Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and fibrosis in chronic hepatitis C virus infection

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Relation Between Hepatocyte G1 Arrest, Impaired Hepatic Regeneration, and Fibrosis in Chronic Hepatitis C Virus Infection AILEEN MARSHALL,* SIMON RUSHBROOK,* SUSAN E. DAVIES,‡ LESLEY S. MORRIS,§ IAN S. SCOTT,§ SARAH L. VOWLER,储 NICHOLAS COLEMAN,§ and GRAEME ALEXANDER* *Departments of Medicine and ‡Pathology, Addenbrooke’s Hospital, University of Cambridge, Cambridge; §MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge; and 储Centre for Applied Medical Statistics, Department of Public Health and Primary Care, University Forvie Site, Cambridge, England

Background & Aims: An increased risk of hepatitis C virus (HCV)-related cirrhosis is associated with hepatic steatosis, older age, and high alcohol consumption, which could be explained by synergistic effects on cell proliferation. We aimed to investigate hepatocyte cell cycle state and phase distribution in chronic HCV infection. Methods: Liver biopsy specimens diagnostic for chronic HCV (70), liver regeneration following transplant-related ischemic-reperfusion injury (15), and “normal” liver adjacent to colorectal cancer metastasis (10) were studied. Immunohistochemistry was used to detect cell cycle phase markers cyclin D1 (maximal in G1), cyclin A (S), cyclin B1 (cytoplasmic during G2) and phosphorylated histone 3 protein (mitosis), mini-chromosome maintenance protein 2 (Mcm-2; present throughout the cell cycle), and cyclindependent kinase inhibitor p21, which inhibits G1/S progression. Results: Hepatocyte Mcm-2 expression was elevated in chronic HCV and liver regeneration (13% vs 26.4%) but negligible in “normal” liver. In proportion to Mcm-2, there was no difference in cyclin D1 between chronic HCV infection and liver regeneration (51.6% of Mcm-2–positive hepatocytes vs 52.6%). In contrast, there was a striking reduction in cyclin A (3% vs 16.3%), cyclin B1 (.4% vs 2.3%), and phosphorylated histone 3 protein (0% vs 3.8%) in chronic HCV infection compared with liver regeneration. In chronic HCV infection, Mcm-2 and p21 expression were associated with fibrosis stage and positive serum HCV RNA. Conclusions: The data are consistent with hepatocyte G1 arrest in chronic HCV infection. This could impair hepatocellular function and limit hepatic regeneration.

hronic infection with hepatitis C virus (HCV) affects 170 million people worldwide. Approximately 20% of affected patients develop cirrhosis, with a significant risk of subsequent hepatocellular carcinoma.1 For reasons that are unclear, older age at infection, the degree of

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hepatic steatosis, and higher alcohol consumption are associated with an increased risk of cirrhosis.2,3 In normal liver, hepatocyte turnover is low, with more than 99% in the quiescent phase of the cell cycle. Following acute injury such as partial hepatectomy, the liver mass is replaced within 7 days by replication of mature hepatocytes. Hepatic progenitor cells may provide further regenerative support in chronic liver diseases, but their exact role remains to be defined. The regenerative potential of the liver is immense, yet the total number of hepatocyte cell divisions is finite. The liver is replaced approximately once a year under physiologic conditions. The capacity of normal liver to regenerate may be reduced by increasing age because delayed and reduced cell replication during liver regeneration has been reported in aged mice.4 It has been suggested that hepatocyte turnover is increased in chronic HCV infection, because markers of cell proliferation such as Ki675 and proliferating cell nuclear antigen6,7 are elevated. In addition, telomere shortening is reported in liver tissue derived from patients with HCV and cirrhosis.8,9 Telomeres are noncoding repetitive DNA sequences at the ends of each chromosome that shorten at each cell division. Critically short telomeres trigger replicative senescence, a state characterized by growth arrest, and are also associated with an increased risk of malignancy.10 We have shown increased hepatocyte cell cycle entry using a novel marker, mini-chromosome maintenance protein 2 (Mcm-2), in liver biopsy specimens from patients with chronic HCV infection.11 Hepatocyte Mcm-2 expression was significantly higher in serum HCV Abbreviations used in this paper: cdk, cyclin-dependent kinase; LI, labeling index; Mcm-2, mini-chromosome maintenance protein 2; PH3, phosphorylated histone 3 protein. © 2005 by the American Gastroenterological Association 0016-5085/05/$30.00 doi:10.1053/j.gastro.2004.09.076

