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Animal: DNA Viruses |
Department of Microbiology, College of Natural Sciences, Pusan National University, Pusan 609-735, Korea1
Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea2
Author for correspondence: Kyung Lib Jang. Fax +82 51 514 1778. e-mail kljang{at}pusan.ac.kr
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Introduction |
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) and is expressed in more than half of HBV-infected human livers and HCCs (Su & Schneider, 1996 ). In addition, HBx deregulates cell-cycle checkpoints (Benn & Schneider, 1994 ) and its overexpression causes uncontrolled cell proliferation (Yen et al., 1996 ). In many cases, failure to arrest cell proliferation generates cells with highly unstable genomes that could evolve into cancer cells (Jackson & Loeb, 1998 ; Martin-Caballero et al., 2001 ; Pruitt & Der, 2001 ).
Interestingly, it has been shown that the function of p53 can be suppressed by HBx via a protein–protein interaction(s) (Wang et al., 1994 ). In addition, transcription of p53 is strongly repressed by HBx (Lee & Rho, 2000 ). Moreover, HBx represses transcription of p21waf1/cip1 via a p53-independent pathway, as demonstrated by Ahn et al. (2001) . A variety of studies reported that p21waf1/cip1 is an universal inhibitor of cyclin–CDK complexes and DNA replication and thus induces cell-cycle arrest at the G1–S checkpoint (Eapen et al., 2001 ; Galbiati et al., 2001 ; Pinski et al., 2001 ). Therefore, the altered expression of p21waf1/cip1 by HBx might be important for the understanding of HBV-mediated HCC. However, it has been reported recently that HBx upregulates the expression of p21waf1/cip1 and prolongs G1
S transition in human hepatoma cells (Han et al., 2000 ; Park et al., 2000 ), arguing that cells respond oppositely to HBx for the expression of p21waf1/cip1 under different situations. Thus, we believe that the choice between cell-cycle arrest and progress through p21waf1/cip1 mediated by HBx must be an important determinant to understand the mechanisms of hepatocellular carcinogenesis. In this study, we have tried to investigate the effect of HBx on the activity of p21waf1/cip1 in either the presence or the absence of its upstream regulator, p53, and to provide evidences that p21waf1/cip1 activities mediated by HBx inhibit or stimulate cell-cycle progression depending on the status of cellular p53.
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Methods |
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Plasmid construction. ). The human p21waf1 promoter construct, WWW-luc (p21P), was a gift from Bert Vogelstein (El-Deiry et al., 1993 ). p21P
2.3 and other deletion mutants of the p21waf1 promoter were described previously (Datto et al., 1995 ).
Transfection and luciferase assays.
HepG2, Hep3B and NIH3T3 cells were transfected by the calcium phosphate precipitation method, as described previously (Ganem & Varmus, 1987 ). To control for the variation in transfection efficiency, 1 µg pCH110 (Pharmacia) containing the Escherichia coli lacZ gene under the control of the SV40 promoter was cotransfected as an internal control. At 48 h after transfection, the level of expression from the reporter gene (luciferase activity) was analysed and values obtained were normalized to the 
-galactosidase activity measured in the corresponding cell extracts. Each experiment was repeated at least three times and the transfection efficiency, as determined by -galactosidase-positive staining using the lacZ control plasmid, was at least 10% under optimal transfection conditions.
Western blot analysis.
Transient transfections of HepG2, Hep3B and NIH3T3 cells for Western blot analysis were performed using the liposome reagent Fugene-6, according to the manufacturer's protocol (Roche). Transfection efficiency was approximately 90%, as determined by -galactosidase staining. Cells were lysed in buffer (50 mM Tris–HC1, pH 8, 150 mM NaCl, 0·1% SDS and 1% NP-40) supplemented with protease inhibitors. The protein concentration of the cell extracts was measured using the BSA Protein Assay kit (Bio-Rad). A 10 µg sample of each cell extract was separated by SDS–PAGE and transferred onto a nitrocellulose membrane (Hybond PVDF; Amersham). Western blotting was performed with either anti-human p53 monoclonal antibody (Santa Cruz), anti-human p21 rabbit polyclonal IgG (Santa Cruz), anti-human actin monoclonal IgG (Santa Cruz) or anti-HA antibody (Roche) and subsequently detected using the chemiluminescent ECL kit (Amersham), as recommended by the manufacturer.
