The nitroxide tempol induces oxidative stress, p21(WAF1/CIP1), and cell death in HL60 cells.
The antiproliferative effect of Tempol, a stable nitroxide free radical, was investigated on the p53-negative human leukemia cell line HL60. A concentration- and time-dependent inhibition of cell growth was observed that appears to be due to induction of apoptosis. Involvement of oxidative stress is indicated by a concentration-dependent increase in intracellular peroxides and a parallel decrease in total cellular glutathione; in addition, increased survival rates were observed in cells simultaneously treated with Tempol and the antioxidant N-acetylcysteine. Tempol did not affect the relative levels of Bax and Bcl2, whereas p21(WAF1/CIP1) was enhanced in a concentration- and time-dependent fashion; this effect was partially inhibited by N-acetylcysteine, was maintained for up to 8 h after Tempol removal, and seemed to depend on continuing protein synthesis. The increase in p21(WAF1/CIP1) was accompanied by a parallel accumulation of cells in the G(1) phase of the cycle and by a decrease in the 110 kDa form of pRb. Our results suggest that p53-independent induction of p21(WAF1/CIP1) mediates the antiproliferative effect of Tempol; on the basis of this observation, the nitroxide could be proposed as an useful adjunct to the treatment of p53-deficient tumors, which are often refractory to standard chemotherapy.
Gariboldi
M B
MB
University of Insubria, Varese, Department of Structural and Functional Biology, Section of Pharmacology, Milan, Italy.
Rimoldi
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V
Supino
R
R
Favini
E
E
Monti
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E
D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003497:Cyclic N-Oxides / Q000633:toxicity*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013113:Spin Labels; D051028:bcl-2-Associated X Protein
D000975:Antioxidants; C494826:BAX protein, human; C493956:CDKN1A protein, human; D003497:Cyclic N-Oxides; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013113:Spin Labels; D051028:bcl-2-Associated X Protein; D005978:Glutathione; C001803:tempol; D000111:Acetylcysteine