Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Room 222 Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107-6799, USA1
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA2
GenWay Biotech, Inc., Suite E-2 Welsh Commons, 1364 Welsh Road, North Wales, PA 19454, USA3
Author for correspondence: Mark Feitelson (at Department of Pathology, Anatomy and Cell Biology). Fax +1 215 503 9982. e-mail Mark.Feitelson{at}mail.tju.edu
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Abstract |
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Introduction |
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On the molecular level, HBxAg may contribute to multistep hepatocarcinogenesis in a number of ways. For example, HBxAg trans-activates many viral and cellular promoters. Virus promoters include those in HBV (Colgrove et al., 1989 ; Nakatake et al., 1993
) and the long-terminal repeat of human immunodeficiency virus-1 (Siddiqui et al., 1989
). Cellular promoters include those for c-fos, c-jun (Natoli et al., 1994a
, b
), c-myc (Balsano et al., 1991
), insulin-like growth factor II (Lee et al., 1998
) and epidermal growth factor receptor (Menzo et al., 1993
). HBxAg trans-activation is also mediated by the binding of HBxAg to a variety of transcription factors in the nucleus (Doria et al., 1995
; Henkler & Koshy, 1996
), and by the stimulation of several signal transduction pathways in the cytoplasm (Haskill et al., 1991
; Kekule et al., 1993
; Doria et al., 1995
). Among the latter, many groups have shown that HBxAg stimulates the nuclear factor kappa B (NF-
B) pathway (Siddiqui et al., 1989
; Mahe et al., 1991
; Doria et al., 1995
; Su & Schneider, 1996
), which regulates a number of genes involved in the immune and inflammatory responses (Baeuerle, 1991
; Grilli et al., 1993
; Liou & Baltimore, 1993
; May & Ghosh, 1998
). HBxAg may also contribute to transformation by functionally inactivating a number of negative growth regulatory pathways (Feitelson et al., 1993a
, 1999
; Lian et al., 1999
), suggesting that HBxAg may participate in several steps in multistep carcinogenesis.
The sustained production of HBxAg during chronic infection (Wang et al., 1991a , b
) is consistent with the hypothesis that it may promote the resistance of infected liver cells to immunologically mediated apoptosis. Recent studies with tissue culture cells have shown that HBxAg modulates apoptosis in a variety of settings (Wang et al., 1995
; Elmore et al., 1997
; Terradillos et al., 1998
). In this context, it is possible that HBxAg physically or functionally interacts with components of signal transduction pathways, thereby blocking the transmission of death signals to the nuclei of infected hepatocytes (Gottlob et al., 1998
). The fact that HBxAg stimulates NF-
B (Doria et al., 1995
; Su & Schneider, 1996
), combined with the centrality of NF-
B activity to liver cell survival in vitro (Bellas et al., 1997
) and in vivo (Beg et al., 1995
; Li et al., 1999
), suggest that HBxAg may promote hepatocellular survival, and their resistance to apoptosis, by an NF-
B-dependent pathway. Certainly, NF-
B activation blocks hepatocellular apoptosis mediated by tumour necrosis factor alpha (TNF-
) (Beg & Baltimore, 1996
; Liu et al., 1996
; Van Antwerp et al., 1996
; Wang et al., 1996
) and transforming growth factor beta (TGF-
) (Bellas et al., 1997
; Arsura et al., 1997
). Although these cytokines may contribute to the pathogenesis of chronic HBV infection, there is increasing evidence that the Fas ligand/receptor system also plays an important role in pathogenesis (Galle & Krammer, 1998
). In particular, there is a significant correlation between levels of Fas antigen and chronic liver disease in HBV carriers (Watanabe-Fukunage et al., 1992
; Galle et al., 1995
; Mochizuki et al., 1996
). Since activated T-cells express the Fas ligand (Rouvier et al., 1993
; Suda et al., 1993
), its binding to Fas receptor on hepatocytes may contribute importantly to the destruction of infected hepatocytes (Ando et al., 1994
; Rouquet et al., 1995
). The exquisite sensitivity of mice to anti-Fas treatment is highlighted by the massive liver cell apoptosis that accompanies such treatments (Ogasawara et al., 1993
). In addition, the fact that HCC cells express little or no Fas receptor (Strand et al., 1996
) may render them resistant to Fas-mediated apoptosis. Hence, experiments were designed to test the hypothesis that the HBxAg-mediated protection of liver cells against anti-Fas-triggered apoptosis is dependent upon HBxAg activation of NF-
B.
