* Graduate Center for Nutritional Sciences, Department of Pathology and Laboratory Medicine,
Graduate Center for Toxicology,
Department of Nutrition and Food Science, and ¶ Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40536
Received January 21, 2004; accepted May 18, 2004
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ABSTRACT |
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Key Words: PCBs; NF-B; hepatocytes; apoptosis; cell proliferation.
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INTRODUCTION |
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Although PCBs are known to bind to a variety of receptors (Robertson and Hansen, 2001), they have been traditionally divided into two groups according to the positions of chlorine atoms. Coplanar PCB congeners substituted in both para positions and at least two meta positions, but not in any of the ortho positions, may assume a more coplanar configuration, similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and they bind strongly to the Ah receptor (AhR) (Bandiera et al., 1982
); binding to the AhR induces expression of AhR-regulated genes including cytochrome P450 1A1 and 1A2 (CYP1A1, CYP1A2; Parkinson et al., 1983
). On the other hand, PCB congeners with chlorine substitution in two ortho positions are non-coplanar and have low affinity for the AhR, but they induce a battery of drug-metabolizing enzymes, including cytochrome P450 2B1 and 2B2 (CYP2B1, CYP2B2), in a pattern that is similar to phenobarbital (Denomme et al., 1983
; Parkinson et al., 1983
).
In rodents pretreated with a genotoxic carcinogen, PCBs promote the development of liver cancer (Glauert et al., 2001), although the exact mechanisms of this promotion are still unknown. Both co-planar PCBs, such as 3,3',4,4'-tetrachlorobiphenyl (PCB-77) and 3,3'4,4',5-pentachlorobiphenyl (PCB-126), and ortho-substituted PCBs, such as 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) have promoting activity in rodent liver carcinogenesis (Glauert et al., 2001
). A number of mechanisms have been investigated, including direct effects on signal transduction pathways, induction of oxidative stress, effects on vitamin A metabolism, and effects on intercellular communication (Glauert et al., 2001
). Recently, hepatic tumor promoters such as peroxisome proliferators and phenobarbital have been found to activate the nuclear transcription factor NF-
B (Li et al., 1996a
, 1996b
, 2000
; Nilakantan et al., 1998
), which regulates the expression of cell proliferationrelated and apoptosis-related genes (Li and Stark, 2002
).
In hepatocytes, the major form of NF-B is a heterodimer consisting of 50-kDa (p50) and 65-kDa (p65/relA) subunits that is complexed to an inhibitory subunit (I
B) in the cytoplasm. Upon stimulation, I
B is phosphorylated, polyubiquitinated, and degraded, allowing translocation of NF-
B into the nucleus (Li and Stark, 2002
). Among the NF-
Bregulated genes, cyclin D1 controls the cell cycle checkpoint at G1/S phase (Guttridge et al., 1999
; Hinz et al., 1999
), and NF-
B is required for the initiation of cyclin D1 transcription and hyperphosphorylation of retinoblastoma protein (pRB), which lead cells to progress through G1 and entry into S phase (Biswas et al., 2000
; Henry et al., 2000
; Joyce et al., 1999
; Kaltschmidt et al., 1999
). NF-
B also regulates expression of certain anti-apoptotic genes, such as the inhibitor of apoptosis proteins (IAPs) (Wang et al., 1998
) and Bcl-2/Bcl-xL (Lee et al., 1999
; Tamatani et al., 1999
; Wang et al., 1999
). Knockout studies revealed that the p65 subunit of NF-
B is essential for hepatocytes to survive tumor necrosis factor alpha (TNF-
)induced apoptosis, because deficiency in p65 results in embryonic lethality from hepatocyte apoptosis (Beg et al., 1995
). Inhibition of NF-
B activation by an I
B
superrepressor results in extensive hepatocyte apoptosis after partial hepatectomy (Iimuro et al., 1998
). Although p65 null mice die during embryogenesis, p50/ mice are viable (Sha et al., 1995
), thus providing a model to study the role of the p65/p50 dimer in the regulation of cell proliferation and cell apoptotic death in the liver. Hepatocyte apoptosis was increased in p50/ mice, and cell proliferation in response to the peroxisome proliferator ciprofibrate was inhibited (Tharappel et al., 2003
). However, DNA synthesis and liver regeneration after partial hepatectomy or carbon tetrachloride treatment were not affected by the absence of the p50 subunit, nor was the hepatic inflammatory response after ischemia/reperfusion (DeAngelis et al., 2001
; Kato et al., 2002
). In addition, B cells lacking p50 show decreased activity in response to the lymphocyte proliferation response (LPS) (Sha et al., 1995
; Snapper et al., 1996
).
