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Molecular biology of hepatitis C virus in hepatocellular carcinoma
Viruses and Liver Cancer
E. Tabor (editor) © 2002ElsevierScienceB.V.All rights reserved
93
Molecular biology of hepatitis C virus in hepatocellular carcinoma Shuichi Kaneko, Kenichi Kobayashi First Department of Internal Medicine, Kanazawa UniversitySchool of Medicine, Takara-machi 13-1, Kanazawa 920-8641 Japan
Abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; PCR, polymerase chain reaction; IRES, internal ribosomal entry site; REF, rat embryo fibroblasts; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated ERK kinase.
Introduction
Since the identification of hepatitis C virus (HCV) as the major causative agent of human non-A, non-B hepatitis, it has been possible to study the link between this chronic viral infection and hepatocellular carcinoma (HCC). Infection with HCV is now known to be a major risk factor for the development of HCC, associated with chronic hepatitis and usually with cirrhosis. The progression induced by chronic inflammation and successive regeneration may overcome DNA repair mechanisms. This may permit DNA mutations to induce cell transformation. Thus, persistent inflammation may be of primary importance in hepatocarcinogenesis. It is clear that hepatitis B virus (HBV)- associated chronic hepatitis and cirrhosis are also a major factor in hepatocarcinogenesis. However, HBV infection is associated with integration of HBV DNA into the host chromosome, raising the possibility that direct HBV interaction with oncogenes, growth factors, or tumor suppressor genes, perhaps accentuated through increased genomic instability, could be important mechanisms. Furthermore, the HBV X protein appears to be a potent transactivator of a wide array of promoters that normally regulate transcription of cellular and viral genes. HCV is a typical RNA virus that does not integrate into the host chromosome, and the mechanisms of carcinogenesis by HCV are poorly understood. This chapter will review the molecular biology of the hepatitis C virus in terms of virus-cell interactions that may contribute to the development of HCC. Status of HCV in HCC
Several viruses have been demonstrated to be involved in the development of human malignancies, for example, Epstein-Barr virus in some types of lymphoma and
94
I Chronic Hepatitis I I Cirrhosis I [ Hepatocellular Carcinoma J.i-, Fig. 1. Role of hepatitis C virus (HCV) in the developmentof hepatocellular carcinoma. nasopharyngeal carcinoma and human papillomavirus in cervical carcinoma. These viruses produce oncoproteins that can transform infected cells. Immunohistologic studies have revealed these oncoproteins to be detectable more frequently in tumors than in normal tissues. The presence of HCV antigens or the presence of HCV RNA in HCCs has been studied using immunohistochemistry, PCR, and in situ hybridization [1-9]. Cells containing HCV were detected more frequently in adjacent nontumorous liver tissues than in HCC tissues, although some HCC samples did contain detectable HCV. Strand-specific PCR and in situ hybridization have been used to detect the presence of replicative intermediates of HCV, showing that active replication of HCV was present in some HCC tissues. Thus, HCV can replicate and express viral proteins in HCC. HCV is an RNA virus that appears to replicate solely in the cytoplasm. Although the detection of HCV core protein in the cytoplasm supports this, several studies suggest that HCV core protein can translocate into the nucleus under certain circumstances, for instance following the experimental truncation of the hydrophobic C-terminal region [10-14]. Although it is not known whether this form exists during naturally occurring HCV infection, the observations suggest that the HCV core protein could have a regulatory role in the nucleus. HCV has been classified into at least six major genotypes on the basis of overall nucleotide sequences [15]; substantial divergence in nucleotide sequences exists between the genotypes. Several investigators have suggested that chronic infection with HCV genotype lb is associated with an increased risk of developing HCC compared with other genotypes [16-19], although other investigators have been unable to show any correlation between genotype and severity of disease including development of HCC [20,21]. Specific nucleotide sequences in HCV isolates have not been associated with HCC, although some of the nucleotide changes in genotype lb have been suggested to be associated with HCC progression [22]. It is not known whether any specific HCV genotype encodes for an oncogenic protein. Cellular proliferation and HCV In normal liver, most hepatocytes are in a resting state (stage GO), but in chronic hepatitis, cirrhosis, and HCC, hepatocytes are in a state of increased cellular
95
Growth
Cytokin~
TN
Apoptosis signals
~
,o~,,utu^,~.=v~,,,7
Cellular proliferation Transformation Fig. 2. Hepatitis C virus infection and reported mechanisms of hepatocarcinogenesis.
