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, wa s investigated on the p53-negative human leukemia cell line HL60. A concent ration- and time-dependent inhibition of cell growth was observed that appe ars 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, increa sed survival rates were observed in cells simultaneously treated with Tempo l and the antioxidant N-acetylcysteine. Tempol did not affect the relative levels of Bar and Bc12, whereas p21(WAF1/CIP1) was enhanced in a concentrat ion- and time-dependent fashion; this effect was partially inhibited by N-a cetylcysteine, was maintained for up to 8 h after Tempol removal, and seeme d to depend on continuing protein synthesis. The increase in p21(WAF1/CLP1) was accompanied by a parallel accumulation of cells in the G(1) phase of t he 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 antiprolifera tive effect of Tempol; on the basis of this observation, the nitroxide coul d be proposed as an useful adjunct to the treatment of p53-deficient tumors , which are often refractory to standard chemotherapy. (C) 2000 Elsevier Sc ience Inc.