We investigated the in vitro effects of arsenic trioxide on cell growth, cell cycle regulation, and apoptosis in As4.1 juxtaglomerular cells. Arsenic trioxide inhibited the growth of As4.1 cells with an IC(50) of approximately 5 microM. Arsenic trioxide induced S phase arrest of the cell cycle and very efficiently stimulated apoptosis in As4.1 cells, as evidenced by flow cytometric detection of sub-G(1) DNA content, annexin V binding assay, and 4'-6-diamidino-2-phenylindole staining. This apoptotic process was accompanied by the loss of mitochondrial transmembrane potential (DeltaPsi(m)), a decrease in Bcl-2, the activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. However, all of the caspase inhibitors tested in this experiment failed to rescue As4.1 cells from arsenic trioxide-induced cell death in view of sub-G(1) cells and annexin V positive-staining cells. However, a caspase-8 inhibitor (Z-IETD-FMK) noticeably decreased the loss of DeltaPsi(m) in arsenic trioxide-treated cells. When we examined the changes in reactive oxygen species (ROS), H(2)O(2), or O(2)(*-) in arsenic trioxide-treated cells, H(2)O(2) was significantly decreased and O(2)(*-) was increased. In addition, we detected a decreased GSH content in arsenic trioxide-treated cells. Taken together, we have demonstrated that arsenic trioxide as a ROS generator potently inhibited the growth of As4.1 JG cells through S phase arrest of the cell cycle and caspase-independent apoptosis.