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Methods |
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Plasmids.
The recombinant retroviral vector plasmids, pSLXCMV-FLAGX and pSLXCMV-CAT, were constructed by inserting the HBV X gene or bacterial chloramphenicol acetyltransferase (CAT) gene into the polylinker of pSLXCMV (Duan et al., 1995 ), which is a murine leukaemia virus-based vector containing the neomycin-resistance gene (Miller & Rosman, 1989
), as described (Lian et al., 1999
). Plasmid pZeoSV-I
B
-HA was constructed by using the PCR-amplified human I
B
gene from plasmid F-I
B
(Haskill et al., 1991
) (kindly provided by D. W. Ballard, Dept of Microbiology and Immunobiology, Vanderbilt University, TN, USA). The PCR product was digested with BamHI and EcoRI, and the resulting 1028 bp fragment was ligated into the polylinker of pZeoSV2(+) (Invitrogen), which carries the zeocin-resistance gene. This recombinant produced a translation product containing the influenza virus haemagglutinin (HA) epitope at the amino terminus of I
B
. Plasmid pGL2-HIV-1-LTR contains full-length HIV-1 LTR, which drives expression of the luciferase reporter gene (a kind gift from Ed Mercer, Kimmel Cancer Center, Thomas Jefferson University, PA, USA).
Transduction of HepG2 cells with pSLXCMV-FLAGX and pSLXCMV-CAT.
Transduction of HepG2 cells with recombinant retroviruses encoding HBxAg or CAT was performed as described (Lian et al., 1999 ). Cultures were selected for growth by addition of G418 to the medium for 14 days. Drug-resistant cells were trypsinized and grown in complete medium without cloning prior to analysis. These HepG2X and HepG2CAT cultures were not only used for prior work (Lian et al., 1999
), but also the studies herein.
CAT assay.
This was done essentially as described (Ausubel et al., 1991 ; Gorman et al., 1992
) with minor modifications (Lian et al., 1999
).
Anti-Fas treatment.
Cells were added to 6-well plates (7x105 cells per well) and incubated overnight in complete medium. Cultures were then treated with 0·5 µg/ml of anti-Fas (Ab-2, monoclonal mouse IgG; Oncogene Research, Cambridge, MA, USA) or 0·5 µg/ml of mouse IgG (Sigma) as control in the presence of actinomycin D (0·3 µg/ml) or cycloheximide (2 µg/ml). These compounds sensitize cells to anti-Fas killing by preventing de novo synthesis of cellular proteins that would otherwise diminish the effects of anti-Fas. After 0, 24, 36, 48 and 60 h treatment, all cells (adherent and floating) in each well were collected by trypsinization, stained with trypan blue, and counted in a haemocytometer. About 800 cells in each sample were counted. The percentage of live cells, as determined by trypan blue exclusion, was calculated as follows: (no. of live cells in anti-Fas or control IgG treated well/no. of total cells in each corresponding well)x100. All tests were done in duplicate. Cell viability was independently determined using the modified tetrazolium salt (MTT) assay, as described by the manufacturers (CellTitre 96 Non-radioactive Cell Proliferation assay, Promega).
TUNEL assay.
Apoptosis was assessed by measuring DNA fragmentation in a standard TUNEL (deoxynucleotidyltransferase-mediated dUTP nick end labelling) assay according to the instructions with the kit (TACS In situ Apoptosis Detection kit, Trevigen, Gaithersburg, MD, USA).
Transient transfection and luciferase assay.