proliferation. The significance of hepatocellular proliferation in the development of HCC has been reported in chronic hepatitis C and cirrhosis [23-28] and in nonmalignant nodules [29,30]. The cellular proliferation is caused by inflammation directed against HCV-infected hepatocytes. The effect on HCC development of HCV infection without concomitant cellular proliferation has been studied in patients with normal aminotransferase levels after interferon therapy. Yoshida et al. reported that the risk of developing HCC was reduced to an equivalent extent by interferon therapy among 260 patients who had normal serum aminotransferase levels with persisting serum HCV RNA compared to 789 patients who eliminated serum HCV RNA after the therapy. This suggests that lowering cellular proliferation might be more important than elimination of HCV in preventing hepatocarcinogenesis. HCV may play a role in hepatocarcinogenesis through interaction with host factors during hepatocellular proliferation. Translation of the HCV polyprotein is mediated by an internal ribosomal entry site (IRES) that is located within the 5'-non-translated region of HCV RNA. The IRES activity is greatest in actively proliferating cells and relatively reduced in resting cells (evaluated using established cell lines), suggesting that host proteins in proliferating cells might interact with the HCV IRES and thus increase HCV translation [32].
96 In addition, HCV itself may influence hepatocellular proliferation through interaction with cellular factors. In one report, studies in stable cell lines that constitutively expressed HCV core protein [33] revealed that the HCV core protein contributed to increased cellular proliferation and sensitized apoptosis to serum starvation. To explain these findings, the authors examined the expression of the c-myc gene, which has been reported to induce apoptosis. In response to serum starvation, expression of the c-myc oncogene was significantly increased in the cell lines. Thus, the HCV core protein appeared to induce apoptosis and impaired regulation of the cell cycle by activating c-myc expression in the cells. Induction of cellular transformation by HCV proteins To investigate the possible oncogenic activity of the HCV proteins, cells transfected with an expression vector containing HCV cDNA have been studied. NIH3T3 cells became transformed after transfection with HCV NS3 cDNA and were tumorigenic in nude mice [34]; this suggested that proteinase activity associated with the NS3 protein might be the cause of transformation, although the precise mechanism is unknown. Ray et al. demonstrated that HCV core protein transformed rat embryo fibroblasts (REF) in cooperation with Ha-ras [35]. However, in contrast, Chang et al. [37] reported that HCV core protein in cooperation with the Ha-ras oncogene did not induce the transformation of REF cells. BALB/3T3 cells expressing HCV core protein have been reported to activate mitogen-activated protein kinase (MAPK) and the serum response element and resulted in cell transformation [36]. It has been reported that HCV core protein expression resulted in a loss of function of LZIP, a subfamily of bZIP proteins, and triggered transformation [38]. Cooperative transformation of NIH3T3 cells with the HCV NS4B gene and Ha-ras by AP1 activation has been shown [39]. Transfection of the HCV NS5A gene into NIH3T3 cells promoted anchorage-independent growth in vitro and tumor formation in nude mice [40]; the authors of this study suggested that the transformation was induced by differential modulation of the cell cycle regulatory gene p21/WAF1 and by PCNA. HCC developed in transgenic mice carrying the HCV core gene, but not in transgenic mice carrying the envelope genes under the same transcription control [41]. The HCV core protein has been shown to be present in the nuclei and mitochondria of some HCC cells. These findings support the idea that the core protein may act as a transcriptional regulator and could possibly affect the proliferative ability of cells, thereby perhaps playing a role in hepatocarcinogenesis. Modulation of apoptosis by HCV Many viruses have evolved gene products to suppress or delay apoptosis. Alterations of apoptosis by HCV infection could result in the inability to prevent the effects of harmful mutations, subsequently leading to the development of HCC. The effect of HCV core protein on TNF~-induced apoptosis has been investigated. HCV core protein suppressed apoptosis induced by cisplatin in human
