Influence of hepatitis G virus infection on liver disease.

The influence of hepatitis G virus (HGV) infection on disease activity in hepatitis C related and unrelated liver disease was investigated in 254 individuals using an EIA polymerase chain reaction assay for HGV. One hundred patients had chronic hepatitis C, 26 primary biliary cirrhosis, and 30 alcoholic liver cirrhosis. In addition, 51 hepatitis B surface antigen (HBsAg)-positive and 47 anti-hepatitis C virus (HCV)-positive blood donors were screened. Hepatitis G virus was detected in 18% of patients with chronic hepatitis C, 13% of patients with alcoholic liver cirrhosis, 11% of patients with primary biliary cirrhosis, 10% of anti-HCV-positive blood donors, and 2% of HBsAg-positive blood donors. Virus load and alanine aminotransferase (ALT) levels did not differ significantly in patients with HCV alone versus patients coinfected with HCV and HGV. However, mild liver fibrosis correlated with HGV coinfection. Hepatitis G virus did not influence ALT levels or liver damage in liver disease unrelated to viral infection.

A new mutational hot-spot in the p53 gene in human hepatocellular carcinoma.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinoma. We have examined 38 hepatocellular carcinoma cases from Taiwan for the presence of p53 alterations in exons 5-8 of the gene using the single-stranded conformational polymorphism method and direct sequencing of polymerase chain reaction products. Using the single-stranded conformational polymorphism method, we found mutations in 16 (42.1%) cases. Twelve mutations were found in exon 5, three in exon 7, and one in exon 8. No mutations were found in exon 6. Sequencing of polymerase chain reaction products showed that all mutations in exon 5 were clustered at codon 166 and were T/A transversions resulting in an amino acid change from serine to threonine, identifying a new hot-spot for point mutations in the p53 gene. The mutations in exon 7 were all at codon 249, and were G/T transversions leading to an amino acid change of arginine to serine. Finally, the mutation at exon 8 was a G-to-T transversion at codon 286 leading to a stop codon. These data indicate that mutations of the p53 gene may be important in the development of human hepatocellular carcinoma and that, in contrast to other tumors, the mutations of the p53 gene in hepatocellular carcinomas can be clustered in a specific codon of the gene.

Detection of hepatitis B and C viruses in liver tissue with hepatocellular carcinoma.

Polymerase chain reaction was used to investigate the presence of the hepatitis B and C viruses in liver tissue from Taiwanese patients with hepatocellular carcinoma by examining paired samples (tumor and non-tumor) from 38 cases. We used a DNA-polymerase chain reaction protocol with primers spanning the regions of the hepatitis B virus genome corresponding to HBs, HBc, and HBx genes and RNA-polymerase chain reaction protocol with primers spanning the 5' untranslated region of the hepatitis C virus. Co-infection with hepatitis B and hepatitis C viruses was seen in nine patients (23%), only three of whom had anti-hepatitis C virus in serum. One of these three was HBsAg-negative in serum while the other two and four of the other six from this group were HBsAg-positive. One of the patients with anti-HCV and no HBsAg in serum had no hepatitis C virus-RNA in liver tissue, while hepatitis B virus-DNA was detectable by using the HBc and HBx specific primers. We detected hepatitis C virus as a single agent in the liver in only one patient. This patient was anti-HCV positive and HBsAg-negative. The remaining 27 patients (71%) had infection with hepatitis B virus only. Twenty-five of 27 patients had HBsAg in their sera. HBs-specific primers detected hepatitis B virus-DNA in non-tumor tissue from 23 patients and in tumor tissue from 25 patients. HBc-specific primers detected hepatitis B virus-DNA in non-tumor tissue from 24 patients and in tumor tissue from 20 patients. Finally, HBx-specific primers detected hepatitis B virus-DNA in non-tumor tissue from 24 patients and in tumor tissue from 25 patients. These data indicate that in a hyperendemic area, hepatitis B virus is closely associated with the development of hepatocellular carcinoma but that infection with hepatitis C virus may play a secondary role.

Hepatitis B X-gene expression in hepatocellular carcinoma.

