title: Mitochondrial and intracellular free-calcium regulation of radiation-induced apoptosis in human leukemic cells.
abstract: OBJECTIVE
  To investigate the mechanisms and pathways of X-ray apoptosis in Molt-4 cells, focusing on mitochondrial and cytosolic Ca2+ ([Ca2+]i) regulation.
  
  
  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(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 24 h. 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 6 h. Predominant Bax reduced deltapsi(m), and low-deltapsi(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 approximately 50%, suggesting a approximately 50% role of caspase-3-activated DNase (CAD). [Ca2+]i was increased after 5 Gy. [Ca2+]i-chelating BAPTA-AM (5 microM) and/or DNase gamma-inhibiting Zn2+ (0.5 mM) inhibited approximately 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 [Ca+2]i-mediated DNase gamma pathway were involved in the regulation of X-ray apoptosis in sensitive Molt-4 cells.
authors.affiliation: Department of Radiation Biophysics and Genetics, Kobe University School of Medicine, Japan.
authors.forename: Q L
authors.initials: QL
authors.lastname: Zhao
authors.forename: T
authors.initials: T
authors.lastname: Kondo
authors.forename: A
authors.initials: A
authors.lastname: Noda
authors.forename: Y
authors.initials: Y
authors.lastname: Fujiwara
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects*; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D006224:Cricetinae; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000378:metabolism / Q000528:radiation effects; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors; D004307:Dose-Response Relationship, Radiation; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007945:Leukemia, Lymphoid / Q000378:metabolism / Q000473:pathology* / Q000532:radiotherapy*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000502:physiology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D014965:X-Rays; D015032:Zinc / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D002614:Chelating Agents; D004273:DNA, Neoplasm; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease; D015032:Zinc; D002118:Calcium