97 cervical epithelial cells, by c-myc over-expression in Chinese hamster ovary cells [42], and by TNFo~ in MCF7 human breast carcinoma cells [43]. Inhibition of TNF~-induced apoptosis in Hep G2 human hepatoblastoma cells by the HCV core protein by activating nuclear factor vd3 (NF-~cB) has been reported [44,45]. DNA chip analysis has demonstrated augmented expression of the NF-lcB gene and anti-apoptotic genes in livers from patients with chronic hepatitis C [46]. There are also some contradictory reports on the effect of the HCV core protein on TNF~-induced apoptosis. Stable expression of the HCV core protein in a mouse cell line or in Hep G2 cells has been reported to sensitize them to TNF-induced apoptosis [47], suggesting that the HCV core protein can promote apoptosis via TNF signaling pathways, possibly as a result of interaction with the cytoplasmic tail of TNF receptor 1 (TNFR1). Others [48] have reported that stable MCF7 cell transfectants expressing the HCV core protein suppressed TNF-induced NF-~zB activation, and, in contrast, constitutively activated AP-1. These discrepancies might have resulted from differences in cells, in transfection systems, or in HCV cDNAs used. Similarly, discrepant reports regarding the effect of HCV core protein on Fas-mediated apoptosis have been published. It has been reported that expression of the core protein made Hep G2 cells prone to Fas-mediated apoptosis [49], and others have reported that the core protein reduced apoptosis [44]. Increased sensitivity to Fas-mediated apoptosis was shown in transgenic mice carrying HCV core and envelope cDNA [50]. The HCV NS5A protein from interferon-resistant strains of HCV can repress the action of the interferon-induced protein kinase PKR [51], suppress TNF~-induced apoptosis in a stable transfectant of Hep G2 cells [52], and can inhibit doublestranded RNA-induced apoptosis via an NS5A-mediated block in elF-2cz phosphorylation [53]. NIH3T3 cells expressing the NS3 protein were reported to be more resistant to actinomycin D-induced apoptosis than control cells [54]. Other cellular targets of HCV proteins
The tumor suppressor protein p53 induces growth arrest or apoptosis in response to a variety of stress signals, thereby eliminating damaged and potentially dangerous cells from the organism. Previous studies showed that p53 protein functionally interacts with several HCV proteins. It has been reported that HCV core protein augmented the transcriptional activity of p53 and increased expression of the p21/WAF1 protein, which is a major target of p53 [55,56]. The HCV core protein increased both the DNA-binding affinity of p53 in a mobility shift assay and the transcriptional activity of p53 itself in a reporter assay [55]. In contrast, Ray et al. [57,58] found that HCV core protein repressed transcriptional activity of the p53 promoter and the p21 promoter. The role of HCV core protein in the regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade has been studied. The MAPK/ERK cascade is involved in the signal transduction of a wide variety of extracellular stimuli, and activates nuclear transcriptional factors to induce
98 the transcription of genes required for cell cycle progression. Hayashi et al. [59] reported that HCV core protein activated the MAPK/ERK cascade including Elk1 probably at or near the gene for mitogen-activated E R K kinase (MEK)1. They also reported finding the HCV core protein bound to 14-3-3 protein and activated Raf-1 kinase, a central component of the M A P K / E R K cascade that phosphorylates MEK1 [60]. However, Fukuda et al. [61] reported that HCV core protein enhances Elkl activation without affecting E R K activity or Elkl phosphorylation. Interactions of HCV core protein with tumor necrosis factor receptor-related lymphotoxin-13 receptor [62-64], nuclear ribonucleoprotein K [65], and RNA helicase [66-68] have also been reported. Transcriptional regulation of cellular and viral promoters by the HCV core protein has also been reported [69]. References
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99 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.
