title: Induction of apoptosis by heat and gamma-radiation in a human lymphoid cell line; role of mitochondrial changes and caspase activation.
abstract: OBJECTIVE
  The aim of the study was to investigate the molecular mechanisms involved in apoptosis of human promyelocytic cells (HL60) induced by hyperthermia and to compare this to radiation-induced apoptosis as a reference model.
  
  
  METHODS
  Apoptosis of HL60 cells was induced by heat-treatment (430C during 1 h) or by gamma-radiation (8 Gy) and followed at increasing time periods after treatment with Annexin V binding to phosphatidylserine (PS). The transition of the mitochondrial membrane potential (delta psim) was estimated by the extent of mitochondrial JC-1 uptake. Bcl-2 and Bax protein expression levels were monitored using fluorescent-labelled antibodies. Caspase activation was studied using a fluorochrome-labelled pan-caspase inhibitor (FLICA), which also allowed one to study the kinetics of the apoptotic cascade.
  
  
  RESULTS
  After heat-treatment or irradiation of HL60 cells, a decreased delta psim as well as PS membrane expression were detectable after 8 h. Bcl-2 and Bax protein expression levels were decreased and increased, respectively, 1 h after heat-treatment or irradiation. The apoptotic rate of HL60 cells, as measured by the FLICA binding, was faster with heat-treatment as compared to gamma-irradiation. Addition of a pan-caspase inhibitor prevented PS externalization after heat-treatment but not after irradiation. The presence of a pan-caspase inhibitor did not influence the decrease of delta psim both after heat-treatment and gamma-irradiation. However, the addition of the specific caspase-2 inhibitor zVDVAD-fmk prevented the mitochondrial breakdown after heat-treatment. Inhibition of caspase-2 had no effect on the gamma-irradiation induced apoptosis.
  
  
  CONCLUSIONS
  These results suggest that the commitment to apoptosis in HL60 cells after heat-treatment is started by mitochondrial membrane transition involving the Bcl-2 family members and is mainly executed in a caspase-dependent pathway. The results suggest that caspase-2 plays a key role in the heat-induced apoptosis.
authors: University of Twente, Faculty of Science and Technology, Polymer Chemistry and Biomaterials Group, Enschede, The Netherlands.
authors: E H A
authors: EH
authors: Nijhuis
authors: A A
authors: AA
authors: Poot
authors: J
authors: J
authors: Feijen
authors: I
authors: I
authors: Vermes
mesh_terms: D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism*; D005720:Gamma Rays; D018922:HL-60 Cells; D006801:Humans; D006979:Hyperthermia, Induced; D053078:Membrane Potential, Mitochondrial / Q000502:physiology / Q000528:radiation effects; D008928:Mitochondria / Q000502:physiology* / Q000528:radiation effects
chemical_list: D017304:Annexin A5; D002135:Calcium-Binding Proteins; D020169:Caspases