A RNA-PCR method and different sets of primers were used to investigate the expression of different hepatitis B (HB) virus genes at the RNA level. We tested paired samples (tumor and non-tumor) from the liver tissues of 48 Taiwanese patients with primary hepatocellular carcinoma (HCC). By using a set of primers which spanned the sequences of the S-gene, we found expression in only 2 patients. In one HBs-RNA was only detected in the tumor tissue and in the other only in the surrounding non-tumor tissue. Using primers covering the C-gene, expression was found in 7 patients. In 2 of these RNA was detected in both the tumor and the surrounding tissue, in 2 in the tumor tissue, and in 3 in the surrounding tissue only. Finally, when primers spanning the X-gene sequences were used, RNA was detected in 40/48 patients. In 33 of these cases HBx-RNA was detected in both tumor and non-tumor tissue, in 3 patients in tumor tissue only, and in 4 in the surrounding tissue. Among the cases in which HBc and HBs-RNA was expressed, all showed HBx expression also. These data indicate that the expression of the HBx gene in HCC may play an important role in hepatocarcinogenesis.

Polymerase chain reaction detects hepatitis B virus DNA in paraffin-embedded liver tissue from patients sero- and histo-negative for active hepatitis B.

The polymerase chain reaction (PCR) was used to analyse tissues from paraffin blocks of liver needle biopsies retrospectively. Biopsies of 29 patients with proven HBsAg and HBcAg expression in liver tissue and of 8 healthy volunteers served as positive (group 1) and negative tissue controls (group 2), respectively. These were compared with 16 patients with proven HBsAg expression in liver but lack of HBcAg (group 3), with 23 patients with anti-HBc as the only hepatitis B virus (HBV)-related marker (group 4) and with 21 patients with liver disease and without HBV markers in tissue or serum (group 5). PCR detected HBV sequences in all cases of the positive control group and in 94% of group 3, in 65% of group 4, and in 71.4% of group 5, whereas all healthy volunteers were negative. Our data show that PCR is able to detect HBV-DNA sequences in virtually all patients with active viral antigen expression but also in a high proportion of hepatitic patients who are silent for active HB but may or may not show signs of a contact with the HBV. Thus, PCR for HBV-DNA in paraffin sections might become a useful tool for identifying patients carrying HBV-DNA but not expressing HBV antigens.

Tumor suppression involves down-regulation of interleukin 3 expression in hybrids between autocrine mastocytoma and interleukin 3-dependent parental mast cells.

Interleukin 3 (IL-3)-dependent PB-3c mouse mastocytes can be transformed by the v-Ha-ras oncogene to generate autocrine IL-3-producing mastocytomas. Hybrid cell lines were constructed by fusing an IL-3-producing mastocytoma cell line with its IL-3-dependent normal parental cell. Unlike the mastocytoma parent cell line, hybrid cell lines required growth factor for in vitro proliferation, indicating that the IL-3-dependent phenotype is dominant. IL-3 mRNA, expressed at high levels in the tumor cells, appeared down-regulated in the cell hybrids. In contrast, p21v-Ha-ras levels were not reduced in the hybrids. The hybrid lines generated tumors in vivo with drastically prolonged latency times when compared to the tumor parent (10 versus 2 weeks). We propose that down-regulation of IL-3 mRNA production after cell fusion is responsible for the loss of growth autonomy in the hybrids and is likely to play a role in the partial suppression of tumor formation in vivo. Our data are consistent with the hypothesis that a tumor suppressor, present in PB-3c cells, acts as a negative regulator of IL-3 expression.

A v-H-ras-dependent hemopoietic tumor model involving progression from a clonal stage of transformation competence to autocrine interleukin 3 production.

Autocrine interleukin 3 (IL-3)-secreting tumors were generated from an IL-3-dependent mouse mast cell line (PB-3c) after introduction of the v-H-ras oncogene. Tumor progression was characterized by four distinct phenotypes. The first corresponded to immortalized mast cells unresponsive to the oncogenic effect of v-H-ras. The second was expressed in a clonable subpopulation of PB-3c cells and was marked by the competence to form v-H-ras-dependent tumors (immortalized transformation competence). The third was a direct effect of v-H-ras expression on all PB-3c cells and was characterized in vitro by a reduced IL-3 requirement. Upon injection of v-H-ras-expressing, transformation-competent cells into mice, the final, fully malignant phenotype developed with a long latency period and was marked in vitro by independence of exogenous IL-3 and by autocrine IL-3 stimulation. Northern (RNA) blot analysis and an RNase A-T1 protection assay showed that IL-3 production was strictly associated with the tumor phenotype. Two of six tumors showed an alteration at the 5' region of the IL-3 gene. We conclude that v-H-ras required complementation by IL-3 gene rearrangement or an alternate event to generate autocrine mastocytomas.