Hyperthermia, a modality of cancer therapy, has been known as a stress to induce apoptosis. However, the molecular mechanism of heat shock-induced apoptosis, especially on roles of intracellular oxidative stress, is not fully understood. First, when human lymphoma U937 cells were treated with heat shock (44 degrees C, 30 min), the fraction of apoptosis, revealed by phosphatidylserine externalization, increased gradually and peaked at 6 hr after the treatment. In contrast, intracellular superoxide formation increased early during the heat shock treatment and peaked at 30 min after the treatment. When the cells were treated with heat shock in the presence of alpha -phenyl-N-tert-butylnitrone (PBN) and its derivatives, which are potent antioxidants, the DNA fragmentation was inhibited in an order according to the agents' hydrophobicity. PBN showing the highest inhibitory effects suppressed not only intracellular superoxide formation but also various apoptosis indicators. cDNA microarray was employed to analyze gene expression associated with heat shock-induced apoptosis, and the time-course microarray analysis revealed 5 groups showing changes in their pattern of gene expression. Among these genes, c-jun mRNA expression showed more than 40 fold increase 2 hr after heat treatment. The expression level of c-jun mRNA verified by quantitative real-time PCR was about 20 fold increase, and c-jun expression was similarly suppressed by PBN and its derivatives. These results suggest that the change of c-jun expression is an excellent molecular marker for apoptosis mediated by intracellular oxidative stress induced by heat shock.