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Abstract

The mitochondrial matrix can be specifically labeled by loading cells with calcein and simultaneous quenching of the non-mitochondrial calcein fluorescence with cobalt (Co2+). Positive staining of mitochondria thus requires that the inner mitochondrial membrane functions as a barrier separating calcein (within the matrix) from Co2+ (outside of the matrix). Upon induction of apoptosis, such calcein/Co2+ -labeled cells, demonstrate a decrease in the overall calcein fluorescence resulting from inner mitochondrial membrane permeabilization. This decrease can be quantified by cytofluorometry and can be dissociated from other apoptosis-associated mitochondrial perturbations such as the loss of the mitochondrial transmembrane potential (delta phi m), the local overproduction of re-active oxygen species, and the mitochondrial release of cytochrome c. In some paradigms of apoptosis the loss of calcein/Co2+ (CC) staining can be dissociated from the delta phi m loss, both of which may occur in a caspase-dependent or caspase-independent fashion, depending on the apoptosis inducer. Importantly, inner membrane permeabilization to CC may occur without a permanent delta phi m dissipation in apoptosis, suggesting that transient permeabilization events could participate at the apoptotic cascade. Altogether, our data demonstrate that inner mitochondrial membrane permeabilization constitutes an early event in the apoptotic cascade.

The permeability transition pore complex in cancer cell death

“…Under PT induction, calcein is released from mitochondria and becomes quenched into the cytosol by Co 2 þ , resulting in a decreased fluorescence. Taking advantage of this methodology, several groups reported that PT occurs in vivo in response to Ca 2 þ overload , after injury such as ischemia/reperfusion , oxidative stress (Nieminen et al, 1995, chemotherapy (Poncet et al, 2003) and iron mitochondrial toxicity (Rauen et al, 2004). In many of the above-mentioned models, an inhibition of the events that happen downstream to mitochondria membrane permeabilization are prevented by CsA and/ or BA.…”

Preapoptotic Chromatin Condensation Upstream of the Mitochondrial Checkpoint

“…Bcl-2 and the unrelated vMIA (from cytomegalovirus) can prevent MMP (28,41). However, neither Bcl-2 nor vMIA did prevent the induction of PACC by short term exposure to STS.…”

Mitochondria as therapeutic targets for cancer chemotherapy

“…The consequence of a partial IM permeabilization, which does not result in the release of matrix proteins but contribute to cell death, is that mitochondria lose their vital metabolic and redox functions (Zamzami et al, 1995;Metivier et al, 1998;Poncet et al, 2003). Mitochondrial permeabilization is therefore recognized as a crucial checkpoint in programmed cell death of both normal and cancer cells.…”