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RNA–positive patients and was linked to fibrosis stage. Mcm proteins 2–7 are part of the prereplicative complex involved in licensing DNA for replication and are highly sensitive and specific markers of cell cycle entry because they are present throughout the cell cycle but rapidly degraded as the cell exits cycle.12 Normal progression through the cell cycle is coordinated by the sequential interaction of phase-specific cyclins and their respective cyclin-dependent kinases (cdk).13 From mid-G1 phase, cyclin D1 interacts with cdk 4 and cdk 6 to phosphorylate retinoblastoma protein, aided at the end of G1 by cyclin E/cdk2. Phosphorylated retinoblastoma protein is required for transition from G1 to S because it releases the transcription factor E2F, allowing transcription of genes required for DNA synthesis. Cyclin A/cdk 2 is active throughout S phase, and cyclin B/cdk1 mediates the transition from G2 to mitosis. Cell cycle progression can be blocked by the cdk inhibitors p21, p27, p57, and INK4 proteins (p16, p15, p18, p19). Several viruses, such as cytomegalovirus, EpsteinBarr virus, and herpes simplex virus, interact with host cell cycle control pathways.14 Viral replication is enhanced by induction of both cell cycle entry and cell cycle arrest by viral factors. A relationship between viral replication and host cell cycle state might also exist for HCV. Several HCV proteins are known to affect cell cycle when transfected into cultured cells. HCV core enhances growth,15,16 can immortalize primary human hepatocytes,17 and inhibits expression of p21.15,16 Nonstructural proteins NS3 and NS4B have been shown to promote growth.18,19 NS5A can promote20 or inhibit growth21 via up-regulation of p21.21 However, single gene transfection experiments do not account for potential interactions that may occur between viral proteins in vivo. In contrast with transfection of single genes, expression of subgenomic or full-length HCV replicons either inhibits growth or has no effect.22 Cdk inhibitors that arrest or slow cell cycle progression are also increased in chronic HCV infection. Elevated expression of p21 has been shown in liver biopsy specimens,23 and up-regulation of p57 and p16 messenger RNA has been found using complementary DNA microarrays.24 Despite elevated expression of proliferation markers in chronic HCV, mitotic activity is usually sparse or absent. Instead of chronic HCV causing increased turnover, hepatocytes expressing “proliferation markers” could have entered cell cycle but have been arrested and unable to complete cell division.

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The aim of this study was to determine whether hepatocyte cell cycle arrest might occur in chronic HCV infection. Cell cycle phase distribution was assessed by immunohistochemical detection of cell cycle phase-specific antigens in chronic HCV infection compared with liver regeneration.

Materials and Methods Liver Biopsy Specimens Archived formalin-fixed, paraffin-embedded liver biopsy specimens from 70 individuals with chronic HCV infection were studied, covering the full range of inflammatory grade and fibrosis stage. All patients were positive in serum for antibodies to HCV determined by a second- or third-generation HCV enzyme-linked immunosorbent assay. Serum HCV RNA was measured by polymerase chain reaction. All patients were negative for hepatitis B virus surface antigen; were negative for serum antimitochondrial, antinuclear, and anti–smooth muscle antibodies; had normal serum ferritin levels or absence of the HFE mutations C282Y or H63D; and had normal serum copper and ceruloplasmin levels. For a control exhibiting hepatocyte cell cycle unaffected by HCV, we used 15 liver biopsy specimens that had been taken during the regenerative phase of acute ischemicreperfusion injury following liver transplantation, when the donor liver is proliferating in response to hepatocyte loss. The liver biopsy specimens showed evidence of regeneration on the H&E-stained section. All liver transplant recipients and donors were negative in serum for antibodies to HCV and hepatitis B virus. Liver specimens from patients who had undergone partial hepatectomy for colorectal cancer metastasis were used as “normal liver” (n ⫽ 10). The specimens were tissue blocks distant to the metastasis and showed normal histologic appearance with no evidence of neoplastic tissue present in the block studied. Table 1 lists the demographic and clinical patient data for HCV and regeneration following ischemic-reperfusion injury. Liver biopsy specimens were used in accordance with local research ethics committee guidelines.

