Paradoxical effects of thiol reagents on Jurkat cells and a new thiol-sensitive mutant form of human mitochondrial superoxide dismutase.
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.
Hernandez-Saavedra
Daniel
D
Webb-Waring Institute for Cancer, Aging, and Antioxidant Research, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
McCord
Joe M
JM
D000483:Alleles; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D019169:Jurkat Cells; D008928:Mitochondria / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000187:drug effects / Q000235:genetics*; D008625:Tiopronin / Q000494:pharmacology*
D017931:DNA Primers; D007527:Isoenzymes; D013438:Sulfhydryl Compounds; D008625:Tiopronin; D013482:Superoxide Dismutase