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1.
Mutat Res ; 452(2): 163-78, 2000 Sep 18.
Article in English | MEDLINE | ID: mdl-11024476

ABSTRACT

Hepatitis B virus (HBV) is a major etiological factor associated with hepatocarcinogenesis, but its role in the transformation process remains unclear. We previously documented the accumulation of genetic alterations in a HBV-transfected cell line. In the present study, we addressed the effect of HBV and its replication on the genome and phenotype of the host cell. Parental HBV-free Hep G2 cells and two HBV-transfected variant lines Hep G2215 and Hep G2T14. 1, which do and do not replicate HBV, respectively, were used to monitor genetic alterations in conjunction with HBV profile in vitro and in vivo. Comparison of in vitro growth rates showed that Hep G2T14.1 cells grew more rapidly, while Hep G2215 cells, replicating HBV, grew slower than parental Hep G2 cells. Molecular analysis confirmed an HBV integration site (s) in both variants, and reverse trancriptase-polymerase chain reaction (RT-PCR) amplification documented expression of transcript for the HBX protein, which has recently been implicated in the compromised efficiency of cellular DNA repair. Tumorigenisity testing indicate a comparable rate of tumor formation in nude mice of both HBV-transfected variants, giving rise to tumors in 3 weeks; parental Hep G2 cells did not form tumors in nude mice. Tumor tissue from nude mice injected with Hep G2T14.1 cells showed no change in HBV status. However, a new HBV integration site was detected in tumor tissue from Hep G2215-injected mice. Two cell lines derived from the respective tumor tissue grew in vitro at rates compatible to those observe before passage in nude mice. The Hep G2215 tumor-derived line continued to replicate HBV, while HBV status remained unchanged in the Hep G2T14.1 tumor-derived line. Unique genetic alterations were detected in both transfected cell lines, and Hep G2215 cells particularly showed cellular mosaicism and clonal selection when analyzed after the passage in nude mice. Further genetic alterations were detected in tumor-derived cell lines. Interestingly, the de novo genetic alterations in the Hep G2215 cells, which maintain the ability to replicate HBV, included a new HBV integration site, several chromosome rearrangements and loss of heterozygosity (LOH) of one p53 allele. Western analyses of p21/Waf1 protein indicate an upregulation of the protein in cells that replicate HBV. Based on the combined data, we hypothesize that the genetic alterations in the cellular genome could also be generated as a function in the presence of HBV and HBV replication. Possible mechanisms that could be implicated in cumulative mutagenetic events are discussed.


Subject(s)
Hepatitis B virus/physiology , Liver/virology , Loss of Heterozygosity , Virus Integration , Virus Replication , Animals , Base Sequence , Blotting, Western , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , DNA Primers , Genes, p53 , Genetic Markers , Hepatitis B virus/genetics , Humans , Karyotyping , Mice , Mice, Nude , Reverse Transcriptase Polymerase Chain Reaction
2.
Cancer Genet Cytogenet ; 116(1): 35-9, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10616529

ABSTRACT

Recent data suggest that Bin1, a novel C-MYC interacting protein, is a suppressor gene whose loss of expression is a frequent aberration associated with several malignancies. The mechanism responsible for loss of BIN1 expression is not understood. The purpose of this study is to investigate DNA profile of the BIN1 gene in human hepatoma Hep G2 cells, previously documented with lack of BIN1 expression. Chromosome and molecular analyses of Hep G2 cells were initiated to exclude the possibility of genetic alterations as a factor affecting BIN1 gene expression in these cells. We used Hep G2 cell line and its hepatitis B virus (HBV) transfected variants--Hep G2T14.1 and Hep G2215 cell lines. The cytogenetic localization of BIN1 was identified in the 2q14 region. Fluorescence in situ hybridization (FISH) with the chromosome 2 whole chromosome painting probe (WCP) demonstrated three or four intact copies of chromosome 2 in all three hepatoma cell lines studied. FISH analyses with the BIN1-specific probe of the Hep G2, Hep G2T14.1, and Hep G2215 metaphase chromosomes document no rearrangement of the BIN1 gene on any of the multiple copies of chromosome 2. FISH with the specific HBV probe did not identify the HBV integration site in Hep G2T14.1 and Hep G2215 cells within the BIN1 locus. Southern blot analyses revealed no genetic rearrangements in the BIN1 gene in any of the cell lines studied. Our RNA analyses (northern blot and RT-PCR) document lack of BIN1 message in Hep G2 cells in contrast to the presence of BIN1 in Hep G2T14.1 and Hep G2215 cells. No difference was identified in other transcripts analyzed, including c-myc. Analyses of BIN1 expression of Hep G2 cells at different passages were initiated and document low levels of BIN1 transcript in Hep G2 cells of passage < 85. Furthermore, BIN1 transcript was identified in additional seven HCC cell lines analyzed. Our data indicate that lack of Bin1 expression in HepG2 cells previously documented is a characteristic of cells of passage > 85 and is not due to genetic loss, or rearrangement within the BIN1 DNA sequence. Loss of the BIN1 transcript is not a characteristic of HCCs analyzed.


Subject(s)
Carcinoma, Hepatocellular/genetics , Carrier Proteins/genetics , Gene Expression , Genes, Tumor Suppressor , Liver Neoplasms/genetics , Nuclear Proteins/genetics , Tumor Suppressor Proteins , Adaptor Proteins, Signal Transducing , Blotting, Northern , Blotting, Southern , Carcinoma, Hepatocellular/virology , Chromosome Painting , Chromosomes, Human, Pair 2 , DNA Restriction Enzymes , Hepatitis B virus/genetics , Humans , In Situ Hybridization, Fluorescence , Liver Neoplasms/virology , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tumor Cells, Cultured
3.
Mutat Res ; 377(2): 187-98, 1997 Jul 03.
Article in English | MEDLINE | ID: mdl-9247614

ABSTRACT

Chromosome and molecular analyses of the hepatitis B virus (HBV)-transfected HepG2T14.1 variant of the HepG2 cell line was conducted. In HepG2T14.1 cells several genetic alterations such as de novo aberrations of chromosomes 9, 14, 15, and 20 were identified that are not present in the parental HepG2 cell line. Furthermore, HepG2T14.1 cells showed loss of heterozygosity (LOH) in the q region of chromosome 14. The single HBV integration site in HepG2T14.1 cells mapped to the 2q35-36 region of one copy of chromosome 2 by fluorescence in situ hybridization (FISH). No genetic changes were identified at or near the HBV integration site at the level of these analyses. In addition, growth rates in vivo and in vitro were dramatically accelerated in HepG2T14.1 cells. These results document that a HBV-transfected hepatoma cell line has de novo genetic mutations at several sites of the host genome, one HBV integration site in an non-rearranged chromosome and an altered phenotype. These findings support our hypothesis that HBV might play a role in cellular transformation by interfering with cellular processes responsible for the stability of the genome.


Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , Hepatitis B/genetics , Animals , Carcinogenicity Tests , Cell Division/genetics , Cell Line , Cell Line, Transformed , Chromosome Aberrations , Chromosome Mapping , DNA Transposable Elements , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Mice , Mice, Nude , Microsatellite Repeats , Transfection
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