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Cell Biology Poster Session






Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References




Discussion
Board

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Multiple negative regulatory elements in 5'-flanking region of the rat catalase gene.


Contact Person: Toshiyuki Takeuchi (taketo@grape.med.tottori-u.ac.jp)


Introduction

Accompanied with tumorigenesis, various genes are observed to be altered in its expression, as well as activation of oncogene or inhibition of tumor-suppressor genes etc. These marked differences in gene expression between the normal cell and tumor are assumed to result in multiple phenotypic change of tumor in morphology, metabolisms, and many often characters. Therefore, elucidation of gene regulation in tumor cells is noteworthy to understand the molecular mechanism of cell transformation.
Catalase [EC1.1.1.6] is a radical scavenging enzyme protecting cells from oxidative stress, together with the other enzymes such as superoxide dismutase and glutathione peroxidase. In mammalian tissues, the highest level of catalase gene expression is found in the liver, kidney and erythrocytes and the lowest level in connective tissues (1). Furthermore, the catalase gene expression is known to be markedly reduced in cultured hepatoma cell lines. It is interesting to analyze the mechanism of the catalase gene expression.
Previously, we reported the G-rich silencer element located -3.5kb upstream of the rat catalase gene (2). Furthermore, we also found another negative regulatory region located in, -2799 to ?2652 bp, downstream from the G-rich silencer element. In this study, we analyzed this region -2799 to -2652 bp by EMSA and CAT assay, to disclose the fine mechanism of negative regulation of the catalase gene in hepatoma cells.

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Materials and Methods

I, Cell lines
AH66 cell is malignant and dedifferentiated hepatoma cell line derived from an ascites rat hepatoma. Reuber cell is the rat minimal deviation hepatoma cell line, and 3Y1 cell is an immortalized cell line from Fischer rat embryonic fibroblast cell. All of these cell lines were maintained in Dulbecco modified Eagle's medium supplemented with 10% fetal calf serum.

II, Electrophoresis mobility shift assay (EMSA)
Preparation of nuclear extract from AH66 and Reuber cell was performed according to Dignam's method (3). Probe DNA was end-labeled with [ganma-32P]ATP and T4 polynucleotide kinase. EMSAs were performed as described previously (4). DNA-protein complexes were resolved in 5% acrylamide:bis-acrylamide-TGE gel containing 2.5% glycerol, and visualized by autoradiogrphy.

III, CAT(Chloramphenicol acetyltransferase) assay
Plasmid bearing truncated the 5'-flanking region of rat catalase gene were prepared with restriction enzymes. CAT plasmid was constructed by insertion of the truncated segments into the polylinker sites of pUC0CAT and pCCAT-P11. pCCAT-P11 was constructed by insertion of the rat catalase gene promoter region (-126 to -26 bp) into the polylinker sites of pUC0CAT.10ug of these CAT constructs were transfected to 3Y1 by the calcium phosphate precipitation procedure (5, 6), and the CAT activities were determined with HPLC as described (7). Each transfection experiment was carried out in triplicate, and the values of CAT activities, which deviated within 5%, were considered valid and are reported here.

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Results

(1) Locating of binding protein in negative regulatory region
To study on negative regulatory element, located on -2799 to -2652 bp and designated F fragment, we determined protein binding to this region by EMSA probed F fragment using nuclear extracts from hepatoma cell lines, Reuber and AH66. The Reuber cell maintains catalase gene expression at significant level as same as normal liver, while the AH66 cell extremely deduced the catalase (2). As a result, a specific shift band was observed in DNA complex with AH66 nuclear extract (Fig.1-B; lanes 2 and 3). Furthermore, the truncated DNA fragments was determined by nuclear protein binding competition assay (Fig.1-A). The DNA protein complex was strongly competed with F-AH fragment (lane 4). Furthermore, F-EH fragment derived from F-AH fragment also inhibited probe-protein complex formation in some extent. However, competition by F-EH fragment was weak compared with F-AH fragment (lanes 5 and 7). Likewise, gel shift of F fragment was competed by F-HA and F-AE fragment at quite low level (lanes 4 and 6). These results suggested that nuclear protein in AH66 hepatoma cells composed DNA-protein complex with F-EH fragment region, but scarcely with F-HA and F-AE fragments.

Fig. 1. EMSA of F fragment and competition with sub-fragments.
(A) Restriction map of the F fragment and DNA sub-fragment used in competition assay. H, A and E indicate restriction sites for HindIII, AluI and EcoRV, respectively. (B) EMSA was performed with F fragment and nuclear extracts from Reuber cell (R, lane 2) and AH66 cell (A, lanes 3 to 7). Arrowhead shows the specific bands found in AH66 cells nuclear extract. Competitors are indicated on the top of panel (lanes 4 to 7).

(2) Transcriptional activity of deletion mutant mutant
Next, to test the transcriptional activity of negative regulatory elements in this region, CAT plasmids carrying various length of negative element were constructed with F fragment and it derivatives. CAT activity of pCCAT-F-EH showed strong repression in the rat catalase promoter. Further, transcriptional activities by pCCAT-F-HA and pCCAT-F-AE were weakly repressed compared with pCCAT-P11. These results suggested that a negative regulatory element was located in AH fragment, paticulary in EH fragment.

Fig. 2. CAT assay of various region in F fragment.

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Discussion and Conclusion

Catalase activity is markedly reduced cultured hepatoma cell lines (8, 9, and 10). Analysis of this phenomenon is assumed to disclose the mechanism of carcinogenesis or dediffereaion of liver cell. From the results of EMSA and CAT assay, we suggested that F-EH binding protein seemed a transcriptional repressor of the rat catalase gene expression in rat hepatoma cell lines.
Recent studies have identified a large number of proteins capable of repressing transcription, and supporting evidence for this mechanisms has been presented (11, 12). Transcriptional repression were classified only two distinct functional mechanisms; position-independent silencing and position-dependent one (13). Transcriptional silencer in the catalase gene was necessary to elucidate the function in hepatocarcinogenesis.

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References

1: Schoenbaum, G. R., et al. (1976) Catalase in The Enzymes 363-408. Academic Press, New York.
2: Sato, K., et al. (1992) Mol. Cell. Biol. 12, 2525-2533.
3: Tamura, T., et al. (1989) Mol. Cell. Biol. 9, 3122-3126.
4: Fridell, Y., et al. (1992) Mol. Cell. Biol. 12, 4571-4577.
5: Chen, C., et al. (1987) Mol. Cell. Biol. 9, 1415-1425.
6: Gorman, C., et al. (1982) Mol. Cell. Biol. 2, 1044-1051.
7: Sato, K., et al. (1986) Mol. Cell. Biol. 6, 1032-1043.
8: Bozzi, A., et al. (1976) Mol. Cell. Biochem. 10, 11-16.
9: Marklund, S.L., et al. (1982) Cancer Res. 42, 1955-1961.
10: Potter, V.R. (1977) Biochemistry of cancer in Cancer Medicine 178-190, Lea and Febiger, Philadelphia.
11: Hanna-Rose., et al. (1996) Trends Genet. 12, 229-234.
12: Johnson, A. D. (1995) Cell. 81, 655-658.
13: Steven, O., et al. (1998) Biochem, J. 331, 1-14

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Takeuchi, T.; Kayasuga, A.; Toda, H.; Sato, K.; (1998). Multiple negative regulatory elements in 5'-flanking region of the rat catalase gene.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/cellbio/takeuchi0634/index.html
© 1998 Author(s) Hold Copyright