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

• Published in: International journal of radiation biology 1999, Vol.75 (4), p.493-504
• Main Author: ZHAO, QING-LI
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis - drug effects; Apoptosis - physiology; Apoptosis - radiation effects; bcl-2-Associated X Protein; Biological and medical sciences; Blotting, Western; Calcium - metabolism; Chelating Agents - pharmacology; CHO Cells; Cricetinae; Deoxyribonucleases - antagonists & inhibitors; DNA Damage; DNA, Neoplasm - metabolism; DNA, Neoplasm - radiation effects; Dose-Response Relationship, Radiation; Egtazic Acid - analogs & derivatives; Egtazic Acid - pharmacology; Enzyme Activation; General aspects (metabolism, cell proliferation, established cell line...); HeLa Cells; Humans; Intracellular Fluid - metabolism; Leukemia, Lymphoid - metabolism; Leukemia, Lymphoid - pathology; Leukemia, Lymphoid - radiotherapy; Medical sciences; Membrane Potentials - drug effects; Membrane Potentials - physiology; Mitochondria - physiology; Proto-Oncogene Proteins - biosynthesis; Proto-Oncogene Proteins - physiology; Proto-Oncogene Proteins c-bcl-2 - biosynthesis; Proto-Oncogene Proteins c-bcl-2 - physiology; Tumor cell; Tumor Cells, Cultured; Tumor Suppressor Protein p53 - physiology; Tumors; X-Rays; Zinc - pharmacology
• ISSN: 0955-3002; 1362-3095
• DOI: 10.1080/095530099140429

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 Deltapsi and (4) [Ca2+]i (flow cytometry), (5) caspasem 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 5Gy induced 57 and 94% apoptosis at 6 and 24h. After 5Gy, p53 was accumulated that upregulated Bax but which repressed Bcl-2 with time, resulting in a 7-fold increase in Bax/Bxl-2 at 6h. Predominant Bax reduced Deltapsi m, and low- Deltapsi m cells increased 45 min earlier than apoptosis after 5Gy. 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 5Gy. [Ca2+]i-chelating BAPTA-AM (5mum) and/or DNase gamma -inhibiting Zn2+ (0.5mm) inhibited ~50% of induced apoptosis and DNA-laddering, 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 i-ray apoptosis in sensitive Molt-4 cells.