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Compensatory apoptosis in preneoplastic liver of a transgenic mouse model for viral hepatocarcinogenesis
Cancer Letters 134 (1998) 181±186
Compensatory apoptosis in preneoplastic liver of a transgenic mouse model for viral hepatocarcinogenesis Kazuhiko Koike*, Kyoji Moriya, Hiroshi Yotsuyanagi, Yoshizumi Shintani, Hajime Fujie, Takeya Tsutsumi, Satoshi Kimura First Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Received 6 July 1998; received in revised form 14 August 1998; accepted 14 August 1998
Abstract The HBx protein of hepatitis B virus has been shown to induce hepatocellular carcinoma in transgenic mice as direct evidence for its involvement in hepatocarcinogenesis. In these transgenic mice, however, it is not clear why hepatocytes do not acquire a neoplastic phenotype by 13 months old despite the continuous growth stimulation by the HBx protein from 2 months old. In this study, we show that the accelerated proliferation of hepatocytes is counterbalanced by apoptosis, which maintains liver homeostasis. A decrease in the extent of apoptosis seems to precede the emergence of neoplasia in the transgenic mouse liver. The disappearance or block of apoptotic signals, which may be the result of additional genetic or epigenetic aberrations, may result in the preneoplastic hepatocytes becoming neoplastic. q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hepatitis B virus; HBx gene; Apoptosis; Hepatocellular carcinoma
1. Introduction Liver homeostasis is maintained by the tight regulation of the balance between the proliferation and apoptosis of hepatocytes [1,2]. Events occurring in the liver during the course of carcinogenesis are thought to be characterized by such rationale, by which the overgrowth of preneoplastic hepatocytes is counterbalanced by apoptosis [1,3±5]. However, little is known about the process underlying the hepatocarcinogenesis associated with hepatitis viral infection, chie¯y due to the limited number of appropriate animal models.
* Corresponding author. Tel.: 181-3-38155411, ext. 3004; fax: 181-3-58008807; e-mail: [email protected].
The mechanism of hepatocarcinogenesis associated with chronic hepatitis B virus (HBV) infection is considered to be accomplished through a multistep process. Among several factors thought to be involved in this process, the HBx gene of the HBV is suggested to have an essential role in the development of hepatocellular carcinoma (HCC); the HBx gene has a transactivating function [6±8], induces HCC in transgenic mice [9,10] and has been shown to be expressed in human HCC samples [11±14]. We previously described, as an animal model for hepatocarcinogenesis occurring in association with hepatitis virus, transgenic mice carrying the HBx gene which develop HCC in the latter half of their life span [9,10,15]. In this mouse model, hepatocytes expressing the HBx protein, which has also been shown to induce cell proliferation in vitro [16,17],
0304-3835/98/$ - see front matter q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0 304-3835(98)002 52-3
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exhibit increased DNA synthesis in their nuclei in mice from 2 months old but the mice do not develop HCC until 13 months old [10,15]. In the present study, we investigated the consequences of the proliferative effect of the HBx protein in the liver. Putative preneoplastic hepatocytes in the HBx transgenic mice succumbed to apoptosis presumably to balance the proliferative effect of the HBx protein.
2. Materials and methods 2.1. Transgenic mice The production of HBx gene transgenic mice has been previously reported [9]. In the transgenic mice in this study, the HBx gene of the HBV is under the control of its own transcriptional control region. Three male mice from the H9 line [10] and three control CD-1 male mice were used at each age when the following analyses were conducted. Tissue sections ®xed in 10% neutral-buffered formalin were used for hematoxylin and eosin staining. 2.2. BrdU and TUNEL assays For bromo-deoxyuridine (BrdU) incorporation studies, mice at various ages were administered BrdU intraperitoneally and killed 2 h later. Liver tissues were ®xed in neutralized formalin for 16 h and stained with anti-BrdU antibody (Amersham International, Buckinghamshire, UK). Age-matched normal CD-1 male mice were analyzed in exactly the same way as control mice. We applied the terminal deoxyribonucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay [18] for the detection of apoptosis using a TACS2 TdT in vitro apoptosis detection kit (Trevigen, Gaithersburg, MD). Paraf®n-embedded sections of the liver were subjected to the TUNEL assay according to the manufacturer's protocol. Western and Northern blotting were carried out as described previously [10]. 2.3. Statistical analysis Student's t-test was used for statistical analysis.