Reid AE, Koziel M J, Aiza I, Jeffers L, Reddy R, Schiff E, et al. Am J Gastroentero11999; 94: 1619-1626. Nagayama K, Kurosaki M, Enomoto N, Miyasaka Y, Marumo F, Sato C. Hepatology 2000; 31: 745-750. Tarao K, Shimizu A, Ohkawa S, Harada M, Ito Y, Tamai S, et al. Gastroenterology 1992; 103: 595-600. Derenzini M, Trere D, Oliveri F, David E, Colombatto P, Bonino F, et al. J Clin Pathol 1993; 46: 727-729. Ballardini G, Groff P, Zoli M, Bianchi G, Giostra F, Francesconi R, et al. J Hepato11994; 20: 218-222. Tarao K, Ohkawa S, Shimizu A, Harada M, Nakamura Y, Ito Y, et al. Cancer 1994; 73: 1149-1154. Dutta U, Kench J, Byth K, Khan MH, Lin R, Liddle C, et al. Hum Pathol 1998; 29: 1279-1284. Kaneko S, Terasaki S, Kobayashi K. In: Srivastava S, et al (eds), Molecular Pathology of Early Cancer. IOS Press; 1999, 255-263. Takayama T, Makuuchi M, Hirohashi S, Sakamoto M, Okazaki N, Takayasu K, et al. Lancet 1990; 336: 1150-1153. Terasaki S, Kaneko S, Kobayashi K, Nonomura A, Nakanuma Y. Gastroenterology 1998; 115: 1216-1222. Yoshida H, Shiratori Y, Moriyama M, Arakawa Y, Ide T, Sata M, et al. Ann Intern Med 1999; 131: 174-181. Honda M, Kaneko S, Matsushita E, Kobayashi K, Abell GA, Lemon SM. Gastroenterology 2000; 118: 152-162. Honda M, Kaneko S, Shimazaki T, Matsushita E, Kobayashi K, Ping LH, et al. Hepatology 2000; 31: 1351-1359. Sakamuro D, Furukawa T, Takegami T. J Virol 1995; 69: 3893-3896. Ray RB, Lagging LM, Meyer K, Ray R. J Virol 1996; 70: 4438-4443. Tsuchihara K, Hijikata M, Fukuda K, Kuroki T, Yamamoto N, Shimotohno K. Virology 1999; 258: 100-107. Chang J, Yang SH, Cho YG, Hwang SB, Hahn YS, Sung YC. J Viro11998; 72: 3060-3065. Jin DY, Wang HL, Zhou Y, Chun AC, Kibler KV, Hou YD, et al. EMBO J 2000; 19: 729-740. Park JS, Yang JM, Min MK. Biochem Biophys Res Commun 2000; 267: 581-587. Ghosh AK, Steele R, Meyer K, Ray R, Ray RB. J Gen Virol 1999; 80: 1179-1183. Moriya K, Fujie H, Shintani Y, Yotsuyanagi H, Tsutsumi T, Ishibashi K, et al. Nat Med 1998; 4: 1065-1067. Ray RB, Meyer K, Ray R. Virology 1996; 226: 176-182. Ray RB, Meyer K, Steele R, Shrivastava A, Aggarwal BB, Ray R. J Biol Chem 1998; 273: 2256-2259. Marusawa H, Hijikata M, Chiba T, Shimotohno K. J Virol 1999; 73: 47134720. Tai DI, Tsai SL, Chen YM, Chuang YL, Peng CY, Sheen IS, et al. Hepatology 2000; 31: 656-664. Honda M, Kaneko S, Kawai H, Shirota Y, Kobayashi K. Gastroenterology (in press). Zhu N, Khoshnan A, Schneider R, Matsumoto M, Dennert G, Ware C, et al. J Virol 1998; 72: 3691-3697. Shrivastava A, Manna SK, Ray R, Aggarwal BB. J Virol 1998; 72: 9722-9728. Ruggieri A, Harada T, Matsuura Y, Miyamura T. Virology 1997; 229: 68-76.
100 50. Honda A, Arai Y, Hirota N, Sato T, Ikegaki J, Koizumi T, et al. J Med Virol 1999; 59: 281-289. 51. Gale M, Blakely CM, Kwieciszewski B, Tan SL, Dossett M, Tang NM, et al. Mol Cell Biol 1998; 18: 5208-5218. 52. Ghosh AK, Majumder M, Steele R, Meyer K, Ray R, Ray RB. Virus Res 2000; 67: 173-178. 53. Gale M, Kwieciszewski B, Dossett M, Nakao H, Katze MG. J Viro11999; 73:6506-6516. 54. Fujita T, Ishido S, Muramatsu S, Itoh M, Hotta H. Biochem Biophys Res Commun 1996; 229: 825-831. 55. Otsuka M, Kato N, Lan KH, Yoshida H, Kato J, Goto T, et al. J Biol Chem 2000; 275: 34122-34130. 56. Lu W, Lo SY, Chen M, Wu Kj, Fung YK, Ou JH. Virology 1999; 264: 134-141. 57. Ray RB, Steele R, Meyer K, Ray R. J Biol Chem 1997; 272: 10983-10986. 58. Ray RB, Steele R, Meyer K, Ray R. Gene 1998; 208: 331-336. 59. Hayashi J, Aoki H, Kajino K, Moriyama M, Arakawa Y, Hino O. Hepatology 2000; 32: 958-961. 60. Aoki H, Hayashi J, Moriyama M, Arakawa Y, Hino O. J Virol 2000; 74: 1736-1741. 61. Fukuda K, Tsuchihara K, Hijikata M, Nishiguchi S, Kuroki T, Shimotohno K. Hepatology 2001; 33: 159-165. 62. Matsumoto M, Hsieh TY, Zhu N, Van Arsdale T, Hwang SB, Jeng KS, et al. J Viro11997; 71: 1301-1309. 63. Chen CM, You LR, Hwang LH, l e e YH. J Virol 1997; 71: 9417-9426. 64. You LR, Chen CM, Lee YH. J Virol 1999; 73: 1672-1681. 65. Hsieh TY, Matsumoto M, Chou HC, Schneider R, Hwang SB, Lee AS, et al. J Biol Chem 1998; 273: 17651-17659. 66. You LR, Chen CM, Yeh TS, Tsai TY, Mai RT, Lin CH, et al. J Viro11999; 73: 2841-2853. 67. Mamiya N, Worman HJ. J Biol Chem 1999; 274: 15751-15756. 68. Owsianka AM, Patel AH. Virology 1999; 257: 330-340. 69. Ray RB, Lagging LM, Meyer K, Steele R, Ray R. Virus Res 1995; 37: 209-220.
