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 bee n 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 c ells was approximately one-fourth of that of the rat liver. Northern hybrid ization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B . DF cells and the rat liver but not in the parental RY121B and H4-II-E ce lls. Reverse transcription-polymerase chain reaction using DAO-specific pri mers amplified a DNA fragment of the expected size in the R-Y121B . DF cell s 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 prot ein 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 RY121B . 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-phenyl alanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.