3. Results and discussion Bromo-deoxyuridine injected intraperitoneally into the transgenic mice was incorporated into the nuclei of pericentral hepatocytes, which expressed high levels of the HBx protein [9,10], indicating that these hepatocytes had a proliferating phenotype (Fig. 1A). In addition, DNA ploidy analysis revealed small aneuploid peaks of nuclear DNA in the liver of the transgenic mice from the age of 7 months, suggesting that these hepatocytes are preneoplastic lesions [10]. In contrast, the nuclei of pericentral hepatocytes of normal CD-1 mice showed no uptake of BrdU. Table 1 shows the time course of BrdU labeling indices in the livers of HBx gene transgenic mice. From 2 months old, labeling indices were signi®cantly higher in the livers of transgenic mice than those in the livers of normal control mice of the same age (P , 0:05). This indicates that the HBx protein has the ability to promote mitosis or cell proliferation in vivo. The mitogenic effect of HBx protein has been con®rmed by in vitro experiments, i.e. hormone-regulated expression of the HBx protein led to the interruption of G1 arrest and subsequently to cell division [16]. Since DNA synthesis is increased from 2 months old in the liver of the HBx gene transgenic mice, it is dif®cult to explain why HCC does not develop until the mice are 13 months old, despite the continuous presence of a cell proliferation signal in the liver. Cell death occurring concurrently with cell proliferation may be a possible explanation for this phenomenon. Therefore, in the present study, we evaluated histologic ®ndings in the liver and in particular the morphology of the hepatocyte nuclei. We also used the TUNEL assay for the detection of cell death in the preneoplastic phase. Upon microscopic examination of liver tissues from the transgenic mice, we found a number of hepatocytes with bizarre nuclei in which the chromatin was condensed at the periphery of the nuclei (Fig. 1B). Such nuclei were scattered throughout the preneoplastic liver. Also observed was a prominent increase in the number of TUNEL-positive cells in samples of preneoplastic liver from the HBx gene transgenic mice compared to in those from non-transgenic littermates. Importantly, the nuclei exhibiting dense chromatin condensation turned out to be TUNEL-positive (Fig. 1C). Before the age of 8 months, the frequency of TUNEL-positive hepato-
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Fig. 1. (A) Increased DNA synthesis in the nuclei of preneoplastic stage hepatocytes of HBx gene transgenic mice. Bromo-deoxyuridine that was injected intraperitoneally was incorporated into the nuclei of hepatocytes chie¯y located around the central veins in a 6-month-old male H9 transgenic mouse. Counterstained by eosin. Magni®cation £200. (B±D) Apoptosis in the preneoplastic liver of the HBx gene transgenic mice. (B) Non-typical apoptotic bodies, but rather hepatocytes with bizarre nuclei in which chromatin was condensed at the periphery (arrows) were observed in the preneoplastic liver of the HBx gene in an 8-month-old male H9 transgenic mouse. Hematoxylin±eosin staining. Magni®cation £300. (C) Nuclei with peripherally condensed chromatin were TUNEL-positive in an 8-month-old male H9 transgenic mouse, indicating that these cells undergo apoptosis. Counterstained by hematoxylin. Magni®cation £300. (D) TUNEL-positive nuclei were absent in the liver tumor (left), while positive cells were frequently observed in adjacent normal liver. A high-magni®cation of a TUNEL-positive cell is shown in the inset (magni®cation £450). The results are from a 16-month-old male H9 transgenic mouse. Counterstained by hematoxylin. Magni®cation £80.