E. Tabor (editor) © 2002ElsevierScienceB.V.All rights reserved
93
Molecular biology of hepatitis C virus in hepatocellular carcinoma Shuichi Kaneko, Kenichi Kobayashi First Department of Internal Medicine, Kanazawa UniversitySchool of Medicine, Takara-machi 13-1, Kanazawa 920-8641 Japan
Abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; PCR, polymerase chain reaction; IRES, internal ribosomal entry site; REF, rat embryo fibroblasts; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated ERK kinase.
Introduction
Since the identification of hepatitis C virus (HCV) as the major causative agent of human non-A, non-B hepatitis, it has been possible to study the link between this chronic viral infection and hepatocellular carcinoma (HCC). Infection with HCV is now known to be a major risk factor for the development of HCC, associated with chronic hepatitis and usually with cirrhosis. The progression induced by chronic inflammation and successive regeneration may overcome DNA repair mechanisms. This may permit DNA mutations to induce cell transformation. Thus, persistent inflammation may be of primary importance in hepatocarcinogenesis. It is clear that hepatitis B virus (HBV)- associated chronic hepatitis and cirrhosis are also a major factor in hepatocarcinogenesis. However, HBV infection is associated with integration of HBV DNA into the host chromosome, raising the possibility that direct HBV interaction with oncogenes, growth factors, or tumor suppressor genes, perhaps accentuated through increased genomic instability, could be important mechanisms. Furthermore, the HBV X protein appears to be a potent transactivator of a wide array of promoters that normally regulate transcription of cellular and viral genes. HCV is a typical RNA virus that does not integrate into the host chromosome, and the mechanisms of carcinogenesis by HCV are poorly understood. This chapter will review the molecular biology of the hepatitis C virus in terms of virus-cell interactions that may contribute to the development of HCC. Status of HCV in HCC
Several viruses have been demonstrated to be involved in the development of human malignancies, for example, Epstein-Barr virus in some types of lymphoma and
94
I Chronic Hepatitis I I Cirrhosis I [ Hepatocellular Carcinoma J.i-, Fig. 1. Role of hepatitis C virus (HCV) in the developmentof hepatocellular carcinoma. nasopharyngeal carcinoma and human papillomavirus in cervical carcinoma. These viruses produce oncoproteins that can transform infected cells. Immunohistologic studies have revealed these oncoproteins to be detectable more frequently in tumors than in normal tissues. The presence of HCV antigens or the presence of HCV RNA in HCCs has been studied using immunohistochemistry, PCR, and in situ hybridization [1-9]. Cells containing HCV were detected more frequently in adjacent nontumorous liver tissues than in HCC tissues, although some HCC samples did contain detectable HCV. Strand-specific PCR and in situ hybridization have been used to detect the presence of replicative intermediates of HCV, showing that active replication of HCV was present in some HCC tissues. Thus, HCV can replicate and express viral proteins in HCC. HCV is an RNA virus that appears to replicate solely in the cytoplasm. Although the detection of HCV core protein in the cytoplasm supports this, several studies suggest that HCV core protein can translocate into the nucleus under certain circumstances, for instance following the experimental truncation of the hydrophobic C-terminal region [10-14]. Although it is not known whether this form exists during naturally occurring HCV infection, the observations suggest that the HCV core protein could have a regulatory role in the nucleus. HCV has been classified into at least six major genotypes on the basis of overall nucleotide sequences [15]; substantial divergence in nucleotide sequences exists between the genotypes. Several investigators have suggested that chronic infection with HCV genotype lb is associated with an increased risk of developing HCC compared with other genotypes [16-19], although other investigators have been unable to show any correlation between genotype and severity of disease including development of HCC [20,21]. Specific nucleotide sequences in HCV isolates have not been associated with HCC, although some of the nucleotide changes in genotype lb have been suggested to be associated with HCC progression [22]. It is not known whether any specific HCV genotype encodes for an oncogenic protein. Cellular proliferation and HCV In normal liver, most hepatocytes are in a resting state (stage GO), but in chronic hepatitis, cirrhosis, and HCC, hepatocytes are in a state of increased cellular
95
Growth
Cytokin~
TN
Apoptosis signals
~
,o~,,utu^,~.=v~,,,7
Cellular proliferation Transformation Fig. 2. Hepatitis C virus infection and reported mechanisms of hepatocarcinogenesis.