Immunohistochemistry Five-micrometer sections were cut onto polylysinecoated slides. The sections were dewaxed in xylene and taken through ethanols to water. Antigen retrieval was performed by pressure cooking for 3 minutes in .1 mol/L citrate buffer for all primary antibodies except cyclin D1. For cyclin D1, the sections were microwaved at 98°C for 30 minutes in high pH antigen retrieval solution (Dako, Ely, England). Endogenous peroxidase activity was quenched by incubating in .6% hydrogen peroxide in Tris-buffered saline for 30 minutes, and then sections were blocked with 10% goat serum. Anti–Mcm-2 was generated as reported previously.11 Cyclin D1 was obtained from Dako, cyclin A

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Table 1. Demographic and Clinical Data for Patients With Chronic HCV and Liver Regeneration Following Transplant-Related Ischemic-Reperfusion Injury Chronic HCV (n ⫽ 70) Age (y) Sex Disease

41 (11–76) 75% male All HCV

Serum HCV status

10 HCV RNA negative 51 HCV RNA positive 9 not done 9 (2–32) 38 (20–45) 85 (39–438) 83 (30–260) —

Serum bilirubin (␮mol/L) Serum albumin (mg/L) Serum alanine aminotransferase (IU/mL) Serum alkaline phosphatase (IU/mL) Posttransplant day of biopsy

Liver regeneration following ischemic-reperfusion injury (n ⫽ 15) 54 (37–63) 40% male 6 primary biliary cirrhosis 4 alcohol-related liver disease 1 autoimmune chronic liver disease 1 primary sclerosing cholangitis 1 cryptogenic cirrhosis 1 congenital hepatic fibrosis All recipients and donors negative for serum antibodies to HCV

27 (16–337) 25 (7–41) 191 (34–1022) 403 (43–1309) 11 (1–44)

NOTE. Values shown are median and range. For the liver regeneration group, “disease” refers to the indication for transplantation.

and cyclin B1 from Novocastra (Newcastle, England), and phosphorylated histone 3 protein (PH3) antibody from Upstate Biotechnology (Lake Placid, NY). Mcm-2 is expressed throughout the cell cycle but not in quiescent cells, cyclin D1 is maximal in G1, cyclin A is maximal during S, cyclin B1 is cytoplasmic during G2, and PH3 is detectable during mitosis. In addition, mouse monoclonal anti-p21 (Dako) was used. Incubation with the primary antibody was performed overnight at 4°C. The following day, biotinylated secondary antibodies were applied (goat anti-rabbit or goat anti-mouse; Dako) followed by a streptavidin/horseradish peroxidase system (Dako) with the substrate diaminobenzidine to develop the stain. For cyclin D1, the mouse envision system (Dako) was used. For negative controls, the primary antibody was omitted.

Immunofluorescence and Double-Labeling Confocal Microscopy The technique was as outlined above for immunohistochemistry. For double labeling, primary antibodies from different species were applied together. For double labeling using 2 mouse monoclonal antibodies, the primary antibodies were applied sequentially. The first primary antibody was applied and the reaction taken through to completion. The sections were then incubated with goat anti-mouse Fab (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes and the second primary applied. Secondary antibodies used were goat anti-mouse 488, goat anti-mouse 543, and goat anti-rabbit 543 (Alexa Fluor; Molecular Probes, Eugene, OR). Images were captured using a Zeiss Axioplan confocal microscope (Zeiss, Welwyn Garden City, England) at wavelengths of 488 and 543 nm.

