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Investigation of the expression of Bin1, a putative suppressor, in human hepatoma cells
K W Livezey1, D Negorev, D Simon
1MCP Hahnemann University, School of Medicine, Department of Pathology, Philadelphia, Pennsylvania, USA.
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.
Insights
Loss of Bin1 suppressor gene expression in HepG2 cells is not due to genetic alterations but is linked to cell passage number. This finding clarifies Bin1 regulation in hepatoma cells.
Area of Science:
- Molecular biology
- Genetics
- Oncology
Background:
- Bin1, a novel C-MYC interacting protein, functions as a suppressor gene.
- Loss of Bin1 expression is frequently observed in various malignancies.
- The mechanism behind Bin1 expression loss remains unclear.
Purpose of the Study:
- To investigate the DNA profile of the Bin1 gene in human hepatoma Hep G2 cells.
- To exclude genetic alterations as a cause for absent Bin1 expression in Hep G2 cells.
Main Methods:
- Cytogenetic and molecular analyses of Hep G2 cells and HBV-transfected variants (Hep G2T14.1, Hep G2215).
- Fluorescence in situ hybridization (FISH) to assess chromosome 2 and Bin1 gene integrity.
- Southern blot analysis for genetic rearrangements.
- RNA analysis (Northern blot, RT-PCR) for Bin1 transcript levels.
Main Results:
- FISH and Southern blot analyses confirmed no genetic alterations or rearrangements of the Bin1 gene in Hep G2 cells.
- Hep G2 cells (passage > 85) showed a lack of Bin1 transcript, while variants (Hep G2T14.1, Hep G2215) and earlier passage Hep G2 cells (< 85) expressed Bin1.
- No integration of HBV was found within the Bin1 locus in transfected cells.
- Bin1 transcript was detected in seven other HCC cell lines.
Conclusions:
- Lack of Bin1 expression in Hep G2 cells is associated with high passage numbers, not genetic loss or rearrangement.
- The absence of Bin1 transcript is not a universal characteristic of hepatocellular carcinoma (HCC).