RNA analysis.
Total RNA was prepared from cells using the guanidinium isothiocyanate procedure (Chomczynski & Sacchi, 1987 ). Total RNA (5 µg) was spotted onto a nitrocellulose membrane (Hybond-N; Amersham) and hybridized with an appropriate probe using the Detection Starter Kit II (Roche). For the preparation of probes, total cellular RNA was amplified by RT–PCR using appropriate pairs of primer and cDNA was labelled using the DIG labelling kit (Roche).
Generation of stable cell lines and determination of cell growth rate.
NIH3T3 cells (2x105 cells per 60 mm diameter plate) transfected with 10 µg pCMV-3xHA1-HBx were selected and amplified to obtain stable cell lines, as described previously (Chang et al., 1998 ). The expression level of HBx was checked by Western blot analysis. For the determination of cell growth rates, 5x104 cells were plated in 6-well plates (Nunc) and the total cell number in each well was counted after incubation under the appropriate conditions.
FACS analysis.
Cell-cycle profiles were analysed using flow cytometry. Briefly, 2x106 cells were trypsinized, washed twice with PBS and fixed in 80% ethanol. Fixed cells were washed with PBS and resuspended in 50 µg/ml propidium iodide containing 125 U/ml RNase A. DNA contents were analysed by flow cytometry using CELL-FIT software (Becton-Dickinson).
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) and cotransfected it with the HA-tagged HBx expression plasmid into a p53-positive hepatoma cell line, HepG2. Consistent with the previous report by Han et al. (2000) , results from this experiment showed that HBx activated the promoter activity of p21waf1/cip1 in p53-positive cells in a dose-dependent fashion (Fig. 1A). A comparable promoter construct, p21P2.3, which is driven by the truncation of the p21waf1/cip1 promoter and thus is not responsive to p53 (Datto et al., 1995 ), was not significantly affected by HBx (Fig. 1A, right panel), suggesting that HBx stimulates the transcription of p21waf1/cip1 via a p53-dependent pathway. A similar experiment was performed in Hep3B cells, in which a functional p53 protein is absent (Park et al., 2000 ). Interestingly, a completely opposite result was obtained, showing that HBx repressed the promoter activity of p21waf1/cip1 in an HBx dose-dependent manner in these p53-negative cells (Fig. 1B, left panel). However, the repression of p21P2.3 reporter activity was largely maintained in p53-negative Hep3B cells (Fig. 1B, right panel). These results imply that, depending on the status of p53 in cells, HBx generates opposite effects on the transcription of p21waf1/cip1 through completely different pathways. Using another luciferase construct, p21Psma1, which contains only 61 bp proximal to the transcription initiation site (Datto et al., 1995 ), was similarly repressed by HBx in Hep3B cells (data not shown), implying that the region of the promoter responsible for the repression by HBx in Hep3B cells might be in a 61 bp region near the transcription initiation site. Therefore, we are currently investigating the binding site responsible for the repression of p21waf1/cip1 by HBx.
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), consistent with the ability of HBx to augment p21waf1/cip1 promoter activity in p53-positive HepG2 cells but not in p53-negative Hep3B cells (Fig. 1A, B). Moreover, repression of p21waf1/cip1 promoter activity by HBx in Hep3B cells was reversed in a p53 dose-dependent fashion (Fig. 1C). Thus, these results clearly indicate that the effect of HBx on the transcription of p21waf1/cip1 is regulated by the status of endogenous p53.
To further demonstrate whether the activation of the p21waf1/cip1 promoter in HepG2 cells is dependent on p53, a control luciferase construct (pT81-luc), containing a basic promoter element (TATA box), was employed. As expected, HBx did not affect basal reporter activity in both HepG2 (Fig. 1D) and Hep3B (data not shown) cells. However, in HepG2 cells, luciferase activity was increased approximately threefold by the presence of 13 copies of the p53-binding site in pTG13 (Lee et al., 1998 ) and was further activated about ninefold by HBx. In contrast, the p53-binding sites in pTG13 were not affected by HBx in p53-negative Hep3B cells. However, the promoter activity of pTG13 in Hep3B cells was increased by the addition of the p53 expression plasmid and was further activated by HBx, whereas the addition of p53 naturally occurring mutants, such as p53 175H and 248Q, each of which has a fatal substitution in the DNA-binding domain at codons 175 (ArgHis) and 248 (ArgGln), respectively (Morgan et al., 2000 ), was not effective to stimulate p53 reporter gene activity mediated by HBx (Fig. 1D). These results confirm that HBx activates transcription of the p21waf1/cip1 gene through a p53-dependent pathway.
