Expression of genes involved in initiation, regulation, and execution of apoptosis in human neutrophils and during neutrophil differentiation of HL-60 cells.
Neutrophils possess a very short lifespan, dying by apoptosis. HL-60 cells undergo apoptosis after neutrophil differentiation with dimethyl sulfoxide (DMSO). We have found that the onset of apoptosis in neutrophil-differentiating HL-60 cells correlates with the achievement of an apoptosis-related gene expression pattern similar to that of peripheral blood mature neutrophils. Using reverse transcriptase-polymerase chain reaction, cloning, and sequencing techniques, we have found that HL-60 cells express bak, bik, bax, bad, bcl-2, bcl-xL, bcl-w, bfl-1, fas, and caspases 1-4 and 7-10. After DMSO treatment, bak, bcl-w, bfl-1, fas, and caspases 1 and 9 were up-regulated, whereas bik, bcl-2, and caspases 2, 3, and 10 were down-regulated at different degrees, achieving mRNA expression levels that correlated with those detected in peripheral blood neutrophils. Caspase-2 mRNA and protein expression was drastically reduced after HL-60 cell differentiation, being absent in both HL-60-differentiated neutrophils and mature neutrophils, whereas caspase-3 and -10 mRNA and protein expression were diminished upon HL-60 cell differentiation until achieving the respective levels found in mature neutrophils. Bak and bfl-1 mRNA levels were largely increased during DMSO-induced differentiation of HL-60 cells, and these genes were the bcl-2 family members that were expressed most abundantly in mature neutrophils. Bcl-2 overexpression or caspase inhibition prevented differentiation-induced apoptosis in HL-60 cells, but not their differentiation capability. Neutrophil spontaneous apoptosis was also blocked by the caspase inhibitor z-Asp-2,6-dichlorobenzoyloxymethylketone. Peripheral blood neutrophils expressed bak, bad, bcl-w, bfl-1, fas, and caspases 1, 3, 4, and 7-10, but hardly expressed bcl-2, bcl-xL, bik, bax, and caspase-2. These results suggest that the above gene expression changes in neutrophil-differentiating HL-60 cells may play a role in the acquisition of the neutrophil apoptotic features.
Santos-Beneit
A M
AM
Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Spain.
Mollinedo
F
F
D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000235:genetics; D020169:Caspases / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005786:Gene Expression Regulation; D019254:Genes, bcl-2; D018922:HL-60 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D015778:Minor Histocompatibility Antigens; D024101:Mitochondrial Proteins; D009504:Neutrophils / Q000166:cytology / Q000502:physiology*; D011506:Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D019014:fas Receptor / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; C494819:BAD protein, human; C494772:BAK1 protein, human; C494826:BAX protein, human; C096816:BCL2-related protein A1; C494811:BCL2L1 protein, human; C096820:BIK protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; D015778:Minor Histocompatibility Antigens; D024101:Mitochondrial Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D019014:fas Receptor; D020169:Caspases; D004121:Dimethyl Sulfoxide