Regulated expression of the prodynorphin gene in the R2C Leydig tumor cell line

We report here that prodynorphin mRNA and prodynorphin-derived peptides are synthesized in the R2C rat Leydig tumor cell line. The size of the prodynorphin transcript found in these cells (approximately 2200 nucleotides) is identical to that found in the intact testis. R2C cells also contain proteolytically processed prodynorphin-derived peptides. In R2C cells, the endogenous prodynorphin gene and cellular levels of prodynophin-derived peptides are positively regulated by cAMP analogs, while phorbol esters exert a slight negative regulation of the prodynorphin mRNA. Using gene transfer techniques, we have identified a 210-basepair fragment of the rat prodynorphin gene which initiates the transcription of the bacterial reporter molecule, chloramphenicol acetyl transferase. The chimeric fusion gene, when transfected into R2C cells, exhibited the same positive response to cAMP analogs as the endogenous gene. The results suggest that a cAMP regulatory element resides within the cloned rat prodynorphin fragment, and that the element is functionally active in R2C cells.