Mitochondrial and intracellular free-calcium regulation of radiation-induced apoptosis in human leukemic cells

Purpose: To investigate the mechanisms and pathways of X-ray apoptosis in M olt-4 cells, focusing on mitochondrial and cytosolic Ca2+ ([Ca2+](i)) regul ation. Materials and methods: X-irradiated Molt-4 cells and cell extract (CE) were used to analyse: (1) induced apoptosis (Giemsa stain), (2) p53, Bcl-2 and Bar expressions (immunoblot), (3) mitochondrial potential Delta psi(m) and (4) [Ca2+](i) (flow cytometry), (5) caspase-3 activity, and (6) roles of [C a2+]- and caspase-3-mediated pathways by inhibiting either or both pathways for induced apoptosis. Results: Molt-4 cells were sensitive to apoptosis since 5 Gy induced 57 and 94% apoptosis at 6 and 24-h. After 5Gy, p53 was accumulated that upregulat ed Bar but which repressed Bcl-2 with time, resulting in a 7-fold increase in Bax/Bxl-2 at 6 h. Predominant Bar reduced Delta psi(m), and low-Delta ps i(m) cells increased 45 min earlier than apoptosis after 5 Gy. Caspase-3 wa s activated in apoptotic CE. The caspase-3 inhibitor Ac-DEVD-CHO inhibited apoptosis and DNA-ladder formation by similar to 50%, suggesting a similar to 50% role of caspase-3-activated DNase (CAD). [Ca2+](i) was increased aft er 5Gy. [Ca2+](i)-chelating BAPTA-AM (5 mu M) and/or DNase gamma-inhibiting Zn2+ (0.5 mM) inhibited similar to 50% of induced apoptosis and DNA-ladder ing, indicating a 50% participation of Ca2+/Mg2+-dependent DNase gamma. Conclusions: The p53-Bax-mitochondria-caspase-3-CAD pathway and the [Ca2+]( i)-mediated DNase gamma pathway were involved in the regulation of X-ray ap optosis in sensitive Molt-4 cells.