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Paradoxical Effects of Thiol Reagents on Jurkat Cells and a New Thiol-sensitive Mutant Form of Human Mitochondrial Superoxide Dismutase¹

Webb-Waring Institute for Cancer, Aging, and Antioxidant Research, University of Colorado Health Sciences Center, Denver, Colorado 80262

The imbalance between oxidants and antioxidants in cells often results in pathological processes and/or diseases. This delicate balance is achieved in part by antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase (SOD), as well as by low-molecular-weight reductants such as glutathione. We evaluated the effect of thiol reagents on the proliferation of the Jurkat human T-cell leukemia-derived cell line. The cells show a multiphasic behavior when grown in the presence of thiols. Low concentrations of N-2-mercaptopropionyl glycine (0.03 mM) cause growth arrest, and intermediate concentrations (0.3–1.0 mM) induce apoptosis. Similarly, 1 mM N-acetylcysteine or glutathione induce apoptosis in more than 40% of the cells. Surprisingly, the cells grow well in higher concentrations (3–10 mM) of these reagents. Because the I58T variant of human SOD2 is thiol-sensitive, we measured SOD in Jurkat cells grown in the presence of thiol agents, observing markedly less SOD activity. In cell-free extracts, thiols quickly eliminated the SOD2 activity. Jurkat cells contain little SOD2 activity, with a different electrophoretic mobility from that of normal lymphocytes. Single-strand conformational polymorphism analysis of the Jurkat sod2 gene revealed a pattern different from the wild-type gene, suggesting a mutation in the sod2 gene. This was confirmed by cloning and sequencing the gene. Jurkat cells are heterozygous for a new mutation, L60F, in exon 3 of the mature protein. Our findings suggest a possible association between decreased SOD2 activity and malignant phenotype.

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