Repression of alkaline phosphatase in human cell cultures by cystine and cysteine.

Regulation of enzyme synthesis in microorganisms in many instances is controlled by the intracellular concentration of small molecular compounds.' Regulation may be positive in the sense that an inducer is required for synthesis, or negative in that synthesis occurs because of release from repression. For example, tyrosinase of Neurospora is regulated by both inductive and.xepressive mechanisms: an increase in the sulfur content of the mediumna represses tyrosinase synthesis and the enzyme is induced by aromatic amino acids.2 A few examples of inductive and repressive effects have been described in cells of higher organisms in tissue culture.3-7 Alkaline phosphatase in certain strains of mammalian cells is induced by adrenal glucocorticoid hormones or by a substrate of the enzyme, such as phenyl phosphate.8-12 In general, skin fibroblasts are induced by substrates but not by hormone, whereas epithelial cell lines are induced by hormone but not by substrates. However, several lines of both epithelial and fibroblastic type are induced to form alkaline phosphatase by both hormone and substrate.9 12 In microorganisms alkaline phosphatase synthesis is repressed by inorganic phosphate in the medium.13 The enzyme in mammalian cells is independent of the concentration of inorganic phosphate.10 However, alkaline phosphatase in certain inducible and constitutive mammalian cell strains is decreased on incubation in fresh growth medium, suggesting that a repressor may be present.9' 10, 14 Other evidence for a repressor in fresh medium may be inferred from the following observations: (1) Medium in which cells have grown for at least 20 days is able to induce high alkaline phosphatase activity within a few days in skin fibroblasts.9 The aged, inducing medium, is referred to as "conditioned" medium. (2) Substrate induction of most skin fibroblastic strains by phenyl phosphate requires 7 to 10 days.9 However, when skin fibroblasts of an inducible strain are inoculated into medium containing phenyl phosphate in which other cells have been previously induced, a more prompt induction of alkaline phosphatase occurs within 3 to 5 days. (3) Dilution of "conditioned" medium with a small amount of fresh medium destroys its inducing activity.9 These findings suggest that "conditioning" of medium both with and without phenyl phosphate involves removal of a repressor present in fresh medium. The following experiments indicate that L cyst(e)ine is the repressor or is converted to one. Methods and Materials.-Cell lines: The methods used have been described previously.9' 10 The skin cell strains are uncloned, diploid, human fibroblast-like cells established in culture following trypsinization of foreskin. The cultures were grown on glass surfaces in Waymouth's medium containing either 10% calf serum or 10% human serum. Two HeLa lines were studied. "Ch" is a constitutive, prednisolone noninducible strain obtained from Mr. Lawrence Chessin and grown in Waymouth's medium with 10% calf serum. HeLa S3, furnished by Dr. Harold Nitowsky, has low alkaline phosphatase activity which is inducible by prednisolone. It was grown in Waymouth's medium containing 10% human serum.