Paradoxical effects of thiol reagents on Jurkat cells and a new thiol-sensitive mutant form of human mitochondrial superoxide dismutase.

Abstract

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.