Interpretation of Slides All chronic HCV biopsy specimens were assessed by a consultant liver histopathologist (S.E.D.) and scored ac-

cording to Ishak et al.25 Histologic activity index represented the sum of interface hepatitis (0 – 4), confluent necrosis (0 – 6), lobular inflammation (0 – 4), and portal inflammation (0 – 4). Fibrosis was scored from 0 (absent) to 6 (cirrhosis), and steatosis was scored from 0 to 3. For assessment of immunohistochemistry, positive and negative hepatocytes were counted in 4 random fields at 40⫻ magnification. Dark brown staining was considered positive. Two observers (A.M. and S.R.) counted the slides, and interobserver and intraobserver variation was ⬍10%. The number of positive hepatocytes was expressed as a percentage of the total to give a labeling index (LI). Cell cycle phase markers were expressed as a percentage of the number of hepatocytes expressing Mcm-2 for each case to produce a labeling fraction.

Statistics Differences between Mcm-2 and p21 in chronic HCV versus liver regeneration following ischemic-reperfusion injury and the fractions of putative phase markers were compared using the Mann–Whitney U test. Increases in Mcm-2 and p21 in the spectrum of fibrosis from stage 0 to 6 and in relation to histologic data were assessed using the Jonckheere–Terpestra test. For association with clinical and demographic data, continuous variables were assessed with Spearman’s rank correlation coefficient and categorical variables with 2 levels by the Mann–Whitney U test.

Results Cell Cycle Phase Markers in Liver Resected for Colorectal Cancer Metastasis Expression of Mcm-2, cyclin D1, cyclin A, cyclin B1, PH3, and p21 was negligible (⬍.01% of hepato-

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Figure 1. Immunofluorescent double labeling for cell cycle phase markers. (A) Cyclin D1 (green) and cyclin A (red) in hepatocellular carcinoma; colocalization not seen. (B) Cyclin A (green) and cytoplasmic cyclin B1 (red) in regenerating liver; colocalization seen in ⬍2% of cyclin A–positive hepatocytes. (C) Cytoplasmic cyclin B1 (green) and PH3 (red) in hepatocellular carcinoma; colocalization only when nuclear cyclin B1 was also present. (D) Cyclin A (green) and PH3 (red) in regenerating liver; colocalization in ⬍5% of cyclin A–positive cells. Arrow indicates cell positive for both cyclin A and PH3.

cytes) in 10 samples of liver resected for colorectal cancer metastasis. Double-Labeling Immunofluorescence for Cell Cycle Phase Markers All markers of cell cycle phase colocalized with Mcm-2. There was no evidence of colocalization between cyclin D1 and cyclin A (Figure 1A) or between cytoplasmic cyclin B1 and PH3 (Figure 1C). Colocalization between cyclin A and cytoplasmic cyclin B1 (Figure 1B) occurred in ⬍2% of cyclin A–positive cells and between cyclin A and PH3 (Figure 1D) occurred in ⬍5% of cyclin A–positive cells. Cell Cycle Phase Markers in Chronic HCV Infection and Postischemic-Reperfusion Injury In chronic HCV, Mcm-2 was expressed predominantly in hepatocytes, although occasional positive lym-

phocytes, sinusoidal lining cells, and bile duct cells were seen. In postischemic-reperfusion injury, positive sinusoidal lining cells and bile duct cells were seen frequently. Figure 2 depicts a representative field showing cell cycle markers for one case of HCV, and Figure 3 shows the same markers for one case with liver regeneration following transplant-related ischemic-reperfusion injury. Hepatocyte Mcm-2 LI was significantly higher in the group with regeneration following ischemic-reperfusion injury compared with HCV (26.4% vs 13%; P ⫽ .002; Figure 4A). There was no evidence of a difference in cyclin D1 labeling fraction (52.6% of Mcm-2–positive hepatocytes vs 51.6%; P ⫽ .2; Figure 4B). However, the labeling fractions for cyclin A, cyclin B1, and PH3 were all significantly higher in regeneration following ischemic-reperfusion injury compared