To measure the effects of anti-Fas treatment upon NF-B activity, 1x106 HepG2X or HepG2CAT cells were plated overnight, and then cotransfected, using calcium phosphate precipitates, with 10 µg of pZeoSV or pZeoSV-I
B
-HA and 2 µg of the reporter plasmid pGL2-HIV-1-LTR. Cells were incubated overnight, washed with PBS, and assayed for luciferase activity at 36 h post-transfection. To determine whether NF-
B activity is sensitive to anti-Fas treatment, cultures were transiently transfected with 2 µg of pGL2-HIV-1-LTR, and then maintained in medium containing mouse IgG (0·5 µg/ml) or anti-Fas (0·5 µg/ml) with or without either actinomycin D (0·3 µg/ml) or cycloheximide (2 µg/ml). Following overnight incubation (17 h), the cells were washed and then lysed in 250 µl of Cell Culture Lysis Reagent from the Luciferase Assay kit (Promega). Protein concentration was determined with the Bio-Rad Protein Assay. For each tube, 30 µg of total protein was mixed with 100 µl of luciferase assay reagent and evaluated in a luminometer. All tests were done in duplicate.
Stable transfection of HepG2X cells with pZeoSV-I
B
-HA.
HepG2X and HepG2CAT cells were plated at 2x106 cells per 100 mm dish, incubated overnight, and then transfected with 15 µg of pZeoSV-IB
-HA or pZeoSV vector in calcium phosphate precipitates (Profection Mammalian Transfection System, Promega). Cells were incubated with DNA precipitate overnight and then selected by addition of zeocin (1 mg/ml final concentration) (Invitrogen) for 3 weeks. All resistant colonies were trypsinized and grown in complete medium.
Western blot analysis.
Western blotting for HBxAg was performed using a mixture of X peptide antibodies raised in rabbits (Lian et al., 1999 ). To detect exogenously expressed I
B
-HA in HepG2X-I
B
cells, Western blotting was performed with a mouse anti-HA monoclonal antibody (clone 12CA5, Boehringer Mannheim) at a final concentration of 2·5 µg/ml. Briefly, 5x106 HepG2X and HepG2CAT cells were washed with ice-cold PBS and lysed in buffer containing 50 mM TrisHCl (pH 7·4), 250 mM NaCl, 5 mM EDTA, phosphatase inhibitors (50 mM NaF, 0·1 mM Na3VO4), protease inhibitors (1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin) and 1% Triton X-100. Cytoplasmic extracts were isolated by centrifugation at 10000 r.p.m. for 10 min at 4 °C. Total protein samples (150 µg, as measured with the Bio-Rad Protein Assay kit) from each cell lysate were analysed by SDSPAGE on 12% gels. Proteins were then transferred to PVDF membranes (Millipore). To detect endogenous I
B
, rabbit anti-I
B
(FL) polyclonal antibody (SC-847, Santa Cruz Biotechnology) was used at 1:1000. Mouse anti-
actin monoclonal antibody (clone AC-15, Sigma) was used at 1:5000 as an internal control. After incubation with horseradish peroxidase-conjugated goat anti-rabbit Ig or goat anti-mouse Ig (Accurate, Westbury, NY, USA), the results were visualized using the ECL detection system (Amersham) and quantified by gel scanning, as described (Lian et al., 1999
).
Northern blot hybridization.
Whole cell RNA from HepG2X-pZeoSV, HepG2X-IB
and HepG2CAT-pZeoSV cells was isolated using the RNeasy Mini kit (Qiagen) according to the enclosed instructions. Total RNA (10 µg) isolated from each cell lysate was then analysed by denaturing agarose gel electrophoresis using formaldehyde. Samples were transferred to a Nytran nylon membrane (Schleicher & Schuell), and Northern blot hybridization was carried out using an I
B
-specific probe. Briefly, pZeoSV-I
B
-HA was digested with BamHI and EcoRI, and the insert isolated by agarose gel electrophoresis followed by extraction. This probe was then radiolabelled with [
-32P]dCTP using the Prime-a-Gene Labelling System (Promega). Hybridization and washing were done under stringent conditions. RNA levels on the resulting autoradiograms were semiquantified by gel scanning. A
-actin probe was used to normalize for loading of the cellular RNA in each sample.
