc: A comparison in rats of nongenotoxic hepatocarcinogens with noncarcinogenic liver mitogens. Fundam. Appl. Toxicol. 40, 129–137.[CrossRef][ISI][Medline]

Hegi, M. E., Ulrich, D., Sagelsdorff, P., Richter, C., and Lutz, W. K. (1990). No measurable increase in thymidine glycol or 8-hydroxydeoxyguanosine in liver DNA of rats treated with nafenopin or choline-devoid low-methionine diet. Mutat. Res. 238, 325–329.[ISI][Medline]

Horwitz, B. H., Zelazowski, P., Shen, Y., Wolcott, K. M., Scott, M. L., Baltimore, D., and Snapper, C. M. (1999). The p65 subunit of NF-kappa B is redundant with p50 during B cell proliferative responses, and is required for germline C-H transcription and class switching to IgG3. J. Immunol. 162, 1941–1946.[Abstract/Free Full Text]

Huang, C. Y., Wilson, M. W., Lay, L. T., Chow, C. K., Robertson, L. W., and Glauert, H. P. (1994). Increased 8-hydroxydeoxyguanosine in hepatic DNA of rats treated with the peroxisome proliferators ciprofibrate and perfluorodecanoic acid. Cancer Lett. 87, 223–228.[ISI][Medline]

Huber, W., Kraupp-Grasl, B., Esterbauer, H., and Schulte-Hermann, R. (1991). Role of oxidative stress in age dependent hepatocarcinogenesis by the peroxisome proliferator nafenopin in the rat. Cancer Res. 51, 1789–1792.[Abstract]

Kasai, H., Okada, Y., Nishimura, S., Rao, M. S., and Reddy, J. K. (1989). Formation of 8-hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator. Cancer Res. 49, 2603–2605.[Abstract]

Kato, A., Edwards, M. J., and Lentsch, A. B. (2002). Gene deletion of NF-kappa B p50 does not alter the hepatic inflammatory response to ischemia/reperfusion. J. Hepatol. 37, 48–55.[CrossRef][ISI][Medline]

Kontgen, F., Grumont, R. J., Strasser, A., Metcalf, D., Li, R., Tarlinton, D., and Gerondakis, S. (1995). Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. Genes Dev. 9, 1965–1977.[Abstract]

Lake, B. G., Gray, T. J., Korosi, S. A., and Walters, D. G. (1989). Nafenopin, a peroxisome proliferator, depletes hepatic vitamin E content and elevates plasma oxidised glutathione levels in rats. Toxicol. Lett. 45, 221–229.[CrossRef][ISI][Medline]

Lake, B. G., Kozlen, S. L., Evans, J. G., Gray, T. J. B., Young, P. J., and Gangolli, S. D. (1987). Effect of prolonged administration of clofibric acid and di-(2-ethylhexyl)phthalate on hepatic enzyme activities and lipid peroxidation in the rat. Toxicology 44, 213–228.[CrossRef][ISI][Medline]

Ledwith, B. J., Johnson, T. E., Wagner, L. K., Pauley, C. J., Manam, S., Galloway, S. M., and Nichols, W. W. (1996). Growth regulation by peroxisome proliferators: Opposing activities in early and late G1. Cancer Res. 56, 3257–3264.[Abstract]

Ledwith, B. J., Manam, S., Troilo, P., Joslyn, D. J., Galloway, S. M., and Nichols, W. W. (1993). Activation of immediate-early gene expression by peroxisome proliferators in vitro. Mol. Carcinog. 8, 20–27.[ISI][Medline]

Li, Y., Leung, L. K., Glauert, H. P., and Spear, B. T. (1996). Treatment of rats with the peroxisome proliferator ciprofibrate results in increased liver NF-kappa B activity. Carcinogenesis 17, 2305–2309.[Abstract]

Li, Y. X., Glauert, H. P., and Spear, B. T. (2000a). Activation of nuclear factor-kappa B by the peroxisome proliferator ciprofibrate in H4IIEC3 rat hepatoma cells and its inhibition by the antioxidants N-acetylcysteine and vitamin E. Biochem. Pharmacol. 59, 427–434.[CrossRef][ISI][Medline]