cytes was nearly 40 times higher in the HBx gene transgenic mice than in non-transgenic littermates (Fig. 2) (P , 0:01, compared with age-matched normal control). In contrast, the number of TUNELpositive cells decreased with mouse age after mice were 12 months old (Fig. 2) and was very low in adenoma or HCC (Fig. 1D). These results indicate that the cell proliferation induced by HBx protein is compensated for by the apoptosis of hepatocytes in the preneoplastic stage in the HBx gene transgenic mice. Nonetheless, there remains a possibility that apoptosis itself might also be induced by HBx protein. Indeed, in vitro induction of apoptosis by the HBx
protein has been reported recently [19]. Maintenance of an equilibrium between hepatocyte proliferation and apoptosis would prevent inappropriate cell expansion. The absence of such an equilibrium might result in unrestrained hepatocyte population growth. Analysis of the HBx gene transgenic mice in which HCC developed after they were 13 months old revealed that (i) HBx protein expression, which induces hepatocyte proliferation in vitro [16], induced cell proliferation in vivo [10], (ii) hepatocyte apoptosis occurred at a high incidence during the preneoplastic phase concomitant with a high mitotic rate and (iii) the apoptotic rate was markedly decreased in the liver
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Table 1 Labeling index of hepatocytes in HBx gene transgenic mice and non-transgenic mice a Age (months) 2 4 6 8 10 12 16 18
HBx transgenic mice 0.88 ^ 0.21 1.56 ^ 0.53 2.11 ^ 0.45 2.21 ^ 0.55 1.98 ^ 0.44 2.42 ^ 0.48 2.11 ^ 0.34 1.56 ^ 0.29
b
Non-transgenic mice 0.33 ^ 0.10 0.11 ^ 0.08 0.10 ^ 0.08 0.12 ^ 0.10 0.11 ^ 0.08 0.10 ^ 0.08 0.12 ^ 0.08 0.10 ^ 0.05
a
The labeling index indicates the number of labeled nuclei per 100 nuclei. Each index was calculated by counting over 100 000 hepatocytes for three independent mice. b Values given are the mean ^ SE.
after mice were 12 months old and in the hepatic tumors. Although we have not identi®ed the pathway via which apoptosis was switched on, the high rate of mitosis in the transgenic mouse liver was apparently compensated for by apoptosis. There are two possible explanations for the occurrence of this transient apoptosis. First, this mechanism might involve p53 protein
Fig. 2. Incidence of TUNEL-positive cells in the liver of transgenic mice. TUNEL-positive cells per approximately 20 000 hepatocytes were calculated at each age for transgenic mice (three mice for each age) or normal control mice. The incidence of TUNEL-positive cells was signi®cantly higher in transgenic mice from 3 to 12 months old than in control mice and declined after the mice were 12 months old. Horizontal bars indicate SE.
which is known to be responsible for one apoptotic pathway. The association of the HBx protein and cellular p53 protein has been reported both in in vitro and in vivo studies [20±22]. According to Ueda et al. [20], by binding with the HBx protein, p53 protein is translocated to the cytoplasm from the nuclei. Thereby, the p53 protein, which normally arrests cell cycle progression in the G1 phase, becomes unable to carry out its function as it is located in the wrong place. If this is the case, p53 cannot induce apoptosis in HBx protein-expressing hepatocytes. Alternatively, the association of p53 with the HBx protein might enhance the function of p53 in causing apoptosis. However, since apoptosis of hepatocytes occurs irrespective of the location in the liver, while the HBx protein expression is con®ned to the pericentral area, it is not likely that the binding of p53 with the HBx protein accounts for apoptosis in the transgenic mouse liver. Second, Fas protein, which is shown to mediate apoptosis in a variety of cell types [23], may also be involved in apoptosis in this transgenic mouse system. Although our preliminary study by Northern blotting shows that the expression levels of the Fas gene are not increased in the HBx transgenic mice when compared with those in normal mice (data not shown), the involvement of the Fas-FasL system cannot be excluded. We also searched for the factors which suppress apoptosis before the development of HCC in our transgenic mice. However, no change was observed in the levels of Bcl-2 or IGF-II in the course of hepatocarcinogenesis (K. Koike et al., unpublished data). In this model for hepatocarcinogenesis, the role of the HBx protein may be to induce mitosis, which results in the development of preneoplastic liver lesions. The occurrence of another event(s) may be necessary for the progression from preneoplastic lesion to carcinoma. This is consistent with the ®nding in transgenic mice carrying known oncogenes such as SV40 T antigen, c-myc or Ha-ras, in which these oncogenes do not cause single-step carcinogenesis in transgenic mice, but induce cell proliferation which leads to hyperplasia or dysplasia [24±27]. Events which confer additional properties onto preneoplastic cells, including the ability to escape the immune system or to recruit feeding vessels, may be necessary for malignant tumor formation in vivo. Interruption of apoptosis may be included as one
K. Koike et al. / Cancer Letters 134 (1998) 181±186
such secondary event in the development of carcinogenesis.