Statistical analysis.
All data points represent the mean±two standard deviations of duplicate determinations. Statistical analysis was done with Students t-test.
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Results |
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HBxAg partially protects HepG2 cells from killing with anti-Fas
HepG2X, HepG2CAT and HepG2.2.15 cells were treated with anti-Fas or an equivalent amount of normal mouse IgG in the presence of cycloheximide or actinomycin D, and cell viability was determined by trypan blue staining 0, 24, 36, 48 and 60 h later. The results for cultures treated with cycloheximide are shown (Fig. 1) and are similar to those obtained in parallel cultures treated with actinomycin D (data not shown). In both cases, anti-Fas killed greater than 80% of HepG2CAT cells within 60 h. In contrast, the fraction of HepG2X cells killed by anti-Fas was significantly less than that of HepG2CAT cells after 24 h (P<0·04) 36 h (P<0·008), 48 h (P<0·006) and 60 h (P<0·002) of treatment. When HepG2.2.15 cells were treated with anti-Fas, protection was observed at 36 h (P<0·05), 48 h (P<0·03) and 60 h (P<0·03) compared to HepG2CAT cells (Fig. 1a
). No killing was observed when HepG2X, HepG2CAT or HepG2.2.15 cells were treated in parallel experiments with an equivalent amount of normal IgG (Fig. 1a
). A representative experiment, presented in Fig. 1(b
e
), shows confluent cultures of HepG2X and HepG2CAT cultures after 48 h treatment with normal mouse IgG, suggesting that under the conditions used cycloheximide was not toxic (Fig. 1b
and c
, respectively). Likewise, cycloheximide was not toxic to HepG2.2.15 cells (data not shown). Treatment of parallel cultures with cycloheximide plus anti-Fas resulted in about 3045% killing of HepG2X cells (Fig. 1d
) and >80% killing of HepG2CAT cells after 48 h treatment (Fig. 1e
). Although these results were obtained using trypan blue staining, very similar observations were made when cell viability was independently determined using the MTT assay (data not shown). Hence, HBxAg partially protected HepG2 cells from anti-Fas killing. Partial protection was also observed in HepG2.2.15 cells compared to HepG2CAT cells, although at lower levels.
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Discussion |
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The finding that HBxAg may directly complex to IB
(Weil et al., 1999
) and/or stimulate I
B
phosphorylation (Su & Schneider, 1996
) suggests mechanisms whereby HBxAg may stimulate NF-
B activity. In this context, the observation of decreased I
B
in HepG2X compared to HepG2CAT cells (Fig. 6
) is consistent with the post-translational degradation of I
B
by HBxAg, as indicated in earlier work (Chirillo et al., 1996
; Su & Schneider, 1996
). However, the decreased levels of endogenous I
B
mRNA in HepG2X compared to HepG2CAT cells (Fig. 6
) imply that HBxAg transcriptionally downregulates the expression of I
B
. This finding is another unique aspect of this work. Recently, transcriptional suppression has been suggested as a mechanism whereby HBxAg may regulate cellular gene expression (Lian et al., 1999
). In this context, it is proposed that low levels of HBxAg may trigger NF-
B, but not fully suppress I
B levels. If this occurs, then infected cells with low levels of HBxAg may have limited resistance to apoptosis compared to uninfected control (HBxAg-negative) cells. On the other hand, in the presence of high levels of HBxAg, I
B would be fully inactivated, resulting in an HBxAg-associated increased resistance to Fas-mediated apoptosis. The finding that HepG2X cells (with high levels of HBxAg) are considerably more resistant to anti-Fas than HepG2.2.15 cells (with lower levels of HBxAg), and that the latter are more resistant than HepG2CAT cells (with no HBxAg), is consistent with this idea. In this model, levels of HBxAg are low early in the course of chronic infection; when HBV DNA integrates into the host chromosomal DNA during the regeneration that follows a bout of hepatitis, the intracellular levels of HBxAg slowly rise. However, further work must be done in order to determine whether I
B levels are depleted in chronically infected livers, whether the I
B
gene is transcriptionally downregulated by HBxAg in natural infection, and whether this correlates with the levels of intrahepatic HBxAg.
