Studies on the Non-Protein Thiols of a Human Prostatic Cancer Cell Line: Glutathione Content

The low molecular weight thiol (-SH) content of a human prostate carcinoma cell line (LNCap), important to the cellular resistance to drugs and irradiation, was investigated using three forms of thiol assay each utilizing different chemistries. The composition of the mixture was examined by derivatization of the thiols with a three-fold excess of the Ellman reagent to give mixed aromatic disulfides. The components were isolated by chromatography on C18 reverse phase silica gel followed by DE52 anion exchange separation, and then analyzed by capillary electrophoresis against prepared standards. The glutathione adduct (GSSE) and an unknown disulfide (RSSE) were the major components isolated on DE52 together with two minor ones. However, from the absorbance at 325 nm, it was found that the GSSE isolated (1.5 ± 0.2 femtomoles/cell) could only account for 28.5 ± 4.3% of the total ASF thiols. It appeared that the bulk of the thiol material had not formed a stable mixed disulfide with Ellman's reagent, and this was confirmed by 35S labeling of the cells. A large proportion of the 35S labeled components, obtained after reaction of the ASF thiols with the Ellman reagent, did not form mixed aromatic disulfides and could therefore not be identified by this labeling method.