Li, Y. X., Tharappel, J. C., Cooper, S., Glenn, M., Glauert, H. P., and Spear, B. T. (2000b). Expression of the hydrogen peroxide-generating enzyme fatty acyl CoA oxidase activates NF-kappa B. DNA Cell Biol. 19, 113–120.[CrossRef][ISI][Medline]

Macdonald, N., Chevalier, S., Tonge, R., Davison, N., Rowlinson, R., Young, J., Rayner, S., and Roberts, R. (2001). Quantitative proteomic analysis of mouse liver response to the peroxisome proliferator diethylhexylphthalate (DEHP). Arch. Toxicol. 75, 415–424.[CrossRef][ISI][Medline]

Marsman, D. S., Cattley, R. C., Conway, J. G., and Popp, J. A. (1988). Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) in rats. Cancer Res. 48, 6739–6744.[Abstract]

Meyer, M., Pahl, H. L., and Baeuerle, P. A. (1994). Regulation of the transcription factors NF-kappa B and AP-1 by redox changes. Chem. Biol. Interact. 91, 91–100.[CrossRef][ISI][Medline]

Mounho, B. J., and Thrall, B. D. (1999). The extracellular signal-regulated kinase pathway contributes to mitogenic and antiapoptotic effects of peroxisome proliferators in vitro. Toxicol. Appl. Pharmacol. 159, 125–133.[CrossRef][ISI][Medline]

Nilakantan, V., Spear, B. T., and Glauert, H. P. (1998). Liver-specific catalase expression in transgenic mice inhibits NF-kappaB activation and DNA synthesis induced by the peroxisome proliferator ciprofibrate. Carcinogenesis 19, 631–637.[Abstract]

Pauley, C. J., Ledwith, B. J., and Kaplanski, C. (2002). Peroxisome proliferators activate growth regulatory pathways largely via peroxisome proliferator-activated receptor alpha-independent mechanisms. Cell. Signal. 14, 351–358.[CrossRef][ISI][Medline]

Poosch, M. S., and Yamazaki, R. K. (1986). Determination of peroxisomal fatty acyl-CoA oxidase activity using a lauroyl-CoA-based fluorometric assay. Biochim. Biophys. Acta 884, 585–593.[ISI][Medline]

Rao, M. S., and Reddy, J. K. (1987). Peroxisome proliferation and hepatocarcinogenesis. Carcinogenesis 8, 631–636.[ISI][Medline]

Reddy, J. K., and Lalwani, N. D. (1983). Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol. 12, 1–58.[ISI]

Reddy, J. K., Lalwani, N. D., Reddy, M. K., and Qureshi, S. A. (1982). Excessive accumulation of autofluorescent lipofuscin in the liver during hepatocarcinogenesis by methyl clofenapate and other hypolipidemic peroxisome proliferators. Cancer Res. 42, 259–266.[ISI][Medline]

Roberts, R. A., Soames, A. R., Gill, J. H., James, N. H., and Wheeldon, E. B. (1995). Non-genotoxic hepatocarcinogens stimulate DNA synthesis and their withdrawal induces apoptosis, but in different hepatocyte populations. Carcinogenesis 16, 1693–1698.[Abstract]

Schoemaker, M. H., Ros, J. E., Homan, M., Trautwein, C., Liston, P., Poelstra, K., vanGoor, H., Jansen, P. L. M., and Moshage, H. (2002). Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-kappa B-regulated inhibitor of apoptosis protein 2 (CIAP2) prevents apoptosis. J. Hepatol. 36, 742–750.[CrossRef][ISI][Medline]

Schoonjans, K., Staels, B., and Auwerx, J. (1996). Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J. Lipid Res. 37, 907–925.[Abstract]

Schulte-Hermann, R., Bursch, W., and Grasl-Kraupp, B. (1995). Active cell death (apoptosis) in liver biology and disease. In Progress in Liver Disease (J. L. Boyer and R. K. Ockner, Eds.), pp. 1–35. Saunders, Philadelphia.