proliferation. The significance of hepatocellular proliferation in the development of HCC has been reported in chronic hepatitis C and cirrhosis [23-28] and in nonmalignant nodules [29,30]. The cellular proliferation is caused by inflammation directed against HCV-infected hepatocytes. The effect on HCC development of HCV infection without concomitant cellular proliferation has been studied in patients with normal aminotransferase levels after interferon therapy. Yoshida et al. reported that the risk of developing HCC was reduced to an equivalent extent by interferon therapy among 260 patients who had normal serum aminotransferase levels with persisting serum HCV RNA compared to 789 patients who eliminated serum HCV RNA after the therapy. This suggests that lowering cellular proliferation might be more important than elimination of HCV in preventing hepatocarcinogenesis. HCV may play a role in hepatocarcinogenesis through interaction with host factors during hepatocellular proliferation. Translation of the HCV polyprotein is mediated by an internal ribosomal entry site (IRES) that is located within the 5'-non-translated region of HCV RNA. The IRES activity is greatest in actively proliferating cells and relatively reduced in resting cells (evaluated using established cell lines), suggesting that host proteins in proliferating cells might interact with the HCV IRES and thus increase HCV translation [32].
96 In addition, HCV itself may influence hepatocellular proliferation through interaction with cellular factors. In one report, studies in stable cell lines that constitutively expressed HCV core protein [33] revealed that the HCV core protein contributed to increased cellular proliferation and sensitized apoptosis to serum starvation. To explain these findings, the authors examined the expression of the c-myc gene, which has been reported to induce apoptosis. In response to serum starvation, expression of the c-myc oncogene was significantly increased in the cell lines. Thus, the HCV core protein appeared to induce apoptosis and impaired regulation of the cell cycle by activating c-myc expression in the cells. Induction of cellular transformation by HCV proteins To investigate the possible oncogenic activity of the HCV proteins, cells transfected with an expression vector containing HCV cDNA have been studied. NIH3T3 cells became transformed after transfection with HCV NS3 cDNA and were tumorigenic in nude mice [34]; this suggested that proteinase activity associated with the NS3 protein might be the cause of transformation, although the precise mechanism is unknown. Ray et al. demonstrated that HCV core protein transformed rat embryo fibroblasts (REF) in cooperation with Ha-ras [35]. However, in contrast, Chang et al. [37] reported that HCV core protein in cooperation with the Ha-ras oncogene did not induce the transformation of REF cells. BALB/3T3 cells expressing HCV core protein have been reported to activate mitogen-activated protein kinase (MAPK) and the serum response element and resulted in cell transformation [36]. It has been reported that HCV core protein expression resulted in a loss of function of LZIP, a subfamily of bZIP proteins, and triggered transformation [38]. Cooperative transformation of NIH3T3 cells with the HCV NS4B gene and Ha-ras by AP1 activation has been shown [39]. Transfection of the HCV NS5A gene into NIH3T3 cells promoted anchorage-independent growth in vitro and tumor formation in nude mice [40]; the authors of this study suggested that the transformation was induced by differential modulation of the cell cycle regulatory gene p21/WAF1 and by PCNA. HCC developed in transgenic mice carrying the HCV core gene, but not in transgenic mice carrying the envelope genes under the same transcription control [41]. The HCV core protein has been shown to be present in the nuclei and mitochondria of some HCC cells. These findings support the idea that the core protein may act as a transcriptional regulator and could possibly affect the proliferative ability of cells, thereby perhaps playing a role in hepatocarcinogenesis. Modulation of apoptosis by HCV Many viruses have evolved gene products to suppress or delay apoptosis. Alterations of apoptosis by HCV infection could result in the inability to prevent the effects of harmful mutations, subsequently leading to the development of HCC. The effect of HCV core protein on TNF~-induced apoptosis has been investigated. HCV core protein suppressed apoptosis induced by cisplatin in human
