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 A. Freeman, 1 S. Hamid, 2 L. Morris, 1 S. Vowler, 3 S. Rushbrook, 2 D. G. D. Wight, 1 N. Coleman 1 and G. J. M. Alexander 2 1 Department of Histopathology, Addenbrooke’s Hospital; 2 Department of Medicine, University of Cambridge School of Clinical Medicine; and 3 Centre for Applied Medical Statistics, Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK Received October 2002; accepted for publication December 2002 SUMMARY. To test the hypothesis that hepatitis C virus (HCV) might induce hepatocyte proliferation directly, thereby pre- disposing HCV carriers to cirrhosis and hepatocellular car- cinoma, we have used a new method to identify proliferating hepatocytes, employing a novel monoclonal antibody to minichromosome maintenance (Mcm) proteins, essential components of the pre-replication complex. Antibody to Ki-67, a conventional marker of cell division, was also studied. Eighty-seven patients with chronic HCV infection and a broad spectrum of histological change were studied. Proliferation was observed rarely in hepatocytes from nor- mal liver from healthy controls (always less than 0.01%). However, proliferating hepatocytes were detected in all HCV- infected patients and the proportion of hepatocytes expres- sing Mcm-2 (3–40%) always exceeded that expressing Ki-67 (1–14%) and correlated positively with increasing stage of fibrosis (P ¼ 0.0001) and viral replication (P ¼ 0.0004). There were weaker but significant associations between the proportion of hepatocytes expressing Mcm-2 and inflam- matory indices including interface hepatitis, portal tract inflammation, lobular inflammation and steatosis. There was no association between the proportion of hepatocytes expressing Mcm-2 and age, gender or past alcohol con- sumption, but there was a weak association with current consumption of alcohol (P ¼ 0.0067). The proportion of Ki-67 hepatocytes did not correlate with any clinical, labor- atory or histological parameter. Mcm-2 was also detected in bile duct cells, sinusoidal lining cells and infiltrating lymphocytes, but at low frequency. These data indicate first, that Mcm-2 is a more sensitive marker of hepatocyte pro- liferation than Ki-67, second that many hepatocytes in chronic HCV infection have entered the cell cycle and third, suggest that interference with the hepatocyte cell cycle might be an alternative approach to therapy. Keywords: hepatitis C; DNA proliferation; cell cycle; disease progression; natural history INTRODUCTION Cell number in human organs is regulated by a balance between cell proliferation and apoptosis [1]. Once stimulated to proliferate, cells enter the cell cycle, comprising several phases. Attention has focused recently on the initiation of DNA replication in eukaryotic cells. The pre-replication complex, comprising ORC proteins, Cdc6 and the mini- chromosome maintenance (Mcm) family of proteins, assembles during the G1 phase of the cell cycle, rendering chromatin competent for replication. Chromatin, thus ÔlicensedÕ for replication, is guided into the S phase by acti- vation of cell-cycle regulating protein kinases. Upon entry into S phase, phosphorylation of Cdc6 and Mcm proteins results in dissociation of the prereplication complex with release of Mcm proteins, thus preventing further DNA rep- lication within the same cell cycle [2]. The highly conserved proteins, Mcms and Cdc6, are essential for the key regula- tory step of initiation of DNA replication in all eukaryotic cells investigated so far. The Mcm proteins can be detected in the nucleus throughout the cell cycle, but are absent from quiescent cells [3,4]. Thus, Cdc6 was absent from fibroblasts in G0, but present throughout the cell cycle [5]. Identical results were Abbreviations: HCV, hepatitis C virus; Mcm, minichromosome maintenance. Correspondence: G.J.M. Alexander, Box 157, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, UK. E-mail: gja1000@doctors.org.uk Journal of Viral Hepatitis, 2003, 10, 345–350 Ó 2003 Blackwell Publishing Ltd