Rad18 is involved in postreplication repair mainly through monoubiquitination of proliferating cell nuclear antigen (PCNA). Here we show that Rad18 protein was detected in human cells as two major bands at 75 and 85 kDa by Western blot. The bands were identified as nonubiquitinated and monoubiquitinated forms of Rad18, respectively, by mass spectrometry. Multiple ubiquitinated bands of Rad18 were detected in vitro in the presence of E1, E2 (Rad6), and methylated ubiquitin, indicating that Rad18 was monoubiquitinated at multiple sites through autoubiquitination. Rad18 self-associates, and this interaction was abolished by replacing one of the conserved cysteine residues with phenylalanine in the zinc finger domain (C207F). In the C207F mutant Rad18, monoubiquitination of Rad18 was not observed in vivo, suggesting that self-association was critical for monoubiquitination. Monoubiquitinated Rad18 was detected mainly in the cytoplasm, whereas nonubiquitinated Rad18 was detected predominantly in the nuclei. Furthermore, Rad18 was shown to be polyubiquitinated in cells treated with proteasome inhibitors. Purified Rad18 was also polyubiquitinated in an in vitro system containing E1, E2 (Rad6), and ubiquitin, and it was degraded by the addition of proteasomes. These results suggest that the amount of Rad18 in the nucleus is regulated differentially by mono-and polyubiquitination.

The combinational use of HBsAg and HBcrAg levels may be useful to predict the 24-month outcome of NUC/IFN-α sequential therapy. Maximal levels of ALT and HBV DNA during post-treatment follow-up may also help monitor responses to IFN-α sequential therapy.

Purpose: We studied the clinical features and the etiology of hepatitis B virus surface antigen (HBsAg)-negative and antibody to hepatitis C virus (anti-HCV) negative patients with hepatocellular carcinoma. Methods: A total of 550 patients, hospitalized with an initial diagnosis of HCC were retrospectively studied. Eighty-one of these patients were HBsAg-positive (HB group), 404 patients were anti-HCV positive (HC group). The other 65 patients were negative for both HBsAg and for anti-HCV (NBNC group). We puri®ed HBV-X gene from HCC or non-tumorous liver tissue of 23 NBNC patients using PCR. Results: Clinical features of NBNC as compared with HB and HC patients were as follows, respectively: non-cirrhosis rate (%): 57,37,15 ( P 0.02 for HB, P < 0.00001 for HC), the proportion of patients with normal ALT concentrations (%): 59,28,10 ( P 0.0002 for HB, P < 0.00001 for HC). Forty of 59 NBNC patients (68%) had anti-HBs and/or anti-HBc (healthy controls: 29%, P < 0.00001) and two of 56 had serum HBV DNA. Twelve of 23 NBNC patients had HBV-X gene in HCC and/or non-cancerous liver tissues (52%). None of 52 had serum HCV RNA. Conclusions: The NBNC patients with HCC had a higher frequency of non-cirrhotic liver without liver injury. The presence of the HBV-X gene in HCC suggests a possible role of past HBV infection in the development of HCC. About half of NBNC HCC is associated with seronegativity for HBsAg and positivity for the HBV-X gene in liver tissue. Hepatitis B virus (HBV) infection is now recognized to be a major risk factor for HCC (1±3). Since the discovery of HCV RNA in 1989, anti-HCV antibodies have been found in a large proportion of HBsAg-negative patients with HCC (4±8) but about 10% of patients in Japan with HCC are negative for HBsAg and for anti-HCV (9). The precise mechanism by which HCV infection results in HCC is not known. HCV is an RNA virus the genome of which does not seem to become integrated into the host DNA (10). Most studies on HCV have focused on its indirect role in causing cancer as a result of cirrhosis (11±14).HBV is thought to induce HCC by two mechanisms. HBV infection contributes to cirrhosis of the liver, which is associated with HCC in 60±80% of patients with this disease. In addition, HBV infection leads to integration of HBV DNA into host chromosomes, with a possible direct carcinogenic effect of HBV through interactions with oncogenes, growth factors, or tumour suppressive genes (15,16).

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PegIFNα-2a plus ribavirin resulted in higher SVR rates than PegIFNα-2b plus ribavirin in Japanese patients. PegIFNα-2a-based treatment should therefore be the preferred choice for women, older or low-weight patients, and those with the IL28B TT genotype.

The combinational use of HBsAg and HBcrAg levels at baseline and their changes throughout sequential therapy may be useful for predicting a response to NUC/peg-IFN sequential therapy.

Virtual touch quantification is a non-invasive, simple method that is more accurate for staging liver fibrosis than the FIB-4 index and APRI. However, when the SCD is longer than 17.5 mm, there may be measurement failures.

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