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Figure 2. Immunoperoxidase stain in a liver biopsy specimen from a patient with chronic hepatitis C infection using cell cycle phase-specific antibodies. (A) Mcm-2, present throughout cell cycle. (B) Cyclin D1, maximal during G1. (C) Cyclin A, maximal during S phase. (D) Cyclin B1, cytoplasmic during G2. (E) PH3, detectable during mitosis.

with HCV (cyclin A, 16.3% vs 3.0% [range, 2.5%– 36.8% vs 0%–14.8%] [Figure 4C]; cyclin B1, 2.3% vs .4% [range, .3%–10.5% vs 0%–10%] [Figure 4D]; PH3, 3.8% vs 0% [range, .3%–10.5% vs 0%–3.3%] [Figure 4E]; all P ⬍ .0001).

There was a significant association between Mcm-2 LI and fibrosis stage in chronic HCV (P ⫽ .001), as shown in Figure 5. Mcm-2 LI was also significantly higher in the liver biopsy specimens from HCV antibody–positive, HCV RNA–positive patients compared with HCV anti-

Figure 3. Immunoperoxidase stain in a liver biopsy specimen from a patient with liver regeneration following transplant-related acute ischemicreperfusion injury using cell cycle phase-specific antibodies. (A) Mcm-2, present throughout cell cycle. (B) Cyclin D1, maximal during G1. (C) Cyclin A, maximal during S phase. (D) Cyclin B1, cytoplasmic during G2 (closed arrow). Cyclin B1 translocates to the nucleus at the start of mitosis (open arrow). (E) PH3, detectable during mitosis.

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Figure 4. Hepatocyte Mcm-2 and cell cycle phase markers comparing chronic HCV and liver regeneration. (A) Percentage Mcm-2–positive hepatocytes in HCV versus regeneration (P ⫽ .002). B–E give the median of results expressed as percentage of Mcm-2–positive hepatocytes for each case, termed labeling fraction. (B) Cyclin D1 (P ⫽ .2). (C) Cyclin A (P ⬍ .0001). (D) Cyclin B1 (P ⬍ .0001). (E) PH3 (P ⬍ .0001). For these and all subsequent figures, the black bar in the middle of the box represents the median, the box stretches between the lower and upper quartile, and the whiskers extend to the range of the data or 1.5 times the box length, whichever is shorter.

body–positive, HCV RNA–negative individuals (Figure 6; median, 14% vs 7%; P ⫽ .001). There was a significant increase in median Mcm-2 LI as interface hepatitis and lobular inflammation increased (P ⫽ .0002 and P ⫽ .012, respectively). There was a borderline significant increase in Mcm-2 LI as portal

inflammation increased (P ⫽ .065). There was no evidence of an association between Mcm-2 LI and histologic activity index, confluent necrosis, hepatic steatosis, patient age, sex, estimated duration of infection, past or current alcohol use, serum bilirubin level, or alanine aminotransferase level.

Figure 5. Hepatocyte Mcm-2 expression in chronic HCV liver biopsy specimens in relation to fibrosis stage according to Ishak’s criteria (P ⫽ .001; Jonckheere–Terpestra test).

Figure 6. Hepatocyte Mcm-2 expression comparing HCV antibody– positive, HCV RNA–negative individuals with HCV antibody positive, HCV RNA–positive patients (P ⫽ .001; Mann–Whitney U test).