Our previous studies indicated that PCB-153, a non-coplanar PCB congener, activated hepatic NF-B after a single injection (Lu et al., 2003
) or during the promotion stage of hepatocarcinogenesis (Tharappel et al., 2002
). In addition, hepatocyte proliferation was induced by a single injection of PCB-153 (Lu et al., 2003
), which correlated with hepatic NF-
B activation in the same animals. Thus, the purpose of this study was to use p50/ mice to determine the role of NF-
B in PCB-induced cell proliferation. The effects on apoptotic cell death after treatment with PCB-153 in these p50/ mice were also studied.
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MATERIALS AND METHODS |
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Experimental design. Eight-week-old male B6.129 mice homozygous for the NF-B (p50) mutation and wild-type controls were obtained from our breeding colony (originally purchased from the Jackson Laboratory, Bar Harbor, ME). Mice were housed 24 animals per cage in a temperature-controlled and light-controlled room and fed an unrefined diet (#5001, Purina Mills, St. Louis, MO) and water ad libitum. In a 2-day study, 16 p50/ mice and 16 wild-type littermates were first injected ip with either corn oil (5 p50/ mice and 6 wild-type mice) or PCB-153 (300 µmol/kg; 11 p50/ mice and 10 wild-type mice) and then euthanized 2 days later. One p50/ mouse (PCB-153 group) and two wild-type mice (one in the corn oil group and one in the PCB-153 group) died before the end of the study. All mice were injected subcutaneously (sc) with BrdU (100 mg/kg) 2 h before euthanasia.
Then, in a 21-day study, mice received ip injections of corn oil (10 wild-type mice and 7 p50/ mice) or PCB-153 (100 µmol/kg, 10 wild-type mice and 7 p50/ mice) on days 0, 3, 7, 10, 14, and 17 and were euthanized on day 21; before the end of the study, 4 p50/ mice (2 in the control group and 2 in the PCB-153 group) and 2 wild-type mice (PCB-153 group) died. Three days before euthanasia mice were surgically implanted with osmotic pumps containing BrdU (20 mg/ml, 10 µl/h). The liver was removed from each mouse, and pieces of each liver were fixed in 10% formalin and processed for histology. The remaining liver was snap frozen in liquid N2 and stored at 80°C.
Nuclear extract preparation. Nuclear extracts were prepared from frozen liver tissue by a modification of the method of Deryckere and Gannon (Deryckere and Gannon, 1994). Approximately 250 mg of tissue was homogenized in 5 ml of buffer [0.6% IGEPAL CA-630; 150 mM NaCl; 10 mM Hepes-KOH, pH 7.9; 1 mM EDTA; and 0.5 mM phenylmethylsulfonyl fluoride (PMFS)] using a Dounce homogenizer. The homogenate was centrifuged at 270 x g for 30 s. The supernatant was transferred into a new tube, incubated on ice for 5 min, and then centrifuged again at 3,000 x g for 20 min. The supernatant was discarded and the pellet was resuspended with 1 ml homogenization buffer, and then transferred into a microcentrifuge tube and centrifuged at 3,000 x g for 5 min. Again, the supernatant was discarded and the pellet was resuspended in 100 µl buffer (20 mM Hepes-KOH, pH 7.9; 420 mM NaCl; 1.2 mM MgCl2; 0.2 mM EDTA; 0.5 mM PMSF; 0.5 mM dithiothreitol; 2 mM benzamidine; 5 µg/ml aprotinin; 5 µg/ml leupeptin; 5 µg/ml pepstatin A; and 25% glycerol), incubated on ice for 1 h and then centrifuged at 11,000 x g for 10 min. The resulting supernatant was then divided into aliquots and stored at 80°C. One aliquot was diluted in PBS to measure the protein concentration using the BCA method (Pierce, Rockford, IL).
Electrophoretic mobility shift assay. Nuclear extract (5µg) from each liver was incubated with 0.5 mg poly (dI-dC) in binding buffer (50 mM KCl; 10 mM Hepes-KOH, pH 7.9; 6.5 mM dithiothreitol; and 10% glycerol) on ice for 5 min, and then for 15 min at room temperature after the addition of radiolabeled probe [20,000 counts per min (cpm)]. The NF-B oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3') was obtained from Promega (Madison, WI) and end-labeled using T4 polynucleotide kinase. After incubation, samples were resolved by electrophoresis on a 7% polyacrylamide gel at 180 volts for 2 h using 0.5x TBE as the running buffer. After the electrophoresis, gels were dried under vacuum and exposed to a phosphorimage screen. The radioactivity was counted with a Storm phosphorimaging system (Amersham Biosciences, Piscataway, NJ).