Acknowledgements We thank Ms C. Ariga for her technical assistance. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan and by a grant from the Sankyo Foundation of Life Science.
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Compensatory apoptosis in preneoplastic liver of a transgenic mouse model for viral hepatocarcinogenesis Kazuhiko Koike*, Kyoji Moriya, Hiroshi Yotsuyanagi, Yoshizumi Shintani, Hajime Fujie, Takeya Tsutsumi, Satoshi Kimura First Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Received 6 July 1998; received in revised form 14 August 1998; accepted 14 August 1998
Abstract The HBx protein of hepatitis B virus has been shown to induce hepatocellular carcinoma in transgenic mice as direct evidence for its involvement in hepatocarcinogenesis. In these transgenic mice, however, it is not clear why hepatocytes do not acquire a neoplastic phenotype by 13 months old despite the continuous growth stimulation by the HBx protein from 2 months old. In this study, we show that the accelerated proliferation of hepatocytes is counterbalanced by apoptosis, which maintains liver homeostasis. A decrease in the extent of apoptosis seems to precede the emergence of neoplasia in the transgenic mouse liver. The disappearance or block of apoptotic signals, which may be the result of additional genetic or epigenetic aberrations, may result in the preneoplastic hepatocytes becoming neoplastic. q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hepatitis B virus; HBx gene; Apoptosis; Hepatocellular carcinoma
1. Introduction Liver homeostasis is maintained by the tight regulation of the balance between the proliferation and apoptosis of hepatocytes [1,2]. Events occurring in the liver during the course of carcinogenesis are thought to be characterized by such rationale, by which the overgrowth of preneoplastic hepatocytes is counterbalanced by apoptosis [1,3±5]. However, little is known about the process underlying the hepatocarcinogenesis associated with hepatitis viral infection, chie¯y due to the limited number of appropriate animal models.
* Corresponding author. Tel.: 181-3-38155411, ext. 3004; fax: 181-3-58008807; e-mail: [email protected].
The mechanism of hepatocarcinogenesis associated with chronic hepatitis B virus (HBV) infection is considered to be accomplished through a multistep process. Among several factors thought to be involved in this process, the HBx gene of the HBV is suggested to have an essential role in the development of hepatocellular carcinoma (HCC); the HBx gene has a transactivating function [6±8], induces HCC in transgenic mice [9,10] and has been shown to be expressed in human HCC samples [11±14]. We previously described, as an animal model for hepatocarcinogenesis occurring in association with hepatitis virus, transgenic mice carrying the HBx gene which develop HCC in the latter half of their life span [9,10,15]. In this mouse model, hepatocytes expressing the HBx protein, which has also been shown to induce cell proliferation in vitro [16,17],
0304-3835/98/$ - see front matter q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0 304-3835(98)002 52-3
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K. Koike et al. / Cancer Letters 134 (1998) 181±186
exhibit increased DNA synthesis in their nuclei in mice from 2 months old but the mice do not develop HCC until 13 months old [10,15]. In the present study, we investigated the consequences of the proliferative effect of the HBx protein in the liver. Putative preneoplastic hepatocytes in the HBx transgenic mice succumbed to apoptosis presumably to balance the proliferative effect of the HBx protein.
2. Materials and methods 2.1. Transgenic mice The production of HBx gene transgenic mice has been previously reported [9]. In the transgenic mice in this study, the HBx gene of the HBV is under the control of its own transcriptional control region. Three male mice from the H9 line [10] and three control CD-1 male mice were used at each age when the following analyses were conducted. Tissue sections ®xed in 10% neutral-buffered formalin were used for hematoxylin and eosin staining. 2.2. BrdU and TUNEL assays For bromo-deoxyuridine (BrdU) incorporation studies, mice at various ages were administered BrdU intraperitoneally and killed 2 h later. Liver tissues were ®xed in neutralized formalin for 16 h and stained with anti-BrdU antibody (Amersham International, Buckinghamshire, UK). Age-matched normal CD-1 male mice were analyzed in exactly the same way as control mice. We applied the terminal deoxyribonucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay [18] for the detection of apoptosis using a TACS2 TdT in vitro apoptosis detection kit (Trevigen, Gaithersburg, MD). Paraf®n-embedded sections of the liver were subjected to the TUNEL assay according to the manufacturer's protocol. Western and Northern blotting were carried out as described previously [10]. 2.3. Statistical analysis Student's t-test was used for statistical analysis.
