First Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan1
Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Tokyo 108-0072, Japan2
Author for correspondence: Kazuhiko Koike. Fax +81 3 5800 8807. e-mail kkoike-tky{at}umin.ac.jp
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Abstract |
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Introduction |
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While overwhelming numbers of clinical and epidemiological studies indicate a relationship between HBV and hepatocellular carcinoma (HCC) (for reviews see Ganem & Varmus, 1987 ; Tiollais et al., 1985
) and the X gene has been implicated in the pathogenesis of HCC, several questions remain regarding the pathogenesis of HCC associated with chronic HBV infection. One of these relates to the long latency before the development of HCC: in general, it takes 4050 years from infection to the development of HCC (Okuda, 1986
). If the X gene of HBV is involved in hepatocarcinogenesis through the mechanisms mentioned above, why is such a long incubation period necessary? A possible explanation involves apoptosis. Most animal viruses possess strategies to block and/or induce apoptosis depending on the intracellular conditions or external environment (for a review see OBrien, 1998
). For example, E1A of adenovirus, Tax of human T-cell leukaemia virus type I and T-antigen of simian virus 40 (SV40) have been shown to have the ability to induce apoptosis, in addition to their ability to induce cell growth. In an animal model for HBV hepatocarcinogenesis, increased apoptosis of hepatocytes in the precancerous liver was noted (Koike et al., 1998
; Terradillos et al., 1998
). Alternatively, apoptosis in HBV infection may be associated with the pathogenesis of hepatitis or may lead to impaired cell-mediated immunity that could allow the virus to evade host inflammatory responses. Therefore, the possibility that HBx can induce apoptosis must be explored.
One of the problems in studying apoptosis in cultured cells lies in the difficulty in establishing cell lines with a sufficient level of apoptosis-inducing product, since a high level of such a protein may result in cell death during the selection process for cell establishment. In fact, we had previously attempted to establish stable cell lines expressing high levels of HBx, but were unable, in spite of repeated trials, to obtain such a cell line: the only cell lines that were obtained had very low levels of HBx. To overcome this problem, we employed the Cre/loxP system in combination with a recombinant adenovirus vector expressing Cre (Kanegae et al., 1995 ). By using this system, one can introduce a potentially toxic gene, in a completely silent state, permanently into cell lines. In order to make the X gene active in our study, the stuffer DNA was excised by a sequence-specific recombinase, Cre, upon infection with recombinant adenovirus. Driven by a strong promoter, the established cells now produced a high level of HBx, allowing us to carry out biological and biochemical analyses.
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Methods |
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For transient transfection of pCAGp53 plasmid by the calcium phosphate method, HepG2 cells were incubated with DNA precipitates for 6 h at 37 °C, followed by AxCANCre infection (see below).
Generation and administration of recombinant adenovirus.
AxCANCre virus, which expresses Cre DNA recombinase, was generated as described previously (Kanegae et al., 1995 ). As a control for adenovirus infection, Adex1w1, not containing any exogenous genes, was used. A concentrated and purified virus stock was prepared and the titre of the purified stock was determined as described previously (Kanegae et al., 1994
). Infection of adenovirus into cultured cells was carried out at the m.o.i. indicated (Kanegae et al., 1995
, 1996
).
Infection with recombinant adenovirus.
We determined the optimal concentration of recombinant adenovirus needed to infect a maximal number of cells with minimal cytopathic effect (Yeh & Perricaudet, 1997 ). For this purpose, recombinant adenovirus, AxCANCre, was infected into HLF-derived Fz and HepG2-derived Gz cell lines, both of which possessed the lacZ gene, at m.o.i. of 0, 0·5, 2·0, 5·0, 30 and 100. A representative experiment is shown in Fig. 2
for Fz3 cells. By infection with AxCANCre at an m.o.i. of 0·5 to 5·0, nearly 100% of the cells were stained with X-Gal. However, a cytopathic effect was observed to a mild degree with infection at an m.o.i. of 2·0, to a moderate degree at an m.o.i. of 5·0 and to a severe degree at an m.o.i. of 30. From these results, we decided to use an m.o.i. of 0·5 for the remaining experiments. It is not surprising that nearly 100% of the cells were infected at an m.o.i <1·0, as an m.o.i. of 1·0 does not mean one virus particle per cell on the basis of the titration method (Kanegae et al., 1996
).
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Southern hybridization of genomic DNA.
Ten micrograms of genomic DNA was digested with the appropriate restriction enzymes, separated through a 0·8% agarose gel and transferred to a nylon membrane (Schleicher & Schuell). The membrane was then hybridized with a digoxigenin-labelled (Boehringer Mannheim), 1·2 kb linearized pUCHBx probe.
Antibody and indirect immunofluorescence.