BrdU immunohistochemical staining. The paraffin-embedded liver tissues were sectioned, stained with an anti-BrdU antibody, and counter-stained with hematoxylin. The staining was carried out using the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA), according to the protocol provided by the manufacturer. Cells that had incorporated BrdU were easily identified as those with brown nuclei. At least 3000 hepatocellular nuclei per slide (1000 in each of three lobes) were counted in random fields, and the labeling index was expressed as a percentage of the number of labeled hepatocyte nuclei out of the total number of hepatocyte nuclei counted.
In situ cell death detection assay. The liver tissue processed for paraffin sections was used for the detection of apoptotic hepatocytes by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The assay was carried out using an in situ cell death detection assay kit (Intergen, Purchase, NY), according to the protocol provided by the manufacturer. At least 3000 hepatocyte nuclei were counted as described above, and the apoptotic index was expressed as a percentage of the number of labeled nuclei out of the total number of nuclei counted.
Western blotting. For analysis of IKB kinases (IKK) and IB proteins, frozen liver pieces were diluted 1:4 with lysis buffer (1% Nonidet P-40, 0.1% SDS (sodium dodecyl sulfate), 0.1 mg/ml PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, and 1x PBS) and homogenized with an Ultra-Turrax homogenizer (Tekmar Co., Cincinnati, OH). The homogenates were centrifuged at 10,000 x g for 20 min, and the supernatants were collected and then centrifuged at 100,000 x g for 1 h; the supernatants (cytosolic fraction) were then collected and divided into aliquots. For the cyclin D1 protein, frozen liver tissues were homogenized in a buffer containing 50 mM Tris-HCl, pH 7.4 l; 150 mM NaCl; 1% IGEPAL; 0.1% SDS; 0.5% sodium deoxycholate; 50 mM NaF; 1 mM Na2VO3; 1 mM DTT; 1 mM phenylmethylsulfonyl fluoride; 5 µg/ml aprotinin; 5 µg/ml leupeptin; and 5 µg/ml pepstatin A. The homogenates were centrifuged at 12,000 rpm for 15 min, and the supernatants were divided into aliquots and stored at 80°C. Protein concentrations of all samples were determined using the BCA method (Pierce). The samples were denatured by boiling for 5 min in 2x gel-loading buffer (17.3% glycerol; 1.25 M ß-mercaptoethanol' 5.2% SDS; 0.22 M Tris, pH 6.8; and 12 mg bromophenol blue). For each sample, 25 µg of the protein was electrophoresed on an 8.5% separating gel and 4% stacking gel at 175 V for 1 h. The proteins in the gels were transferred to nitrocellulose membranes (Life Technologies, Carlsbad, CA) at 100 V for 1 h. The membranes were incubated with a blocking buffer (5% fat-free dry instant powdered milk, 1 mM Tris-base, 15 mM NaCl, and 0.05% Tween-20) for 1 h at room temperature with shaking. The primary and secondary antibodies were diluted in the blocking buffer and incubated with the membrane for 1 h with shaking at room temperature. The membranes were washed three times with a wash buffer (1 mM Tris-base, 15 mM NaCl, and 0.05% Tween-20) between the primary and secondary antibodies. All primary and secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA), and the SuperSignal Chemiluminescent Substrate Kit (Pierce) was used to detect proteins.
RNA isolation and ribonuclease protection assays (RPA). Frozen liver tissues were homogenized in 1 ml Trizol reagent (Life Technologies) according to the manufacturer's instructions. The concentration and purity of RNA were determined by absorption at 260 nm and 280 nm using a spectrophotometer. The RPA were carried out using the RiboQuant RNase Protection Assay Kit from Becton Dickinson Pharmingen (San Diego, CA), according to the protocol provided by manufacturer. The RNA template used was MCYC-1 RiboQuant Mouse Cyclin Multi-Probe Template Set (Becton Dickinson Pharmingen).
Statistical analysis. Results were first analyzed by a two-way analysis of varianceANOVA (the two factors being genotype and PCB treatment). If a significant interaction was observed, individual differences between means were determined using Tukey's post hoc test. The results were reported as means ± standard error of mean (SEM).
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RESULTS |
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DISCUSSION |
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In the 2-day study, PCB-153 increased the hepatic DNA binding activity of NF-B in wild-type mice, but not in p50/ mice, consistent with the idea that p50 is important for full NF-
B activity in the liver. The levels of IKK and I
B proteins were not changed by PCB-153, although I
B
protein levels were lower in p50/ livers than in wild-type livers. Unlike the 2-day study, hepatic NF-
B activity in wild-type mice was not induced by PCB-153 in the 21-day study. The increased NF-
B activity in corn oiltreated wild-type mice may be due to increased stress caused by the multiple ip injections.
