title: Enhancement of heme oxygenase-1 synthesis by glutathione depletion in Chinese hamster ovary cells.
abstract: Chinese hamster ovary cells cultured in vitro were used to assess the role of glutathione metabolism in the induction of the 32-kDa stress protein. Enhanced synthesis of the 32-kDa protein was observed after cells were incubated with CdCl2 or diethylmaleate and protein was subjected to SDS-PAGE followed by fluorography. Concomitantly, in both cell preparations an increase in heme oxygenase activity was observed. Proteins from CdCl2- and diethylmaleate-treated cells were subjected to Western blotting and protein crossreacting with either rabbit antibody to rat liver heme oxygenase-1 (32,000 Mr) or rat testis heme oxygenase-2 (36,000 Mr) quantitated. The analysis indicated that the CdCl2 treatment increased the intensity of the HO-1 band 5.5-fold while the diethylmaleate treatment increased it three-fold relative to control. Neither treatment affected the intensity of HO-2 antibody binding. Incubation of cells with buthionine sulfoximine, under conditions which resulted in greater than or equal to 90% of the intracellular glutathione being depleted, enhanced synthesis of a 32-kDa protein when assayed by SDS-PAGE. This protein exhibited a Mr similar to the 32-kDa protein induced by either CdCl2 or diethylmaleate treatment. Proteins from buthionine sulfoximine and diethylmaleate-treated cells were mixed together and subjected to 2D PAGE. The resulting fluorograph demonstrated that both treatments produced identical patterns. In contrast, incubation of cells in diamide, a thiol oxidizing compound, resulted in enhanced synthesis of the 110-, 90-, and 73-kDa heat shock proteins but not the 32-kDa protein. The data presented have shown that depletion of glutathione by two independent methods, conjugation and inhibition of synthesis, enhances the synthesis of a 32-kDa protein identified as heme oxygenase-1; oxidation of glutathione, on the other hand did not. We interpret this to indicate that glutathione depletion rather than conjugation or oxidation represents one pathway for induction of heme oxygenase-1.
authors.affiliation: Vanderbilt Center for Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors.forename: E L
authors.initials: EL
authors.lastname: Saunders
authors.forename: M D
authors.initials: MD
authors.lastname: Maines
authors.forename: M J
authors.initials: MJ
authors.lastname: Meredith
authors.forename: M L
authors.initials: ML
authors.lastname: Freeman
mesh_terms: D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D015153:Blotting, Western; D019328:Buthionine Sulfoximine; D002104:Cadmium / Q000494:pharmacology; D019256:Cadmium Chloride; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008298:Maleates / Q000494:pharmacology; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D008970:Molecular Weight; D010053:Ovary
chemical_list: D000963:Antimetabolites; D006360:Heat-Shock Proteins; D008298:Maleates; D002104:Cadmium; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D006419:Heme Oxygenase (Decyclizing); C014476:diethyl maleate; D005978:Glutathione; D019256:Cadmium Chloride