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Figure 7. Hepatocyte p21 expression in chronic HCV liver biopsy specimens according to fibrosis stage (P ⬍ .0001; Jonckheere– Terpestra test).

p21 Expression in Chronic HCV Infection and Postischemic-Reperfusion Injury In chronic HCV, p21 was expressed predominantly in hepatocytes, although occasional positive lymphocytes, sinusoidal lining cells, and bile duct cells were seen. Hepatocyte p21 LI was increased both in regeneration following ischemic-reperfusion injury and in chronic HCV (expressed in 9.5% of hepatocytes vs 10.4%; P ⫽ .53). The ratio of p21 to Mcm-2 was higher for chronic HCV compared with liver regeneration following ischemic-reperfusion injury (.95 vs .42; P ⫽ .002). There was a significant association between p21 LI and fibrosis stage in chronic HCV (P ⬍ .0001), as shown in Figure 7. p21 LI was also significantly higher in the liver biopsy specimens from HCV antibody–positive, HCV RNA–positive patients compared with HCV antibody–positive, HCV RNA–negative individuals (Figure 8; median, 11.6% vs 5.1%; P ⫽ .004). There was a significant increase in median p21 LI as interface hepatitis (P ⫽ .001), portal inflammation (P ⫽ .022), and steatosis increased (P ⫽ .013). There was no evidence of an association between p21 LI and histologic activity index, patient age, sex, estimated duration of infection, past or current alcohol use, bilirubin level, alanine aminotransferase level, or other histologic features.

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chronic HCV infection using an immunohistochemical method; significantly fewer hepatocytes express markers present from S phase onward in chronic HCV infection, consistent with G1 arrest. In addition, p21, a cdk inhibitor able to block progression from G1 to S phase, is elevated disproportionately in chronic HCV infection. An increased proportion of Mcm-2– and p21-positive hepatocytes was associated with advanced fibrosis and viremia. Cell cycle phase distribution can be measured by flow cytometry, but this would be difficult to interpret for liver tissue affected by chronic HCV infection given the large proportion of nonhepatocyte cell types present and the variable proportion of infected hepatocytes. Cell cycle phase analysis by immunohistochemical staining for the markers used in this study gave similar results to flow cytometry in a study of colorectal cancer, a tissue largely composed of a single cell type.26 Flow cytometry also allows distinction to be made only between pre-DNA and post-DNA replication, whereas the immunohistochemical method used here permits estimation of each individual phase. Immunofluorescent double labeling for markers of adjacent cell cycle phases in this study showed that coexpression was minimal. Increased hepatocyte expression of proliferation markers Ki67,5 proliferating cell nuclear antigen,6,7 and Mcm-211 has been shown in chronic HCV infection. One explanation for this finding could be regeneration in response to hepatocyte loss, causing increased cell turnover. However, our observation of a striking reduction in markers of late cell cycle (ie, S phase and beyond) suggests that although hepatocytes have entered cell cycle, progression to S phase is

Discussion These data show marked differences in the phase distribution of cycling hepatocytes in liver regeneration following ischemic-reperfusion injury compared with

Figure 8. Hepatocyte p21 expression comparing HCV antibody–positive, HCV RNA–negative individuals with HCV antibody– positive, HCV RNA–positive patients (P ⫽ .004; Mann–Whitney U test).

