Establishment of a rat hepatoma-derived cell line proliferating in d-phenylalanine medium and expressing d-amino-acid oxidase

Summary.

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.