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 Molt-4 cells, focusing on mitochondrial and cytosolic Ca²⁺ ([Ca²⁺](i)) regulation. 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 Bax expressions (immunoblot), (3) mitochondrial potential Δψ(m) and (4) [Ca²⁺](i) (flow cytometry), (5) caspase-3 activity, and (6) roles of [Ca²⁺]- 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 24h. After 5 Gy, p53 was accumulated that upregulated Bax but which repressed Bcl-2 with time, resulting in a 7-fold increase in Bax/Bxl-2 at 6 h. Predominant Bax reduced Δψ(m) and low-Δψ(m) cells increased 45 min earlier than apoptosis after 5 Gy. Caspase-3 was activated in apoptotic CE. The caspase-3 inhibitor Ac- DEVD-CHO inhibited apoptosis and DNA-ladder formation by ~50%, suggesting a ~50% role of caspase-3-activated DNase (CAD). [Ca²⁺](i) was increased after 5 Gy. [Ca²⁺](i)-chelating BAPTA-AM (5 μM) and/or DNase γ-inhibiting Zn²⁺ (0.5 mM) inhibited ~50% of induced apoptosis and DNA-laddering, indicating a 50% participation of Ca²⁺/Mg²⁺-dependent DNase γ. Conclusions: The p53-Bax-mitochondria-caspase-3-CAD pathway and the [Ca⁺²](i)-mediated DNase γ pathway were involved in the regulation of X- ray apoptosis in sensitive Molt-4 cells.