Involvement of c-Jun NH2-terminal kinase-1 in heat-induced apoptotic cell death of human monoblastic leukaemia U937 cells.
OBJECTIVE
To determine the involvement of c-Jun NH(2)-terminal kinase-1 (JNK1) and possibly of HSP27 in heat-induced apoptosis of human monoblastic leukaemia U937 cells.


METHODS
Dominant negative JNK1 (APF), in which the phosphorylation sites Thr-Pro-Tyr were changed to Ala-Pro-Phe, was overexpressed in U937 cells. Cell viability and DNA fragmentation were analysed by the erythrosin-B dye exclusion test and by agarose gel electrophoresis, respectively. Expression of activated caspase-9, phosphorylated JNK1, JNK2, p38 and HSP27 was examined by Western blotting. JNK1 kinase assay was also performed using c-Jun as a substrate.


RESULTS
Loss of viability, activated cleavage form of caspase-9 and DNA fragmentation were rapid in U937 cells after 44 degrees C hyperthermia, while overexpression of dominant negative JNK1 interfered with phosphorylation or activation of JNK1 without affecting that of JNK2 or p38/SAPK, and apparently delayed or reduced cleavage and activation of caspase-9, DNA fragmentation and cell death. Heat-induced phosphorylation of HSP27, observed in parental U937 cells, was suppressed and only slightly detectable in jnk1 mutant cells.


CONCLUSIONS
Prolonged phosphorylation or activation of JNK1 was considered important for heat-induced apoptosis and JNK1 may control the process possibly through phosphorylation of HSP27 and caspase-9 activation in U937 cells.
Enomoto
A
A
Department of Radiation Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Toyko 113-0033, Japan.
Suzuki
N
N
Liu
C
C
Kang
Y
Y
Zhu
J
J
Serizawa
S
S
Matsumoto
Y
Y
Morita
A
A
Ito
M
M
Hosoi
Y
Y
D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D053938:DNA Fragmentation; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D006358:Hot Temperature; D006801:Humans; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D010766:Phosphorylation; D020298:U937 Cells
D017931:DNA Primers; D004792:Enzyme Precursors; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases