Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and fibrosis in chronic hepatitis C virus infection
2005; Elsevier BV; Volume: 128; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2004.09.076
ISSN1528-0012
AutoresAileen Marshall, Simon Rushbrook, Susan E. Davies, Lesley S. Morris, Ian Scott, Sarah L. Vowler, Nicholas Coleman, Graeme Alexander,
Tópico(s)Hepatitis B Virus Studies
ResumoBackgrounds & 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 cyclin-dependent 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. Backgrounds & 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 cyclin-dependent 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. Chronic 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.1Seeff L.B. Natural history of chronic hepatitis C.Hepatology. 2002; 36: S35-S46Crossref PubMed Google Scholar For reasons that are unclear, older age at infection, the degree of hepatic steatosis, and higher alcohol consumption are associated with an increased risk of cirrhosis.2Adinolfi L.E. Gambardella M. Andreana A. Tripodi M.F. 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: 1358-1364Crossref PubMed Scopus (977) Google Scholar, 3Poynard 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: 730-739Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar 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.4Fry M. Silber J. Loeb L.A. Martin G.M. Delayed and reduced cell replication and diminishing levels of DNA polymerase-alpha in regenerating liver of aging mice.J Cell Physiol. 1984; 118: 225-232Crossref PubMed Scopus (67) Google Scholar It has been suggested that hepatocyte turnover is increased in chronic HCV infection, because markers of cell proliferation such as Ki675Farinati 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: 1468-1475Crossref PubMed Google Scholar and proliferating cell nuclear antigen6Lake-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: 1644-1648Crossref PubMed Scopus (15) Google Scholar, 7Donato M.F. Arosio E. Del Ninno E. Ronchi G. Lampertico P. Morabito A. Balestrieri M.R. Colombo M. High rates of hepatocellular carcinoma in cirrhotic patients with high liver cell proliferative activity.Hepatology. 2001; 34: 523-528Crossref PubMed Scopus (104) Google Scholar are elevated. In addition, telomere shortening is reported in liver tissue derived from patients with HCV and cirrhosis.8Miura N. Horikawa I. Nishimoto A. Ohmura H. Ito H. Hirohashi S. Shay J.W. Oshimura M. Progressive telomere shortening and telomerase reactivation during hepatocellular carcinogenesis.Cancer Genet Cytogenet. 1997; 93: 56-62Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 9Ohashi 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: 1747-1751PubMed Google Scholar 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.10Wu X. Amos C.I. Zhu Y. Zhao H. Grossman B.H. Shay J.W. Luo S. Hong W.K. Spitz M.R. Telomere dysfunction a potential cancer predisposition factor.J Natl Cancer Inst. 2003; 95: 1211-1218Crossref PubMed Scopus (415) Google Scholar 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.11Freeman A. Hamid S. Morris L. Vowler S. Rushbrook S. Wight D.G. Coleman N. Alexander G.J. 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: 345-350Crossref PubMed Scopus (30) Google Scholar Hepatocyte Mcm-2 expression was significantly higher in serum HCV 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.12Musahl C. Holthoff H.P. Lesch R. Knippers R. Stability of the replicative Mcm3 protein in proliferating and differentiating human cells.Exp Cell Res. 1998; 241: 260-264Crossref PubMed Scopus (76) Google Scholar Normal progression through the cell cycle is coordinated by the sequential interaction of phase-specific cyclins and their respective cyclin-dependent kinases (cdk).13Sherr C.J. Cancer cell cycles.Science. 1996; 274: 1672-1677Crossref PubMed Scopus (5042) Google Scholar 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, Epstein-Barr virus, and herpes simplex virus, interact with host cell cycle control pathways.14Flemington E.K. Herpesvirus lytic replication and the cell cycle arresting new developments.J Virol. 