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blocked. A similar scenario is observed in vitro with herpes simplex virus, Epstein-Barr virus, and cytomegalovirus; host cell cycle entry is induced and cell cycle progression blocked by individual viral proteins. Viral replication is enhanced under these conditions and may be inhibited by inhibiting the interaction between viral proteins and host cell cycle control.14 The cellular changes that take place on entry to the cell cycle are likely to be advantageous for viral replication. In general, the host cell cycle machinery can be used and factors required for RNA or DNA replication are more plentiful. Specifically for HCV, the activity of the HCV internal ribosomal entry site, which mediates translation of the HCV polyprotein, has been shown to vary between cell cycle phases in vitro; separate studies have shown up-regulation of HCV internal ribosomal entry site activity during mitosis and G1/S, respectively.27,28 The difference between these 2 studies could be due to the different cell culture systems used. Evidence for variation in HCV replication according to cell cycle state is shown by the HCV replicon, whereby cells are transfected stably with part or all of the HCV genome. It has been reported in this model that proliferating cells carried the highest level of HCV RNA and produced more viral proteins, whereas there was a sharp decline as cells entered the resting phase.22 Induction of hepatocyte cell cycle entry in chronic HCV infection could be caused directly by HCV; transfection of individual HCV proteins (core,15,17 NS3,18 NS4B19) promoted host cell growth in vitro and HCV core alone immortalized primary human hepatocytes.17 p21 and other cdk inhibitors such as p16 and p57 that are increased in human chronic HCV infection may mediate inhibition of cell cycle progression.23,24 p21mediated cell cycle arrest might also be a direct viral effect because NS5A can up-regulate p21 in vitro.21 Alternatively, p21-mediated cell cycle arrest could be part of the host response to viral infection; p21 transcription is regulated by p53, and expression is induced under conditions of oxidative stress and DNA damage and is up-regulated by interferon gamma and transforming growth factor ␤. In this study, there was a significant association between the number of hepatocytes expressing p21 and the degree of steatosis. p21 may be involved in fatty liver disease; p53-dependent induction of p21 occurred in mouse models of fatty liver (leptin-deficient ob/ob and a transgenic mouse model that overexpresses an active form of the key transcriptional regulator of lipogenesis, sterol regulatory element-binding protein 1, in the liver)29; in ob/ob mice with fatty livers, p21 was increased following partial hepatectomy and liver regener-

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ation impaired.30 Similarly, mice exposed to ethanol show increased p21 and impaired liver regeneration following partial hepatectomy.31 Up-regulation of p21 may therefore be a common mechanism that explains the adverse impact of alcohol and steatosis in patients with HCV. The effect of slowed hepatocyte cell cycle or prolonged cell cycle arrest on the liver is not known. Potentially, cells could be more sensitive to apoptotic stimuli or enter a senescence-like state. Cellular senescence is characterized by lack of proliferation in response to external mitogens, altered morphology, expression of p21 or p16, and increased activity of the lysosomal form of ␤-galactosidase, detectable at pH 6.32 Senescence occurs in vitro when cells have undergone a certain number of cell divisions (termed replicative senescence), and it is assumed that a similar replicative limit applies to somatic cells in vivo. Senescence can be precipitated prematurely by other factors, such as oxidative stress. Cytomegalovirus was reported to induce cell cycle arrest and cause a premature senescence-like state in human fibroblasts, as determined by the presence of senescence-associated ␤-galactosidase activity and overexpression of plasminogen activator inhibitor type 1 gene.33 Indeed, hepatocytes showing senescence-associated ␤-galactosidase activity have been detected in chronic hepatitis due to HCV,34 and hepatocytes with senescence-associated ␤-galactosidase activity and reduction in telomere length were reported in cirrhosis caused by viral hepatitis, autoimmune hepatitis, alcohol, primary biliary cirrhosis, and primary sclerosing cholangitis.35 There are several potential consequences of cell cycle arrest and senescence for the liver. Firstly, impaired liver regeneration would limit the response to additional insults such as alcohol, steatosis, or acute hepatitis A virus infection. Secondly, cell cycle entry is associated with changes in metabolic function, including decreased glycolysis and increased gluconeogenesis36 and switching from expression of the apical biliary transport protein MRP2 to basolaterally located MRP1.37 This change could result in transport of bile constituents back into the bloodstream instead of excretion into bile. Accumulation of cells in cycle could therefore cause a gradual loss of hepatocellular function. Thirdly, p21 expression induces up-regulation of transcription of profibrotic factors such as connective tissue growth factor and fibronectin 1.38 This could be a mechanism linking hepatocyte infection to hepatic stellate cell activation and fibrosis development in chronic HCV. Finally, cellular senescence is a risk factor for cancer development. Senescent fibroblasts promote proliferation of epithelial cells in coculture39; thus, senescent hepato-

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cytes might act synergistically with oncogenic mutations in neighboring hepatocytes, leading to the development of hepatocellular carcinoma. In conclusion, this study has shown that hepatocyte cell cycle phase distribution is altered in chronic HCV infection compared with liver regeneration following ischemic-reperfusion injury consistent with G1/S cell cycle arrest. p21 was elevated and may mediate hepatocyte cell cycle arrest. Accumulation of growth-arrested hepatocytes could impair hepatocellular function and limit hepatic regeneration.

