417 The ATM-Chk2 kinase pathway: molecular interaction maps and therapeutic rationale

Abstract

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 p21WAF1/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 p21WAF1/CIP1 was accompanied by a parallel accumulation of cells in the G1 phase of the cycle and by a decrease in the 110 kDa form of pRb. Our results suggest that p53-independent induction of p21WAF1/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.