HLF or HepG2 cells were grown on Lab-Tek cell culture chambers (Nunc) and infected with adenovirus as described above. Cells were washed with PBS after an appropriate interval after the infection and then fixed with fresh acetonemethanol (70:30) at -20 °C for 7 min. The cells were then reacted with rabbit anti-HBx antibody (Koike et al., 1988 ), washed and incubated with FITC-conjugated secondary antibody (Organon Teknika). For the detection of p53 protein, mouse monoclonal antibody PAb 1801 (Oncogene Science) was used as the first antibody and rhodamine-conjugated goat anti-mouse IgG (Organon Teknika) was used as the second antibody. Stained cells were examined and photographed by using a fluorescent UV microscope.
Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and nuclear condensation assays.
We applied the TUNEL assay (Gavrieli et al., 1992 ) by using a TACS2 TdT in vitro apoptosis detection kit (Trevigen). Fixed cells were subjected to the TUNEL assay according to the manufacturers protocol. To detect nuclear condensation, cells were fixed and stained with Hoechst 33258 (Wako) in PBS at 37 °C for 1 h. After washing with PBS, the cells were visualized under a fluorescent UV microscope.
Flow cytometry.
After the indicated interval following infection with adenovirus, cells were harvested and resuspended in DMSOsucrose buffer. After being stained with propidium iodide, the cells were analysed by FACScan (Becton Dickinson) with the CellFIT program.
Statistical analysis.
Fishers exact test was used for statistical evaluation.
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Results |
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Cre-mediated recombination and switch-on of the X gene
A series of Fx and Gx cell lines was infected with AxCANCre (m.o.i. of 0·5). After incubation at 37 °C for 60 min, the cells were cultured for an additional 2 days and harvested for the determination of gene recombination and HBx protein expression. Firstly, genomic DNA was prepared from the cells and analysed by Southern blot analysis. Recombination of the introduced plasmid, pCALNLX, had occurred in all the cell lines analysed. The result of a representative experiment on the Fx19 cell line is shown in Fig. 1(B). The size of the band detected after HindIII digestion confirmed the expected HBx-on structure, as shown by the shift of the specific band from 6·5 to 5·2 kb. Immunofluorescence staining demonstrated the expression of HBx upon infection with AxCANCre (Fig. 3B
), while no expression was noted upon infection with a control adenovirus, Adex1w1 (Fig. 3A
). The pattern of HBx staining was chiefly perinuclear and cytoplasmic, which is consistent with previous reports (Takada et al., 1997
). The mRNA expression levels of control genes such as the HCV envelope genes or the lacZ gene were comparable to that of the X gene (data not shown).
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Firstly, Fx cell lines Fx2, Fx19 and Fx23, which had the X gene in the silent state, and Fz cell lines Fz3 and Fz5, which had a silent lacZ gene, were infected with recombinant adenoviruses and analysed by TUNEL at 4 days after infection. While the cells infected with the control Adex1w1 showed only slight TUNEL positivity (Fig. 4A), those infected with AxCANCre showed a marked increase in the number of TUNEL-positive cells (Fig. 4B
). The results together with those of the control cell lines are summarized in Fig. 4(C)
. The Fx cell line contained 2938% TUNEL-positive cells after induction of the X gene, while only 58% TUNEL-positive cells were observed following infection with control Adex1w1. Also, the control cell lines showed only 79% positive cells, irrespective of the expression of the lacZ gene. Thus, the HBx protein induced cell death, which was not due either to the effect of adenovirus or to the Cre recombinase itself, in hepatoma-derived cells. Importantly, even after more than 15 passages, Fx or Gx cells could still be induced to express the same levels of HBx protein after infection with AxCANCre recombinant adenovirus, suggesting that the potentially detrimental HBx protein is completely silent unless the stuffer is removed.
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Discussion |
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To overcome the aforementioned problems, several inducible expression systems have been developed and used to analyse the functions of proteins. These include (i) hormone- or metal ion-inducible expression vectors such as those containing the mouse mammary tumour virus (MMTV) long terminal repeat (LTR) or the regulatory region of metallothionein gene, (ii) the tetracycline (Tc)-inducible expression system and (iii) the Cre/loxP switch-on system. To date, only the first two systems have been applied to the assessment of the function of HBx. In our earlier study, we utilized an MMTV LTR-driven HBV X gene construct and demonstrated the promotion of cell cycle progression by HBx in NIH3T3 cells (Koike et al., 1994b ). Recently, Chirillo et al. (1997)
employed the same system and showed that HBx induced apoptosis in NIH3T3 cells in addition to its effect of promoting cell cycle progression. A potential problem in this system lies in the leaky character of the MMTV promoter: i.e. HBx was expressed at a basal level even before induction with glucocorticoids (Koike et al., 1994b
). Therefore, it is possible that specific cells such as those tolerant of or resistant to death signals are selected during the process of establishment of stable cell lines. This may explain the failure of HBx to induce apoptosis in our previous study (Koike et al., 1994b
) despite the experimental conditions being the same as those used in the study of Chirillo et al. (1997)
.