Since HBx has opposite effects on p21waf1/cip1 promoter activity depending on the status of p53, we examined the level of endogenous p21waf1/cip1 protein. Western blot analysis showed that the level of p21waf1/cip1 was increased by HBx in HepG2 cells (Fig. 2A, lanes 1 and 2) but was repressed in Hep3B cells (Fig. 2A, lanes 3 and 4). Furthermore, similarly to the promoter activity, the ability of HBx to induce the level of p21waf1/cip1 protein was restored when Hep3B cells were complemented with p53 (Fig. 2A, lanes 5 and 6), thus supporting the fact that the dual roles of HBx in the regulation of p21waf1/cip1 expression depend on the status of p53 in cells. Interestingly, the level of p53 was slightly increased in the presence of HBx in HepG2 cells (Fig. 2A, lanes 1 and 2). Apparently, it might not result from the effect of HBx on the promoter of p53 because a similar effect was also observed in which the p53 protein was introduced exogenously into Hep3B cells. To further elucidate whether the level of p21waf1/cip1 by HBx is influenced by transcription or translation, we isolated RNA from HepG2 cells and performed dot blot analysis showing that the level of p21waf1/cip1 RNA was clearly increased by the presence of HBx, whereas the level of p53 RNA was not significantly affected by HBx (Fig. 2B). These results imply that HBx activates transcription of p21waf1/cip1 not by the increased synthesis of p53 but by either the interaction or the modification of the p53 protein.
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). We thus argued that this effect might be due to the very low expression level of p53 in NIH3T3 cells; a similar effect of HBx was observed in Hep3B cells when complemented with a low level of p53 (0·5 µg) (Fig. 1C). To demonstrate this possibility, we tried to increase the protein level of p53 in NIH3T3 cells by transient transfection of the p53-expressing plasmid and tested the effect of HBx on the p21 promoter. As the level of p53 increased, the repression fold of the p21waf1/cip1 promoter by HBx gradually decreased and was eventually reversed to activation when more than 1 µg of p53-expressing plasmid was added (Fig. 3A). A similar result was also obtained when the level of endogenous p53 was elevated by treatment with a DNA-damaging agent, cisplatin, which is widely used to induce the level of p53 (Fig. 3B). Western blot analysis showed that the level of p21waf1/cip1 protein in NIH3T3 cells was decreased by HBx but was increased when p53 was activated by treatment with cisplatin (Fig. 3C). The level of p53 in NIH3T3 cells seemed to be too low to show the stabilization effect by HBx but was clearly increased by HBx when the basal level was elevated by treatment with cisplatin.
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; McDonald & El-Deiry, 2001 ); the altered expression of p21waf1/cip1 by HBx may effect cell growth. Thus, we prepared several NIH3T3 cell lines that stably expressed HBx (NIH3T3/HBx); five different cell lines were selected and tested to show the differences in the growth rate. As expected, the growth rate of HBx-expressing cell lines, as presented by a representative one in Fig. 4(A, B), was approximately twofold faster compared with that of the parental NIH3T3 cells, indicating that the repression of p21waf1/cip1 by HBx in NIH3T3 cells could be properly reflected by the stimulation of cell growth. When cells were treated with 5 µM cisplatin, both the parent NIH3T3 cells and the HBx-expressing cells progressively decreased in total cell number. However, the growth rate of HBx-expressing cells decreased more dramatically compared with that of the parent cells (Fig. 4A). Furthermore, the percentage difference in the total cell number between HBx-expressing cells and control cells increased as more cisplatin was added (Fig. 4B). From the data presented here, the rapid reduction in total cell number observed in the HBx-expressing cells could be explained by the effect of the increased levels of p21waf1/cip1 in HBx-expressing cells to induce cell-cycle arrest at the G1–S checkpoint and/or to induce apoptosis caused by prolonged cell-cycle arrest. To further explore the effect of HBx on the progress of the cell cycle, we performed flow cytometry experiments on NIH3T3 cells in which the level of p21waf1/cip1 was increased by HBx when p53 was activated by cisplatin treatment. As shown in Fig. 4(C), in the presence of cisplatin, the proportion of cells in the G1 phase clearly increased in HBx-expressing NIH3T3 cells compared to the parent cells. When cells were treated with cisplatin for a longer period or at a higher concentration, the G1 proportion of HBx-expressing cells increased up to 80% compared to the parent cells in their percentage difference, whereas cells in other stages, S and G2/M, decreased significantly (Fig. 1C). Therefore, the kinetics of the cell-cycle profile clearly demonstrates that HBx can effect cell growth at the G1/S phase depending on the status of p53.