97 cervical epithelial cells, by c-myc over-expression in Chinese hamster ovary cells [42], and by TNFo~ in MCF7 human breast carcinoma cells [43]. Inhibition of TNF~-induced apoptosis in Hep G2 human hepatoblastoma cells by the HCV core protein by activating nuclear factor vd3 (NF-~cB) has been reported [44,45]. DNA chip analysis has demonstrated augmented expression of the NF-lcB gene and anti-apoptotic genes in livers from patients with chronic hepatitis C [46]. There are also some contradictory reports on the effect of the HCV core protein on TNF~-induced apoptosis. Stable expression of the HCV core protein in a mouse cell line or in Hep G2 cells has been reported to sensitize them to TNF-induced apoptosis [47], suggesting that the HCV core protein can promote apoptosis via TNF signaling pathways, possibly as a result of interaction with the cytoplasmic tail of TNF receptor 1 (TNFR1). Others [48] have reported that stable MCF7 cell transfectants expressing the HCV core protein suppressed TNF-induced NF-~zB activation, and, in contrast, constitutively activated AP-1. These discrepancies might have resulted from differences in cells, in transfection systems, or in HCV cDNAs used. Similarly, discrepant reports regarding the effect of HCV core protein on Fas-mediated apoptosis have been published. It has been reported that expression of the core protein made Hep G2 cells prone to Fas-mediated apoptosis [49], and others have reported that the core protein reduced apoptosis [44]. Increased sensitivity to Fas-mediated apoptosis was shown in transgenic mice carrying HCV core and envelope cDNA [50]. The HCV NS5A protein from interferon-resistant strains of HCV can repress the action of the interferon-induced protein kinase PKR [51], suppress TNF~-induced apoptosis in a stable transfectant of Hep G2 cells [52], and can inhibit doublestranded RNA-induced apoptosis via an NS5A-mediated block in elF-2cz phosphorylation [53]. NIH3T3 cells expressing the NS3 protein were reported to be more resistant to actinomycin D-induced apoptosis than control cells [54]. Other cellular targets of HCV proteins
The tumor suppressor protein p53 induces growth arrest or apoptosis in response to a variety of stress signals, thereby eliminating damaged and potentially dangerous cells from the organism. Previous studies showed that p53 protein functionally interacts with several HCV proteins. It has been reported that HCV core protein augmented the transcriptional activity of p53 and increased expression of the p21/WAF1 protein, which is a major target of p53 [55,56]. The HCV core protein increased both the DNA-binding affinity of p53 in a mobility shift assay and the transcriptional activity of p53 itself in a reporter assay [55]. In contrast, Ray et al. [57,58] found that HCV core protein repressed transcriptional activity of the p53 promoter and the p21 promoter. The role of HCV core protein in the regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade has been studied. The MAPK/ERK cascade is involved in the signal transduction of a wide variety of extracellular stimuli, and activates nuclear transcriptional factors to induce
98 the transcription of genes required for cell cycle progression. Hayashi et al. [59] reported that HCV core protein activated the MAPK/ERK cascade including Elk1 probably at or near the gene for mitogen-activated E R K kinase (MEK)1. They also reported finding the HCV core protein bound to 14-3-3 protein and activated Raf-1 kinase, a central component of the M A P K / E R K cascade that phosphorylates MEK1 [60]. However, Fukuda et al. [61] reported that HCV core protein enhances Elkl activation without affecting E R K activity or Elkl phosphorylation. Interactions of HCV core protein with tumor necrosis factor receptor-related lymphotoxin-13 receptor [62-64], nuclear ribonucleoprotein K [65], and RNA helicase [66-68] have also been reported. Transcriptional regulation of cellular and viral promoters by the HCV core protein has also been reported [69]. References
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