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Loss of mitochondrial membrane potential is dependent on the apoptotic program activated: prevention by R-2HMP
D Zhang1, M D Berry, I A Paterson
1Neuropsychiatry Research Unit, Department of Psychiatry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. daz212@mail.usask.ca
Abstract:
Recent evidence suggests that the mitochondrial membrane potential begins to decrease well before the cells commit to apoptotic death. By using cultured cerebellar granule cells, two types of apoptosis can be induced, one by adding cytosine arabinoside (Ara-c; p53-dependent apoptosis) and one by lowering the K(+) concentrations of the medium (p53-independent apoptosis). Cultures show clear signs of increased apoptosis (chromatin condensation as visualized with bis-benzamide) after 12 hr which increases with time up to 24 hr. A fluorescent probe, chloromethyl-tetramethylrhodamine methyl ester (CMTMR), a lipophilic, potentiometric dye, which when introduced into the media accumulates within mitochondria in proportion to the mitochondrial membrane potential, was added at various time points after the induction of apoptosis. In Ara-c-induced apoptosis, there was a shift in the distribution of cell populations towards low-intensity CMTMR fluorescence, whereas in control and low-K(+) cultures, there was no such shift. This effect was observed as early as 6 hr after adding Ara-c. The antiapoptotic drug R-N-2-heptyl-N-methylpropargylamine hydrochloride (R-2HMP) reversed this loss of mitochondrial membrane potential in Ara-c-induced apoptosis; the effect was antagonized by the S-2HMP.
Insights
Mitochondrial membrane potential loss precedes cell death in apoptosis. This early event was detected using a fluorescent probe in p53-dependent apoptosis induced by cytosine arabinoside (Ara-c).
Area of Science:
- Cell Biology
- Neuroscience
- Biochemistry
Background:
- Mitochondrial membrane potential (MMP) decline is an early event in apoptosis.
- Apoptosis can be induced via p53-dependent (cytosine arabinoside, Ara-c) or p53-independent (low K+) pathways in cerebellar granule cells.
- Chromatin condensation is a late marker of apoptosis, observable after 12 hours.
Purpose of the Study:
- To investigate the temporal relationship between MMP changes and apoptosis.
- To differentiate MMP alterations in p53-dependent versus p53-independent apoptosis.
- To assess the effect of an antiapoptotic drug on MMP during Ara-c-induced apoptosis.
Main Methods:
- Cultured cerebellar granule cells were subjected to Ara-c or low K+ to induce apoptosis.
- Mitochondrial membrane potential was assessed using the fluorescent probe chloromethyl-tetramethylrhodamine methyl ester (CMTMR).
- CMTMR fluorescence intensity was measured at various time points post-apoptosis induction.
Main Results:
- A significant decrease in CMTMR fluorescence (indicating reduced MMP) was observed as early as 6 hours in Ara-c-induced apoptosis.
- No such decrease in MMP was detected in control or low K+-induced (p53-independent) apoptosis.
- The antiapoptotic drug R-N-2-heptyl-N-methylpropargylamine hydrochloride (R-2HMP) reversed the MMP loss in Ara-c-treated cells.
Conclusions:
- Early loss of mitochondrial membrane potential is a hallmark of p53-dependent apoptosis.
- This MMP decrease precedes observable chromatin condensation.
- R-2HMP demonstrates potential in mitigating early apoptotic mitochondrial dysfunction.