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Discussion |
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). Furthermore, a significant increase of apoptotic liver cells was found in severe chronic viral hepatitis cases compared to patients with mild steatohepatitis (Papakyriakou et al., 2002 ), suggesting that apoptotic cell death might be involved in the pathogenesis of hepatocellular damage of viral hepatitis. In the meantime, expression of HBx may enhance liver cell susceptibility to carcinogen-induced mutagenesis, potentially through downregulation of DNA excision repair by the HBx protein (Jia et al., 1999 ; Prost et al., 1998 ), thus increasing the chance that p53 is inactivated. Actually, mutational inactivation of the p53 gene is very common (30–55%) in HCCs (Sohn et al., 2000 ). Now, transcriptional repression of p21waf1/cip1 by HBx in the absence of p53 may induce rapid and uncontrolled cell proliferation and thus make it easy to accumulate more mutations on other oncogenes or tumour suppressor genes, ultimately leading to HCC. Therefore, we suggest here that conversion of the effect of HBx on p21waf1/cip1 from activation to repression by the loss of functional p53 might be a critical event during HBV-mediated hepatocellular carcinogenesis. |
Acknowledgments |
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References |
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Benn, J. & Schneider, R. J. (1994). Hepatitis B virus HBx protein activates Ras–GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proceedings of the National Academy of Sciences, USA 91, 10350-10354.
Chang, J., Yang, S. H., Cho, Y. G., Hwang, S. B., Hahn, Y. S. & Sung, Y. C. (1998). Hepatitis C virus core from two different genotypes has an oncogenic potential but not sufficient for transforming primary rat embryo fibroblasts in cooperation with the H-ras oncogene. Journal of Virology 72, 3060-3065.
Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry 162, 156-159.[Medline]
Datto, M. B., Yu, Y. & Wang, X. F. (1995). Functional analysis of the transforming growth factor responsive elements in the WAF1/Cip1/p21 promoter. Journal of Biological Chemistry 270, 28623-28628.
Eapen, A. K., Henry, M. K., Quelle, D. E. & Quelle, F. W. (2001). DNA damage-induced G1 arrest in hematopoietic cells is overridden following phosphatidylinositol 3-kinase-dependent activation of cyclin-dependent kinase 2. Molecular and Cellular Biology 21, 6113-6121.
El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W. & Vogelstein, B. (1993). WAF1, a potential mediator of p53 tumor suppression. Cell 4, 817-825.
El-Deiry, W. S., Harper, J. W., O'Connor, P. M., Velculescu, V. E., Canman, C. E., Jackman, J., Pietenpol, J. A., Burrell, M., Hill, D. E., Wang, Y., Wiman, K. G., Mercer, W. E., Kastan, M. B., Kohn, K. W., Elledge, S. J., Kinzler, K. W. & Vogelstein, B. (1994). WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Research 54, 1169-1174.
Galbiati, F., Volonte, D., Liu, J., Capozza, F., Frank, P. G., Zhu, L., Pestell, R. G. & Lisanti, M. P. (2001). Caveolin-1 expression negatively regulates cell cycle progression by inducing G0/G1 arrest via a p53/p21WAF1/Cip1-dependent mechanism. Molecular Biology of the Cell 12, 2229-2244.
Ganem, D. & Varmus, H. E. (1987). The molecular biology of the hepatitis B viruses. Annual Review of Biochemistry 56, 651-693.[Medline]
Han, J., Yoo, H. Y., Choi, B. H. & Rho, H. M. (2000). Selective transcriptional regulations in the human liver cell by hepatitis B viral X protein. Biochemical and Biophysical Research Communications 272, 525-530.[Medline]
Jackson, A. L. & Loeb, L. A. (1998). On the origin of multiple mutations in human cancers. Seminars in Cancer Biology 8, 421-429.[Medline]
Jia, L., Wang, X. W. & Harris, C. C. (1999). Hepatitis B virus X protein inhibits nucleotide excision repair. International Journal of Cancer 80, 875-879.