3. Results and discussion Bromo-deoxyuridine injected intraperitoneally into the transgenic mice was incorporated into the nuclei of pericentral hepatocytes, which expressed high levels of the HBx protein [9,10], indicating that these hepatocytes had a proliferating phenotype (Fig. 1A). In addition, DNA ploidy analysis revealed small aneuploid peaks of nuclear DNA in the liver of the transgenic mice from the age of 7 months, suggesting that these hepatocytes are preneoplastic lesions [10]. In contrast, the nuclei of pericentral hepatocytes of normal CD-1 mice showed no uptake of BrdU. Table 1 shows the time course of BrdU labeling indices in the livers of HBx gene transgenic mice. From 2 months old, labeling indices were signi®cantly higher in the livers of transgenic mice than those in the livers of normal control mice of the same age (P , 0:05). This indicates that the HBx protein has the ability to promote mitosis or cell proliferation in vivo. The mitogenic effect of HBx protein has been con®rmed by in vitro experiments, i.e. hormone-regulated expression of the HBx protein led to the interruption of G1 arrest and subsequently to cell division [16]. Since DNA synthesis is increased from 2 months old in the liver of the HBx gene transgenic mice, it is dif®cult to explain why HCC does not develop until the mice are 13 months old, despite the continuous presence of a cell proliferation signal in the liver. Cell death occurring concurrently with cell proliferation may be a possible explanation for this phenomenon. Therefore, in the present study, we evaluated histologic ®ndings in the liver and in particular the morphology of the hepatocyte nuclei. We also used the TUNEL assay for the detection of cell death in the preneoplastic phase. Upon microscopic examination of liver tissues from the transgenic mice, we found a number of hepatocytes with bizarre nuclei in which the chromatin was condensed at the periphery of the nuclei (Fig. 1B). Such nuclei were scattered throughout the preneoplastic liver. Also observed was a prominent increase in the number of TUNEL-positive cells in samples of preneoplastic liver from the HBx gene transgenic mice compared to in those from non-transgenic littermates. Importantly, the nuclei exhibiting dense chromatin condensation turned out to be TUNEL-positive (Fig. 1C). Before the age of 8 months, the frequency of TUNEL-positive hepato-
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Fig. 1. (A) Increased DNA synthesis in the nuclei of preneoplastic stage hepatocytes of HBx gene transgenic mice. Bromo-deoxyuridine that was injected intraperitoneally was incorporated into the nuclei of hepatocytes chie¯y located around the central veins in a 6-month-old male H9 transgenic mouse. Counterstained by eosin. Magni®cation £200. (B±D) Apoptosis in the preneoplastic liver of the HBx gene transgenic mice. (B) Non-typical apoptotic bodies, but rather hepatocytes with bizarre nuclei in which chromatin was condensed at the periphery (arrows) were observed in the preneoplastic liver of the HBx gene in an 8-month-old male H9 transgenic mouse. Hematoxylin±eosin staining. Magni®cation £300. (C) Nuclei with peripherally condensed chromatin were TUNEL-positive in an 8-month-old male H9 transgenic mouse, indicating that these cells undergo apoptosis. Counterstained by hematoxylin. Magni®cation £300. (D) TUNEL-positive nuclei were absent in the liver tumor (left), while positive cells were frequently observed in adjacent normal liver. A high-magni®cation of a TUNEL-positive cell is shown in the inset (magni®cation £450). The results are from a 16-month-old male H9 transgenic mouse. Counterstained by hematoxylin. Magni®cation £80.
