Maintenance of intracellular levels of glutathione(GSH), glutathione disulfide (GSSG), and protein:glutathione mixed disulfides (Pr-SSG) s well as the relative GSH/GSSG is required for normal cell function. Biological thiols undergo reversible oxidation as essential reactions in numerous cellular systems including DNA synthesis, protein synthesis, protein secretion, enzyme regulation, hormone metabolism nd detoxication. The interaction between both high and low molecular weight monothiols or dithiols and disulfides are normally too slow at physiological pH to take place to any significant extent. A group of enzymes that catalyze these exchange reactions have been identified and are known as thiol protein disulfide isomerase, thioredoxin and thioltransferase (also known as glutaredoxin) (TT,GRX). Agents that affect the GSH/GSSG status of a cell, e.g., peroxides, may influence vital cellular functions provided the cells are equipped with normal levels of the exchange enzymes. Recently, evidence was acquired that transformed human fibroblasts (clone HT 1080) are deficient in TT,GRX activity when compared with normal human fibroblasts. This phenomenon will be characterized with respect to effects of cell density, cell cycle and growth factor requirements using cloned cDNA for human TT,GRX mRNA Northern analysis, antibodies against human TT,GRX for Western blot analysis, and enzymatic activity as probes. Additional matched sets of transformed human fibroblasts and their parental lines will be similarly studied. In addition, malignant rat mammary cells (Huggins model) will be investigated by analogous protocols. the antitumor drug, cisplatin, was shown to strongly inhibit pure pig liver TT,GRX and to decrease normal human fibroblast levels by 75% after incubation of cells for 1 hr with 1 mM cisplatin. The possibility that cisplatin, which is more cytotoxic to transformed cells than normal, may cause malignant cells to be more vulnerable to its action, because of the lack of expression of the gene for TT,GRX, will be a major hypothesis to be tested in this proposal. In addition, this approach should lead to a better understanding of the cellular functions of TT,GRX, especially if repression is found to be correlated with various unusual properties of transformed cells, such as sensitivity to extracellular matrix, loss of contact inhibition, etc.
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