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Negative regulation of catalase gene expression in hepatoma cells
1Department of Molecular Biology, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Japan.
Abstract:
For an understanding of the molecular basis of the marked decrease in catalase activity of various tumor cells, expression of the catalase gene was studied in rat and human hepatoma cell lines and in rat liver, which was used as a control with high activity. RNA blot hybridization profiles and run-on assays indicated that the decrease in catalase activity was due to depression of catalase gene transcription. Chloramphenicol acetyltransferase (CAT) assays for the fragments with various lengths of the 5'-flanking region (up to -4.5 kb from the ATG codon) of the catalase gene revealed the presence of several cis-acting elements involved in the negative regulation of transcription. The most-upstream element with the strongest activity (-3504 to -3364 bp), when linked to the catalase promoter region (-126 bp) of the CAT construct and subjected to an in vitro transcription assay, did not yield transcripts in experiments with the hepatoma nuclear extract, whereas the unlinked template did yield transcripts. A gel shift competition assay using hepatoma nuclear extract showed the core sequence of the silencer element to be 5'-TGGGGGGAG-3'. A homology search found that the same core sequence was also present in 5'-flanking regions of the albumin gene and of some other liver enzyme genes, the expression of which has been reported to be down regulated in some hepatoma cells. Southwestern (DNA-protein) analysis demonstrated that an approximately 35-kDa nuclear protein bound to the silencer element was present in hepatoma cells but not in rat liver cells.
Insights
Tumor cells show decreased catalase activity due to reduced gene transcription. A specific DNA sequence acts as a silencer, binding a protein found in hepatoma cells but not normal liver cells.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Catalase activity is significantly reduced in various tumor cells.
- Understanding the molecular mechanisms behind this decrease is crucial for cancer research.
Purpose of the Study:
- To investigate the molecular basis for the decreased catalase activity in hepatoma cells.
- To identify regulatory elements and proteins involved in catalase gene expression.
Main Methods:
- RNA blot hybridization and run-on assays to assess gene transcription.
- Chloramphenicol acetyltransferase (CAT) assays to analyze 5'-flanking regions.
- Gel shift competition and Southwestern analysis to identify DNA-binding proteins.
Main Results:
- Decreased catalase activity in hepatoma cells is caused by suppressed catalase gene transcription.
- A novel silencer element (-3504 to -3364 bp) in the catalase gene's 5'-flanking region negatively regulates transcription.
- A 35-kDa nuclear protein binds to this silencer element and is present in hepatoma cells but absent in rat liver cells.
Conclusions:
- A specific silencer element and its binding protein contribute to the downregulation of catalase gene expression in hepatoma cells.
- This mechanism may be involved in the altered gene expression observed in other liver enzyme genes in hepatoma cells.