Establishment of a rat hepatoma-derived cell line proliferating in D-phenylalanine medium and expressing D-amino-acid oxidase.
A cell line (R-Y121B.DF) has been established from a cell line (R-Y121B) derived from a rat hepatoma line (H4-II-E). The R-Y121B.DF cells have been continuously cultured in a serum-free modified Eagle's minimum essential medium in which L-phenylalanine was replaced by D-phenylalanine. They had D-amino-acid oxidase (DAO) activity which is essential for the growth in the medium containing D-amino acids. The enzyme activity of the R-Y121B.DF cells was approximately one-fourth of that of the rat liver. Northern hybridization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B.DF cells and the rat liver but not in the parental R-Y121B and H4-II-E cells. Reverse transcription-polymerase chain reaction using DAO-specific primers amplified a DNA fragment of the expected size in the R-Y121B.DF cells but not in the R-Y121B and H4-II-E cells. This fragment was confirmed to be DAO cDNA by nucleotide sequencing. Western blotting showed that DAO protein was present in the R-Y121B.DF cells and the rat liver but not in the R-Y121B and H4-II-E cells. Southern hybridization showed that the DAO gene structure was not different among the R-Y121B.DF cells, R-Y121B cells, H4-II-E cells, and the rat liver. These results indicate that the R-Y121B.DF is a unique cell line which proliferates in the medium containing D-phenylalanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.
Yoda
N
N
Department of Microbiology, Dokkyo University School of Medicine, Tochigi, Japan.
Konno
R
R
Nagashima
S
S
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology*; D002455:Cell Division; D003470:Culture Media; D003605:D-Amino-Acid Oxidase / Q000235:genetics* / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D003470:Culture Media; D011994:Recombinant Proteins; D010649:Phenylalanine; D003605:D-Amino-Acid Oxidase