2001; 75: 4475-4481Crossref PubMed Scopus (144) Google Scholar 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,15Ray R.B. Steele R. Meyer K. Ray R. Hepatitis C virus core protein represses p21WAF1/Cip1/Sid1 promoter activity.Gene. 1998; 208: 331-336Crossref PubMed Scopus (147) Google Scholar, 16Dubourdeau M. Miyamura T. Matsuura Y. Alric L. Pipy B. Rousseau D. Infection of HepG2 cells with recombinant adenovirus encoding the HCV core protein induces p21(WAF1) down-regulation—effect of transforming growth factor beta.J Hepatol. 2002; 37: 486Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar can immortalize primary human hepatocytes,17Basu A. Meyer K. Ray R.B. Ray R. Hepatitis C virus core protein is necessary for the maintenance of immortalized human hepatocytes.Virology. 2002; 298: 53-62Crossref PubMed Scopus (50) Google Scholar and inhibits expression of p21.15Ray R.B. Steele R. Meyer K. Ray R. Hepatitis C virus core protein represses p21WAF1/Cip1/Sid1 promoter activity.Gene. 1998; 208: 331-336Crossref PubMed Scopus (147) Google Scholar, 16Dubourdeau M. Miyamura T. Matsuura Y. Alric L. Pipy B. Rousseau D. Infection of HepG2 cells with recombinant adenovirus encoding the HCV core protein induces p21(WAF1) down-regulation—effect of transforming growth factor beta.J Hepatol. 2002; 37: 486Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Nonstructural proteins NS3 and NS4B have been shown to promote growth.18Kwun H.J. Jung E.Y. Ahn J.Y. Lee M.N. Jang K.L. p53-dependent transcriptional repression of p21(waf1) by hepatitis C virus NS3.J Gen Virol. 2001; 82: 2235-2241Crossref PubMed Scopus (89) Google Scholar, 19Park J.S. Yang J.M. Min M.K. Hepatitis C virus nonstructural protein NS4B transforms NIH3T3 cells in cooperation with the Ha-ras oncogene.Biochem Biophys Res Commun. 2000; 267: 581-587Crossref PubMed Scopus (84) Google Scholar NS5A can promote20Ghosh A.K. Steele R. Meyer K. Ray R. Ray R.B. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth.J Gen Virol. 1999; 80: 1179-1183Crossref PubMed Scopus (176) Google Scholar or inhibit growth21Arima N. Kao C.Y. Licht T. Padmanabhan R. Sasaguri Y. Padmanabhan R. Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A.J Biol Chem. 2001; 276: 12675-12684Crossref PubMed Scopus (105) Google Scholar via up-regulation of p21.21Arima N. Kao C.Y. Licht T. Padmanabhan R. Sasaguri Y. Padmanabhan R. Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A.J Biol Chem. 2001; 276: 12675-12684Crossref PubMed Scopus (105) Google Scholar 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.22Pietschmann T. Lohmann V. Rutter G. Kurpanek K. Bartenschlager R. Characterization of cell lines carrying self-replicating hepatitis C virus RNAs.J Virol. 2001; 75: 1252-1264Crossref PubMed Scopus (324) Google Scholar 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,23Wagayama H. Shiraki K. Yamanaka T. Sugimoto K. Ito T. Fujikawa K. Takase K. Nakano T. p21WAF1/CTP1 expression and hepatitis virus type.Dig Dis Sci. 2001; 46: 2074-2079Crossref PubMed Scopus (26) Google Scholar and up-regulation of p57 and p16 messenger RNA has been found using complementary DNA microarrays.24Shackel N.A. McGuinness P.H. Abbott C.A. Gorrell M.D. McCaughan G.W. Insights into the pathobiology of hepatitis C virus-associated cirrhosis analysis of intrahepatic differential gene expression.Am J Pathol. 2002; 160: 641-654Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar 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. 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. 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 ischemic-reperfusion 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.Table 1Demographic and Clinical Data for Patients With Chronic HCV and Liver Regeneration Following Transplant-Related Ischemic-Reperfusion InjuryChronic HCV (n = 70)Liver regeneration following ischemic-reperfusion injury (n = 15)Age (y)41 (11–76)54 (37–63)Sex75% male40% maleDiseaseAll HCV6 primary biliary cirrhosis4 alcohol-related liver disease1 autoimmune chronic liver disease1 primary sclerosing cholangitis1 cryptogenic cirrhosis1 congenital hepatic fibrosisSerum HCV status10 HCV RNA negativeAll recipients and donors negative for serum