In the 2-day study, PCB-153 increased hepatocyte proliferation approximately twofold in wild-type mice but not in p50/ mice, indicating that NF-B (p50/p65) is important in PCB-153induced cell proliferation. An increase in cell proliferation was seen in both the wild-type and p50/ mice after 21 days of PCB treatment, although the increased proliferation was greater in the wild-type animals. An increase in p65 or other NF-
B family members, decreased amounts of I
B
, or alterations in other signal transduction pathways in p50/ mice could compensate for the lack of p50 and explain the increase in DNA synthesis at the later time point. Studies have shown normal liver regeneration in p50/ mice after partial hepatectomy or carbon tetrachloride administration (DeAngelis et al., 2001
), and it was suggested that this was due to a compensatory increase in the p65 NF-
B subunit.
Hepatocytes typically proliferate at a very low rate, yet increased cellular proliferation can occur in response to physical, infectious, or toxic injury (Kitamura et al., 1998). The cell cycle control system is regulated by the expression of cyclins, CDKs, and CKIs (Bartek et al., 1996
; Beijersbergen and Bernards, 1996
). In the present study, we compared the changes in the mRNA level of cyclin A2, B1, B2, C, D1, and D2. There was no significant change in the cyclin D1 mRNA level between wild-type and p50/ livers, or after PCB-153 treatments, but there was less cyclin D1 protein in p50/ livers than in the wild-type livers. This lack of concomitant changes between cyclin D1 mRNA and protein levels has been described early in regenerating liver after partial hepatectomy (Albrecht et al., 1995
) and in adult rat liver (Awad and Gruppuso, 2000
). Cyclin D1 mRNA was highly induced in rat liver after partial hepatectomy, whereas protein levels changed less than twofold and did not parallel changes in the mRNA (Albrecht et al., 1995
). In adult rat livers, cyclin D1 mRNA levels are present at a higher level than in fetal livers, whereas the protein is not detectable in adult livers, suggesting posttranscriptional regulation (Awad and Gruppuso, 2000
). How the cyclin D1 protein level was decreased while the mRNA level remained unchanged in the p50/ livers is unclear, but our data do suggest a role for posttranslational control of cyclin D1 in hepatocyte growth regulation.
The suppression of apoptosis is another mechanism by which nongenotoxic hepatocarcinogens may induce cancer. Phenobarbital and non-coplanar PCBs, both of which are tumor promoters, have been shown to inhibit UV-induced apoptosis in primary hepatocytes (Bohnenberger et al., 2001). Tharappel et al. have shown inhibition of apoptosis by PCB-153 in placental glutathione S-transferase (PGST)-positive hepatocytes in an initiation-promotion study (Tharappel et al., 2002
). Salvi and Toniello have shown that PCBs inhibit the mitochondrial permeability transition and the consequent release of cytochrome c from mitochondria (Salvi and Toninello, 2001
). Considering the pivotal role of cytochrome c in caspase activation (Liu et al., 1996
; Wang, 2001
), inhibition of its release would indicate an interruption of the apoptotic pathway. However, little is known about the function of the p50 NF-
B subunit in the regulation of apoptotic pathways in hepatocytes. In the studies by DeAngelis et al. (2001)
, in which Fas antibody was used to activate Fas receptormediated apoptosis, p50/ livers showed a small increase in apoptosis compared to wild-type mice. Unfortunately, apoptosis in untreated livers was not reported in that study. In our study, we showed that apoptosis in the liver was increased in p50/ mice compared to wild-type mice regardless of PCB-153 treatment. Although this increase in spontaneous apoptosis was not as severe as that seen in the p65-deficient mice, in which massive hepatic apoptosis leads to embryonic death (Beg et al., 1995
), it demonstrates the importance of NF-
B (p50/p65) as an anti-apoptotic mediator. PCB-153 lowered apoptosis in p50/ mice but not in wild-type mice. Because the apoptotic index in control wild-type mice was very low, it would be difficult to detect a PCB-153 effect unless large numbers of mice were used. The inhibition of apoptosis by PCB-153 in the p50/ mice is likely related to an alteration in a p50-independent signal transduction pathway.
In summary, our data support the hypothesis that NF-B activation plays a critical role in regulation of cell proliferation in liver in response to PCB-153 treatment. Apoptosis was increased in p50/ mice; PCB-153 inhibited this increase, implying that the inhibition of apoptosis may be an important property of non-coplanar PCBs. These results raise the possibility that NF-
B may contribute to the tumor-promoting activity of non-coplanar PCB congeners, including PCB-153. Further studies will be required to determine the molecular mechanisms of these NF-
Bmediated effects.
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ACKNOWLEDGMENTS |
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NOTES |
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2 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 50536-0298. Fax: 859-257-8994. E-mail: bspear{at}uky.edu.
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