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.

(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.

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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