antibodies to HCV51 HCV RNA positive9 not doneSerum bilirubin (μmol/L)9 (2–32)27 (16–337)Serum albumin (mg/L)38 (20–45)25 (7–41)Serum alanine aminotransferase (IU/mL)85 (39–438)191 (34–1022)Serum alkaline phosphatase (IU/mL)83 (30–260)403 (43–1309)Posttransplant day of biopsy—11 (1–44)NOTE. Values shown are median and range. For the liver regeneration group, "disease" refers to the indication for transplantation. Open table in a new tab NOTE. Values shown are median and range. For the liver regeneration group, "disease" refers to the indication for transplantation. Five-micrometer sections were cut onto polylysine-coated 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.11Freeman A. Hamid S. Morris L. Vowler S. Rushbrook S. Wight D.G. Coleman N. Alexander G.J. 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: 345-350Crossref PubMed Scopus (30) Google Scholar Cyclin D1 was obtained from Dako, cyclin A 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. 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. All chronic HCV biopsy specimens were assessed by a consultant liver histopathologist (S.E.D.) and scored according to Ishak et al.25Ishak K. Baptista A. Bianchi L. Callea F. De Groote J. Gudat F. Denk H. Desmet V. Korb G. MacSween R.N. et al.Histological grading and staging of chronic hepatitis.J Hepatol. 1995; 22: 696-699Abstract Full Text PDF PubMed Scopus (4366) Google Scholar 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. 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. Expression of Mcm-2, cyclin D1, cyclin A, cyclin B1, PH3, and p21 was negligible (<.01% of hepatocytes) in 10 samples of liver resected for colorectal cancer metastasis. 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. In chronic HCV, Mcm-2 was expressed predominantly in hepatocytes, although occasional positive lymphocytes, 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.Figure 3Immunoperoxidase stain in a liver biopsy specimen from a patient with liver regeneration following transplant-related acute ischemic-reperfusion 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.View Large Image Figure ViewerDownload (PPT) 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 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 antibody-positive, HCV RNA-negative individuals (Figure 6; median, 14% vs 7%; P = .001).Figure 6Hepatocyte 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).View Large Image Figure ViewerDownload (PPT) 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. 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.Figure 8Hepatocyte p21 expression comparing HCV antibody-positive, HCV RNA-negative individuals with HCV antibody- positive, HCV RNA-positive patients (P = .004; Mann-Whitney U test).View Large Image Figure ViewerDownload (PPT) These data show marked differences in the phase distribution of cycling hepatocytes in liver regeneration following ischemic-reperfusion injury compared with 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.26Scott I.S. Morris L.S. Bird K. Davies R.J. Vowler S.L. Rushbrook S.M. Marshall A.E. Laskey R.A. Miller R. Arends M.J. Coleman N. A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue implications for the prediction of outcome in colorectal cancer.J Pathol. 2003; 201: 187-197Crossref PubMed Scopus (85) Google Scholar 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 Ki67,5Farinati 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: 1468-1475Crossref PubMed Google Scholar proliferating cell nuclear antigen,6Lake-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: 1644-1648Crossref PubMed Scopus (15) Google Scholar, 7Donato M.F. Arosio E. Del Ninno E. Ronchi G. Lampertico P. Morabito A. Balestrieri M.R. Colombo M. High rates of hepatocellular carcinoma in cirrhotic patients with high liver cell proliferative activity.Hepatology. 2001; 34: 523-528Crossref PubMed Scopus (104) Google Scholar and Mcm-211Freeman A. Hamid S. Morris L. Vowler S. Rushbrook S. Wight D.G. Coleman N. Alexander G.J. 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: 345-350Crossref PubMed Scopus (30) Google
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