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Title 

Intracellular pH is a critical element in apoptosis triggered by GM-CSF deprivation in TF1 cells

Authors 

Suk Ran YoonIn Pyo Choi

Issue Date 

2003

Citation 

Immune Network, vol. 3, no. 4, pp. 268-275

Keywords 

apoptosiscaspasesintracellularpHmitochondrial potential

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 mitochondriondependent or independent pathway, is central to caspase activation and apoptosis.

ISSN 

1598-2629

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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