Sha, W. C., Liou, H. C., Tuomanen, E. I., and Baltimore, D. (1995). Targeted disruption of the p50 subunit of NF-kB leads to multifocal defects in immune responses. Cell 80, 321–330.[ISI][Medline]

Snapper, C. M., Rosas, F. R., Zelazowski, P., Moorman, M. A., Kehry, M. R., Bravo, R., and Weih, F. (1996a). B cells lacking RelB are defective in proliferative responses, but undergo normal B cell maturation to Ig secretion and Ig class switching. J. Exp. Med. 184, 1537–1541.[Abstract]

Snapper, C. M., Zelazowski, P., Rosas, F. R., Kehry, M. R., Tian, M., Baltimore, D., and Sha, W. C. (1996b). B cells from p50/NF-kappa B knockout mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching. J. Immunol. 156, 183–191.[Abstract]

Srinivasan, S. R., Chow, C. K., and Glauert, H. P. (1990). Effect of the peroxisome proliferator ciprofibrate on hepatic DNA synthesis and hepatic composition following partial hepatectomy in rats. Toxicology 62, 321–332.[CrossRef][ISI][Medline]

Strange, J., and Roberts, R. A. (1996). Reduced expression of mature TGF beta 1 correlates with the suppression of rat hepatocyte apoptosis by the peroxisome proliferator, nafenopin. Mutat. Res. 372, 107–113.[ISI][Medline]

Takagi, A., Sai, K., Umemura, T., Hasegawa, R., and Kurokawa, Y. (1990a). Relationship between hepatic peroxisome proliferation and 8-hydroxydeoxyguanosine formation in liver DNA of rats following long-term exposure to three peroxisome proliferators: Di(2-ethylhexyl)phthalate, aluminium clofibrate and simfibrate. Cancer Lett. 53, 33–38.[ISI][Medline]

Takagi, A., Sai, K., Umemura, T., Hasegawa, R., and Kurokawa, Y. (1990b). Signifigant increase of 8-hydroxydeoxyguanosine in liver DNA of rats following short-term exposure to the peroxisome proliferators di(2-ethylhexyl)phthalate and di(2-ethylhexyl)adipate. Jpn. J. Cancer Res. 81, 213–215.[ISI][Medline]

Tharappel, J. C., Cunningham, M. L., Spear, B. T., and Glauert, H. P. (2001). Differential activation of hepatic NF-kappa B in rats and hamsters by the peroxisome proliferators Wy-14,643, gemfibrozil, and dibutyl phthalate. Toxicol. Sci. 62, 20–27.[Abstract/Free Full Text]

Vanantwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. (1996). Suppression of TNF-alpha-induced apoptosis by NF-kappa B. Science 274, 787–789.[Abstract/Free Full Text]

Verma, I. M., Stevenson, J. K., Schwarz, E. M., Van Antwerp, D., and Miyamoto, S. (1995). Rel/NF-kB/IkB family: Intimate tales of association and dissociation. Genes Dev. 9, 2723–2735.[CrossRef][ISI][Medline]

Wang, C. Y., Mayo, M. W., and Baldwin, A. S. (1996). TNF-and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-kappa B. Science 274, 784–787.[Abstract/Free Full Text]

Xu, Y., Bialik, S., Jones, B. E., Iimuro, Y., Kitsis, R. N., Srinivasan, A., Brenner, D. A., and Czaja, M. J. (1998a). NF-kappa B inactivation converts a hepatocyte cell line TNF-alpha response from proliferation to apoptosis. Amer. J. Physiol. Cell Physiol. 44, C1058–C1066.

Xu, Y., Bialik, S., Jones, B. E., Iimuro, Y., Kitsis, R. N., Srinivasan, A., Brenner, D. A., and Czaja, M. J. (1998b). NF-kappaB inactivation converts a hepatocyte cell line TNF-alpha response from proliferation to apoptosis. Am. J. Physiol. 275, C1058–C1066.[ISI][Medline]

Yeldandi, A. V., Milano, M., Subbarao, V., Reddy, J. K., and Rao, M. S. (1989). Evaluation of liver cell proliferation during ciprofibrate-induced hepatocarcinogenesis. Cancer Lett. 47, 21–27.[ISI][Medline]