Immune Netw.  2003 Dec;3(4):268-275. 10.4110/in.2003.3.4.268.

Intracellular pH is a Critical Element in Apoptosis Triggered by GM-CSF Deprivation in TF1 Cells

• Yoon SR
• Choi IP

• KMID: 2054230
• DOI: http://doi.org/10.4110/in.2003.3.4.268

Abstract

BACKGROUND
Hemopoietic cells require the constant presence of growth factors for survival in vitro and in vivo. Caspases have been known as central executors of apoptotic cell death. We have, therefore, investigated the pathways that regulate caspase activity and apoptosis using the CD34+ cell line, TF-1 which requires GM-CSF for survival. METHODS: Apoptosis was measured by annexin V staining and mitochondrial membrane potential was measured by DiOC6 labelling. Intracellular pH was measured using pH sensitive fluorochrome, BCECF or SNARF-1, followed by flow cytometry analysis. Caspase activation was analyzed by PARP cleavage using anti-PARP antibody. RESULTS: Removal of GM-CSF induceed PARP cleavage, a hallmark of caspase activity, concomitant with pHi acidification and a drop in mitochondrial potential. Treatment with ZVAD, a competitive inhibitor of caspases, partially rescued cell death without affecting pHi acidification and the reduction of mitochondrial potential, suggesting that both these events act upstream of caspases. Overexpression of Bcl-2 prevented cell death induced by GM-CSF deprivation as well as pHi acidification and the reduction in mitochondrial membrane potential. In parental cells maintained with GM-CSF, EIPA, a competitive inhibitor of Na+/H+ antiporter induced apoptosis, accompanied by a drastic reduction in mitochondrial potential. In contrast, EIPA induced apoptosis in Bcl-2 transfectants without causing mitochondrial membrane depolarization. CONCLUSION: Taken together, our results suggest that the regulation of H fluxes, either through a mitochondrion- dependent or independent pathway, is central to caspase activation and apoptosis.

Keyword

Apoptosis; caspases; intracellular pH; mitochondrial potential

MeSH Terms

Annexin A5
Apoptosis*
Caspases
Cell Death
Cell Line
Flow Cytometry
Granulocyte-Macrophage Colony-Stimulating Factor*
Humans
Hydrogen-Ion Concentration*
Intercellular Signaling Peptides and Proteins
Ion Transport
Membrane Potential, Mitochondrial
Mitochondrial Membranes
Parents
Annexin A5
Caspases
Granulocyte-Macrophage Colony-Stimulating Factor
Intercellular Signaling Peptides and Proteins