Clinical Studies Gene expression in HCV-associated hepatocellular carcinoma – upregulation of a gene encoding a protein related to the ubiquitin-conjugating enzyme Ghada M. El-Nady, Roger Ling and Tim J. Harrison Centre for Hepatology, University College London, London, UK El-Nady GM, Ling R, Harrison TJ. Gene Expression in HCV-Associated Hepatocellular Carcinoma – Upregulation of a Gene Encoding a Protein Related to Ubiquitin-Conjugating Enzyme. Liver International 2003: 23: 329–337. r Blackwell Munksgaard, 2003 Abstract: Background/Aims: To investigate gene expression in HCV- associated human hepatocellular carcinomas (HCC) by identifying up- and down-regulated genes. Methods: Differential display RT-PCR was used to compare levels of gene expression in tumorous and non-tumorous tissues from the same livers. Differential expression was confirmed using a ribonuclease protection assay (RPA). The relative expression levels of one candidate gene were studied in various normal tissues and malignant cell lines using a multiple tissue expression (MTE) array. Further characterisation of this gene was carried out using nucleotide sequence analysis programmes and Northern hybridisation. Results: Fifty-two differentially expressed cDNA fragments were identified and 29 were cloned, sequenced and compared with the nucleotide sequence database. RPA confirmed reproducibly that one particular cDNA was upregulated in the tumour cells. Analysis using the MTE array revealed that this selected candidate gene is expressed at high levels in various human tumour cell lines. The expression levels in HCV-associated HCC were higher than in other tumours. Investigation revealed that this novel gene lies on chromosome 17. The transcript is approximately 2.5 kb in size and encodes a protein similar to the ubiquitin-conjugating enzyme. Conclusions: The ubiquitin system may be involved in HCV-related hepatocarcinogenesis and in the development of other cancers. Key words: hepatitis C virus – hepatocellular carcinoma – differential display RT-PCR – ribonuclease protection assay – multiple tissue expression array – ubiquitin-conjugating enzyme Tim J Harrison, DSc FRCPath, Centre for Hepatology, University College London, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK. Tel: 14420 7433 2882. Fax: 14420 7433 2747. e-mail: T.Harrison@rfc.ucl.ac.uk Received 20 January 2003, accepted 20 May 2003                                                                                       Hepatocellular carcinoma (HCC) develops most frequently as a consequence of chronic hepatitis B or C. Around 80% of hepatitis C virus (HCV) infections of healthy adults become chronic and up to 20% of these may develop to cirrhosis or HCC over one or two decades (1). However, in contrast to chronic hepatitis B, where the development of HCC is associated with chromo- somal integration of viral DNA in the hepato- cytes, integration of viral nucleic acid does not occur in hepatitis C. HCV RNA replication and expression of virus-specific proteins have been detected in HCV-associated HCC, but the precise mechanisms underlying the oncogenic process remain unclear. The rare occurrence of patients with chronic HCV infection who develop HCC without underlying cirrhosis (2) suggests that HCV may be directly oncogenic and viral gene products may be involved in inducing liver cell proliferation. Particular attention has been focused on the HCV nucleocapsid (core) protein, which is able to transform primary rat embryo fibroblasts in combination with cellular oncogenes such as H- ras or c-myc (3). The HCV nucleocapsid protein has been shown to repress p53 promoter activity (4), and the nuclear transcription factor LZIP, which has activities consistent with those of a tumour suppressor, has been shown to bind the HCV nucleocapsid protein and to be a cofactor in cellular transformation. The loss of endogen- ous LZIP activity is correlated with abnormal cellular proliferation (5). Moreover, expression of the HCV nucleocapsid protein promoted cell Liver International 2003, 23, 329–337 Printed in Denmark. All rights reserved Copyright r Blackwell Munksgaard 2003 329