Induction of apoptosis by heat and gamma-radiation in a human lymphoid cell line; role of mitochondrial changes and caspase activation.
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.
Nijhuis
E H A
EH
University of Twente, Faculty of Science and Technology, Polymer Chemistry and Biomaterials Group, Enschede, The Netherlands.
Poot
A A
AA
Feijen
J
J
Vermes
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I
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
D017304:Annexin A5; D002135:Calcium-Binding Proteins; D020169:Caspases