The second system, the Tc-inducible gene, has been established rather recently. This system is a very useful one, in which gene transcription is regulated by the use of a Tc-inducible promoter and a mutated Tc repressor. Recently, Kim et al. (1998) have shown the induction of apoptosis by HBx by the use of this system. By obtaining cell lines that were initially silent for the X gene, they showed directly that HBx induced apoptosis in Chang liver cells, although they used no controls to exclude the possibility that proteins other than HBx may also induce apoptosis when expressed in that system. In addition, as they described in their paper, the Tc-inducible promoter was leaky and allowed a low level of expression of the X gene even without induction; after multiple passages, the expression level of the X gene in their cell lines decreased rapidly to an undetectable level (Kim et al., 1998
).
These problems may be circumvented by the use of the Cre/loxP-mediated conditional expression system, which has already been successfully applied to cultured cells and transgenic animals. We employed a replication-deficient recombinant adenovirus system harbouring Cre recombinase tagged with a nuclear localization signal (Kanegae et al., 1995 , 1996
). The advantages of this system are that (i) almost 100% of the culture cells are infected, (ii) the silent transgene is efficiently switched on in infected cells and (iii) the transgene is completely silent before infection with Cre recombinase-expressing adenovirus (Kanegae et al., 1995
, 1996
). However, this system suffers from the disadvantage of the cytotoxic effect of adenovirus (Yeh & Perricaudet, 1997
). To minimize this effect, we carried out the titration of recombinant adenovirus using lacZ gene-carrying cells and used adenovirus for the X gene experiment at the lowest m.o.i. that gave 100% efficiency of switching on of the gene. We also utilized controls for the adenoviruses and the cell lines: wild-type adenovirus Adex1w1 and a number of cell lines that carried the lacZ gene, the lacZ gene tagged with a nuclear localization signal and the HCV envelope genes.
Our data demonstrate that the HBx protein specifically induced apoptosis in human liver-derived cells when X gene expression was turned on. Selection of cell lines that had a completely silent objective gene (Kanegae et al. 1995 ) by the use of the Cre/loxP system avoided the possibility that cell lines were selected that were more resistant or susceptible to signals from HBx. Our observation that X gene expression was activated reproducibly even after more than 15 passages by the use of the Cre/loxP system is concordant with this notion. We could not observe the cell cycle progression induced by the HBx protein in the current study that we demonstrated previously by using the MMTV inducible system and NIH3T3 cells (Koike et al., 1994b
). Both of the cell lines used in the present study, HLF and HepG2, are hepatoma-derived and do not become quiescent upon the depletion of serum from the culture medium. This may be the reason why the effect of the HBx protein of promoting cell cycle progression was not detectable in the present study.
HBx-induced apoptosis described in previous studies was reported to be either p53-dependent (Chirillo et al., 1997 ) or p53-independent (Terradillos et al., 1998
). Our data indicate that HBx-induced apoptosis occurs via the p53-independent pathway, since apoptosis occurred in cell lines with both mutant and wild-type p53 genes, in which the p53 protein was inactivated by HBx. The mutant p53 in HLF, which contained a mutation from Ala to Gly at codon 244 in the DNA-binding domain, retained its binding capability for HBx and was inactivated by HBx, as was also shown for HuH7 cells by Takada et al. (1997)
. Nonetheless, apoptosis occurred in HLF-derived clones and in HepG2-derived clones with the wild-type p53. Thus, it is considered that HBx inactivated p53 and induced apoptosis concurrently in our system.
Our results show a discrepancy from the previously reported results of Wang et al. (1995) , in which HBx was shown to block apoptosis induced by the overexpression of p53 in primary fibroblasts. It is known that p53 modulates the apoptotic response in various ways. It may exert a protective effect by the induction of cell cycle arrest or induce apoptosis through a transcription-dependent pathway (Amundson et al., 1998
; Evan & Littlewood, 1998
). Thus, differences in cell type and culture conditions may account for the differences. However, it should be noted that Wang et al. (1995)
could not detect HBx in their system: in contrast, in our system, HBx protein was easily detectable after induction.
The precise role of HBx-induced apoptosis in the virus life cycle is not clear. There are some examples of viral or cellular oncoproteins involved in both cell death and oncogenesis. Adenovirus E1A, human T-cell leukaemia virus type I Tax and human papillomavirus E7 stimulate cell proliferation and apoptosis in both p53-dependent and p53-independent manners (Debbas & White, 1993 ; Pan & Griep, 1994
; Yamada et al., 1994
). Having the ability to promote both cell proliferation and cell death, HBx may give the virus an advantage in persistence by boosting both virus and host DNA replication and thereafter destroying host cells in order to release virions. Also, these two apparently opposing functions of HBx, inducing both cell death and proliferation, might explain the long incubation period of 4050 years from HBV infection to the development of HCC in humans, given that HBx plays a central role in hepatocarcinogenesis (Paterlini et al., 1995
; Koike, 1995
; Koike & Takada, 1995
; Caselmann, 1995
; Feitelson & Duan, 1997
).
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Acknowledgments |
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Footnotes |
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References |
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Received 19 July 1999;
accepted 3 August 1999.