AN OXIDATIVE STRESS-MEDIATED DEATH PATHWAY IN IRRADIATED HUMAN LEUKEMIA-CELLS MAPPED USING MULTILASER FLOW-CYTOMETRY

OCI/AML-2 acute myeloid leukemia cells were found to undergo apoptosis after treatment with gamma rays from a Cs-137 source. Multilaser flow cytometry techniques using probes for live cell function were used to monitor the biochemical changes that occurred prior to the loss of su rface membrane integrity. These showed increases in the generation of reactive oxygen species (ROS) and in the glutathione (GSH) content of irradiated cells. An additional population of cells that showed a furt her increase in ROS and depletion of GSH was seen in irradiated cells but not in controls. This population showed loss of mitochondrial memb rane potential (Delta Psi(m)), indicative of the mitochondrial permeab ility transition, and exposure of phosphatidylserine on the cell surfa ce. Increases in intracellular calcium were observed in a proportion o f these low-Delta Psi(m)/high-ROS cells. Similar findings were seen us ing the antileukemia drug cytosine arabinoside (ara-C), although cell cycle analysis showed that the loss of Delta Psi(m) occurred mainly in G(1) phase with ara-C treatment, and mainly in G(1) phase with irradi ation. Furthermore, the protective effect of overexpression of BCL2 wa s more pronounced after ara-C treatment than with radiation. Cells of the TP53 (formerly known as p53)-null human AML line OCI M2 showed gro wth arrest in G(2) phase after radiation treatment, with no loss of De lta Psi(m) or morphological changes indicative of apoptosis. The flavi ne-dependent oxidoreductase inhibitor diphenylene iodonium failed to i nhibit generation of ROS in irradiated OCI/AML-2 cells, indicating tha t the mechanism is unlikely to involve the TP53-induced gene PIG3. The se results show that oxidative stress can occur in irradiated human le ukemia ''blasts'', and may play a direct role in radiation-induced apo ptosis, (C) 1998 by Radiation Research Society.