The findings that anti-Fas inhibits NF-B function (Fig. 5
), and that HBxAg stimulates NF-
B activity, suggest that HBxAg may modulate the effects of Fas-mediated killing during chronic infection. Further, the fact that apoptosis is commonly observed in chronic viral hepatitis (Galle & Krammer, 1998
; Lau et al., 1998
), and that HBxAg is frequently detected in chronically infected liver (Wang et al., 1991a
, b
), may explain the close correlation between X antigen expression and chronic liver disease (Feitelson et al., 1993a
). HBxAg has been shown to promote apoptosis in some systems (Kim et al., 1998
; Terradillos et al., 1998
), while providing resistance to apoptosis in others (Wang et al., 1995
), suggesting that HBxAg may modulate the response of infected cells to different types of apoptotic stimuli (Elmore et al., 1997
). The ability of HBxAg to promote or inhibit apoptosis may also depend upon the state of cell differentiation and/or whether hepatocytes are quiescent or regenerating. For example, NF-
B may be antiapoptotic in thymocytes but proapoptotic in mature peripheral T-cells (Lin et al., 1999
). Although not addressed in this study, HBxAg also appears to stimulate the expression of Fas ligand (Shin et al., 1999
), which is a natural effector of NF-
B (Kasibhatla et al., 1999
). If this occurs in vivo, it would promote the lysis of effector T-cells expressing Fas receptor, resulting in enhanced survival of HBxAg-positive hepatocytes. The fact that NF-
B stimulates multiple immune response genes, and that it also protects a variety of cell types against apoptosis, provides a potential link between inflammation and the survival of HBxAg-positive cells.
In chronic hepatitis B, apoptosis is mediated by activated T-cells that have increased expression of Fas ligand upon hepatocytes that constitutively express Fas receptor (Mochizuki et al., 1996 ). These observations suggest that activated T-cells kill HBV antigen-expressing hepatocytes by Fas ligandreceptor interaction, thereby mediating virus clearance. The ability of HBxAg to prevent Fas-mediated killing, at least in part, would provide some protection to infected cells replicating virus, thereby promoting the development and persistence of the chronic carrier state. This idea is supported by the results in Fig. 1(a)
, showing that HepG2.2.15 cells are partially resistant to anti-Fas-mediated killing. However, the fact that the levels of resistance of HepG2215 cells to anti-Fas killing are considerably lower than that of HepG2X cells may be due to the relative levels of HBxAg in both cell types, with low, undetectable levels in HepG2.2.15 cells, and easily detectable levels in HepG2X cells. Although the protection afforded by HBxAg in HepG2.2.15 cells is not impressive, small differences in protection over the course of many years (or decades) of chronic infection may significantly contribute to the persistence of virus-infected cells. After the clearance of virus from blood and liver, the increased resistance of HBxAg-positive hepatocytes to Fas-mediated apoptosis would contribute to the persistence of such cells in the chronically infected host. These cells would then be ripe for additional steps in multistep carcinogenesis, such as the HBxAg-mediated inactivation of p53 (Feitelson et al., 1993b
; Truant et al., 1995
) its stimulation of the cell cycle (Benn & Schneider, 1995
) and its putative interruption of DNA repair machinery (Lee et al., 1995
). Hence, the HBxAg-mediated resistance to Fas killing may serve to sustain virus replication by promoting the survival of infected cells, as well as contribute to the pathogenesis of HCC.
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Acknowledgments |
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Received 5 June 2000;
accepted 20 September 2000.