References 1. Seeff LB. Natural history of chronic hepatitis C. Hepatology 2002;36(Suppl 1):S35–S46. 2. Adinolfi LE, Gambardella M, Andreana A, Tripodi MF, Utili R, Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity. Hepatology 2001;33:1358–1364. 3. Poynard T, Ratziu V, Charlotte F, Goodman Z, McHutchison J, Albrecht J. Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis C. J Hepatol 2001;34:730 –739. 4. Fry M, Silber J, Loeb LA, Martin GM. Delayed and reduced cell replication and diminishing levels of DNA polymerase-alpha in regenerating liver of aging mice. J Cell Physiol 1984;118:225–232. 5. Farinati F, Cardin R, D’Errico A, De Maria N, Naccarato R, Cecchetto A, Grigioni W. Hepatocyte proliferative activity in chronic liver damage as assessed by the monoclonal antibody MIB1 Ki67 in archival material: the role of etiology, disease activity, iron, and lipid peroxidation. Hepatology 1996;23:1468 –1475. 6. Lake-Bakaar G, Mazzoccoli V, Ruffini L. Digital image analysis of the distribution of proliferating cell nuclear antigen in hepatitis C virus-related chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Dig Dis Sci 2002;47:1644 –1648. 7. Donato MF, Arosio E, Del Ninno E, Ronchi G, Lampertico P, Morabito A, Balestrieri MR, Colombo M. High rates of hepatocellular carcinoma in cirrhotic patients with high liver cell proliferative activity. Hepatology 2001;34:523–528. 8. Miura N, Horikawa I, Nishimoto A, Ohmura H, Ito H, Hirohashi S, Shay JW, Oshimura M. Progressive telomere shortening and telomerase reactivation during hepatocellular carcinogenesis. Cancer Genet Cytogenet 1997;93:56 – 62. 9. Ohashi K, Tsutsumi M, Nakajima Y, Kobitsu K, Nakano H, Konishi Y. Telomere changes in human hepatocellular carcinomas and hepatitis virus infected noncancerous livers. Cancer 1996;77(Suppl 8): 1747–1751. 10. Wu X, Amos CI, Zhu Y, Zhao H, Grossman BH, Shay JW, Luo S, Hong WK, Spitz MR. Telomere dysfunction: a potential cancer predisposition factor. J Natl Cancer Inst 2003;95:1211–1218. 11. Freeman A, Hamid S, Morris L, Vowler S, Rushbrook S, Wight DG, Coleman N, Alexander GJ. Improved detection of hepatocyte proliferation using antibody to the pre-replication complex: an association with hepatic fibrosis and viral replication in chronic hepatitis C virus infection. J Viral Hepat 2003;10:345–350. 12. Musahl C, Holthoff HP, Lesch R, Knippers R. Stability of the replicative Mcm3 protein in proliferating and differentiating human cells. Exp Cell Res 1998;241:260 –264. 13. Sherr CJ. Cancer cell cycles. Science 1996;274:1672–1677. 14. Flemington EK. Herpesvirus lytic replication and the cell cycle: arresting new developments. J Virol 2001;75:4475– 4481. 15. Ray RB, Steele R, Meyer K, Ray R. Hepatitis C virus core protein represses p21WAF1/Cip1/Sid1 promoter activity. Gene 1998; 208:331–336.

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Received April 6, 2004. Accepted September 16, 2004. Address requests for reprints to: Graeme Alexander, MD, Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, Box 157, Hills Road, Cambridge CB2 2QQ England. e-mail: [email protected]; fax: (44) 1223-216111. A.M. is supported by the National Blood Service and the Raymond & Beverley Sackler fund, and N.C. is supported by the Medical Research Council and Cancer Research UK.