cytes was nearly 40 times higher in the HBx gene transgenic mice than in non-transgenic littermates (Fig. 2) (P , 0:01, compared with age-matched normal control). In contrast, the number of TUNELpositive cells decreased with mouse age after mice were 12 months old (Fig. 2) and was very low in adenoma or HCC (Fig. 1D). These results indicate that the cell proliferation induced by HBx protein is compensated for by the apoptosis of hepatocytes in the preneoplastic stage in the HBx gene transgenic mice. Nonetheless, there remains a possibility that apoptosis itself might also be induced by HBx protein. Indeed, in vitro induction of apoptosis by the HBx
protein has been reported recently [19]. Maintenance of an equilibrium between hepatocyte proliferation and apoptosis would prevent inappropriate cell expansion. The absence of such an equilibrium might result in unrestrained hepatocyte population growth. Analysis of the HBx gene transgenic mice in which HCC developed after they were 13 months old revealed that (i) HBx protein expression, which induces hepatocyte proliferation in vitro [16], induced cell proliferation in vivo [10], (ii) hepatocyte apoptosis occurred at a high incidence during the preneoplastic phase concomitant with a high mitotic rate and (iii) the apoptotic rate was markedly decreased in the liver
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Table 1 Labeling index of hepatocytes in HBx gene transgenic mice and non-transgenic mice a Age (months) 2 4 6 8 10 12 16 18
HBx transgenic mice 0.88 ^ 0.21 1.56 ^ 0.53 2.11 ^ 0.45 2.21 ^ 0.55 1.98 ^ 0.44 2.42 ^ 0.48 2.11 ^ 0.34 1.56 ^ 0.29
b
Non-transgenic mice 0.33 ^ 0.10 0.11 ^ 0.08 0.10 ^ 0.08 0.12 ^ 0.10 0.11 ^ 0.08 0.10 ^ 0.08 0.12 ^ 0.08 0.10 ^ 0.05
a
The labeling index indicates the number of labeled nuclei per 100 nuclei. Each index was calculated by counting over 100 000 hepatocytes for three independent mice. b Values given are the mean ^ SE.
after mice were 12 months old and in the hepatic tumors. Although we have not identi®ed the pathway via which apoptosis was switched on, the high rate of mitosis in the transgenic mouse liver was apparently compensated for by apoptosis. There are two possible explanations for the occurrence of this transient apoptosis. First, this mechanism might involve p53 protein
Fig. 2. Incidence of TUNEL-positive cells in the liver of transgenic mice. TUNEL-positive cells per approximately 20 000 hepatocytes were calculated at each age for transgenic mice (three mice for each age) or normal control mice. The incidence of TUNEL-positive cells was signi®cantly higher in transgenic mice from 3 to 12 months old than in control mice and declined after the mice were 12 months old. Horizontal bars indicate SE.
which is known to be responsible for one apoptotic pathway. The association of the HBx protein and cellular p53 protein has been reported both in in vitro and in vivo studies [20±22]. According to Ueda et al. [20], by binding with the HBx protein, p53 protein is translocated to the cytoplasm from the nuclei. Thereby, the p53 protein, which normally arrests cell cycle progression in the G1 phase, becomes unable to carry out its function as it is located in the wrong place. If this is the case, p53 cannot induce apoptosis in HBx protein-expressing hepatocytes. Alternatively, the association of p53 with the HBx protein might enhance the function of p53 in causing apoptosis. However, since apoptosis of hepatocytes occurs irrespective of the location in the liver, while the HBx protein expression is con®ned to the pericentral area, it is not likely that the binding of p53 with the HBx protein accounts for apoptosis in the transgenic mouse liver. Second, Fas protein, which is shown to mediate apoptosis in a variety of cell types [23], may also be involved in apoptosis in this transgenic mouse system. Although our preliminary study by Northern blotting shows that the expression levels of the Fas gene are not increased in the HBx transgenic mice when compared with those in normal mice (data not shown), the involvement of the Fas-FasL system cannot be excluded. We also searched for the factors which suppress apoptosis before the development of HCC in our transgenic mice. However, no change was observed in the levels of Bcl-2 or IGF-II in the course of hepatocarcinogenesis (K. Koike et al., unpublished data). In this model for hepatocarcinogenesis, the role of the HBx protein may be to induce mitosis, which results in the development of preneoplastic liver lesions. The occurrence of another event(s) may be necessary for the progression from preneoplastic lesion to carcinoma. This is consistent with the ®nding in transgenic mice carrying known oncogenes such as SV40 T antigen, c-myc or Ha-ras, in which these oncogenes do not cause single-step carcinogenesis in transgenic mice, but induce cell proliferation which leads to hyperplasia or dysplasia [24±27]. Events which confer additional properties onto preneoplastic cells, including the ability to escape the immune system or to recruit feeding vessels, may be necessary for malignant tumor formation in vivo. Interruption of apoptosis may be included as one
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such secondary event in the development of carcinogenesis.
Acknowledgements We thank Ms C. Ariga for her technical assistance. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan and by a grant from the Sankyo Foundation of Life Science.
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