Kaufmann, W. K., Behe, C. I., Golubovskaya, V. M., Byrd, L. L., Albright, C. D., Borchet, K. M., Presnell, S. C., Coleman, W. B., Grisham, J. W. & Smith, G. J. (2001). Aberrant cell cycle checkpoint function in transformed hepatocytes and WB-F344 hepatic epithelial stem-like cells. Carcinogenesis 8, 1257-1269.
Kim, C. M., Koike, K., Saito, I., Miyamura, T. & Gilbert, J. (1991). HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 351, 317-320.[Medline]
Lee, S. G. & Rho, H. M. (2000). Transcriptional repression of the human p53 gene by hepatitis B viral X protein. Oncogene 19, 468-471.[Medline]
Lee, C. W., Sørensen, T. S., Shikama, N. & La Thangue, N. B. (1998). Functional interplay between p53 and E2F through co-activator p300. Oncogene 16, 2695-2710.[Medline]
McDonald, E. R.III & El-Deiry, W. S. (2001). Checkpoint genes in cancer. Annals of Medicine 33, 113-122.[Medline]
Martin-Caballero, J., Flores, J. M., Garcia-Palencia, P. & Serrano, M. (2001). Tumor susceptibility of p21Waf1 /Cip1-deficient mice. Cancer Research 61, 6234-6238.
Morgan, S. E., Kim, R., Wang, P. C., Bhat, U. G., Kusumoto, H., Lu, T. & Beck, W. T. (2000). Differences in mutant p53 protein stability and functional activity in teniposide-sensitive and -resistant human leukemic CEM cells. Oncogene 19, 5010-5019.[Medline]
Papakyriakou, P., Tzardi, M., Valatas, V., Kanavaros, P., Karydi, E., Notas, G., Xidakis, C. & Kouroumalis, E. (2002). Apoptosis and apoptosis related proteins in chronic viral liver disease. Apoptosis 7, 133-141.[Medline]
Park, U. S., Park, S. K., Lee, Y. I., Park, J. G. & Lee, Y. I. (2000). Hepatitis B virus-X protein upregulates the expression of p21waf1/cip1 and prolongs G1S transition via a p53-independent pathway in human hepatoma cells. Oncogene 19, 3384-3394.[Medline]
Pinski, J., Parikh, A., Bova, G. S. & Isaacs, J. T. (2001). Therapeutic implications of enhanced G0/G1 checkpoint control induced by coculture of prostate cancer cells with osteoblasts. Cancer Research 61, 6372-6376.
Prost, S., Ford, J. M., Taylor, C., Doig, J. & Harrison, D. J. (1998). Hepatitis B x protein inhibits p53-dependent DNA repair in primary mouse hepatocytes. Journal of Biological Chemistry 273, 33327-33332.
Pruitt, K. & Der, C. J. (2001). Ras and Rho regulation of the cell cycle and oncogenesis. Cancer Letters 171, 1-10.[Medline]
Sohn, S., Jaitovitch-Groisman, I., Benlimame, N., Galipeau, J., Batist, G. & Alaoui-Jamli, M. A. (2000). Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis. Mutation Research 460, 17-28.[Medline]
Su, F. & Schneider, R. J. (1996). Hepatitis B virus HBx protein activates transcription factor NF-
B by acting on multiple cytoplasmic inhibitors of related proteins. Journal of Virology 70, 4558-4566.[Abstract]
Wang, X. W., Forrester, K., Yeh, H., Feitelson, M. A., Gu, J. R. & Harris, C. C. (1994). Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proceedings of the National Academy of Sciences, USA 91, 2230-2234.
Yen, A., Sturgill, R., Varvayanis, S. & Chern, R. (1996). FMS(CSF-1 receptor) prolongs cell cycle and promotes retinoic acid-induced hypophosphorylation of retinoblastoma protein, G1 arrest, and cell differentiation. Experimental Cell Research 229, 111-125.[Medline]
Received 14 February 2002;
accepted 26 June 2002.
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, 
) or HBx-expressing stable cells (NIH3T3/HBx, 
,