Hepatitis C virus (HCV) is a nonretroviral oncogenic RNA virus, which is frequently associated with hepatocellular carcinoma (HCC) and B cell lymphoma. We demonstrated here that acute and chronic HCV infection caused a 5-to 10-fold increase in mutation frequency in Ig heavy chain, BCL-6, p53, and ␤-catenin genes of in vitro HCV-infected B cell lines and HCV-associated peripheral blood mononuclear cells, lymphomas, and HCCs. The nucleotide-substitution pattern of p53 and ␤-catenin was different from that of Ig heavy chain in HCVinfected cells, suggesting two different mechanisms of mutation. In addition, the mutated protooncogenes were amplified in HCV-associated lymphomas and HCCs, but not in lymphomas of nonviral origin or HBV-associated HCC. HCV induced error-prone DNA polymerase , polymerase , and activation-induced cytidine deaminase, which together, contributed to the enhancement of mutation frequency, as demonstrated by the RNA interference experiments. These results indicate that HCV induces a mutator phenotype and may transform cells by a hit-and-run mechanism. This finding provides a mechanism of oncogenesis for an RNA virus. Hepatitis C is an emerging infectious disease, affecting 200 million people worldwide. Hepatitis C virus (HCV) causes chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC), and occasionally, non-Hodgkin's B cell lymphoma (1). HCV contains an RNA genome, which replicates in the cytoplasm, does not contain an obvious oncogene, and does not integrate into host genomes; the mechanism of its oncogenesis remains unclear. Some HCV-associated HCCs have mutations in the tumor suppressor p53, the protooncogene ␤-catenin (2, 3) and several other genes. However, the long latency period of HCV infection makes it difficult to demonstrate the causal association between protooncogene mutations and HCV infection. Furthermore, many HCVassociated HCCs do not have detectable HCV RNA (1, 4), suggesting that HCV-induced tumorigenesis may employ a hit-and-run mechanism. HCV infects not only hepatocytes, but also B cells in vitro and in vivo (5, 6). Significantly, the HCV envelope protein E2 can bind CD81 in the CD21͞CD19͞CD81 costimulatory complex (7), suggesting the ability of HCV to alter the intracellular signaling of B cells. In HCV-infected individuals, oligoclonal lymphoproliferative disorders (8) and chromosomal translocation (9) have frequently been observed in B lymphocytes, suggesting that HCV may cause chromosomal instability. The hypervariable region of V(D)J in heavy chain of Ig (V H ) in HCV-associated lymphoma has frequent somatic mutations and intraclonal diversity (10), suggesting the strong association between HCV infection and activation of somatic hypermutation. To understand the molecular mechanism of these changes, we set out to investigate whether HCV infection enhances the mutation frequencies of cellular genes. Materials and Methods Cloning and Sequencing of Peripheral Blood Mononuclear Cell (PBMC),Cellular, and Tumor DNA. Genomic DNA was extracted from PBMCs, cell l...

scribed in frame, and coded mutant FLT3 with a long JM

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells Hepatitis C virus (HCV) has been the major etiological agent of posttransfusion non-A, non-B hepatitis and currently afflicts Ͼ100 million people worldwide. Acute HCV infection is usually subclinical without obvious symptoms. About 15 to 20% of patients can mount a successful immune response to clear the virus in the acute phase; however, 80 to 85% of patients become chronic carriers, and these patients are at high risk of developing liver cirrhosis and/or hepatocellular carcinoma.Besides causing liver pathology, HCV infection is frequently associated with mixed cryoglobulinemia, non-Hodgkin's B-cell lymphoma, and Sjögren's syndrome, all of which involve B-cell proliferation (8, 10, 27, 37, 49; P. Pioltelli, G. Zehender, G. Minti, A. Monteverde, and M. Galli, Letter, Lancet 347:624-625, 1996), suggesting that HCV may infect B cells or affect B-cell functions in natural infection. Negative-strand HCV RNA has been detected by reverse transcriptase (RT) PCR in the peripheral lymphocytes, bone marrow, lymph nodes, and central nervous system of some HCV patients (23,30,34). Analysis of positive-strand HCV RNA sequences and quasispecies patterns suggested that HCV RNAs in these cells are different from those in the serum (22). However, the possibility that HCV replicates in extrahepatic cells remains controversial because of the lack of isolation and characterization of viruses from the infected cells. Further, the use of RT-PCR for detection of viral RNA in these studies could not rigorously rule out possible contamination by the virus from the serum. Several laboratories have also shown that HCV can infect B-cell (30), T-cell (18, 32, 39), and hepatoma cell (14, 41) lines in culture, but the infection is usually transient and inefficient. Nevertheless, these studies suggested that B or T cells could support HCV replication, albeit inefficiently, at least in vitro.The molecular cloning of the HCV genome has made possible the delineation of the gene functions and the potential mechanism of pathogenesis of this virus. Recently, establishment of self-replicating HCV subgenomic (2, 25) and genomic (13, 33) replicons in Huh-7 cells has also provided an important new tool for the study...

Purpose: We performed a phase I trial to investigate the safety, clinical responses, and Wilms' tumor 1 (WT1)-specific immune responses following treatment with dendritic cells (DC) pulsed with a mixture of three types of WT1 peptides, including both MHC class I and II-restricted epitopes, in combination with chemotherapy.