Pinocytic loading of cytochrome c into intact cells specifically induces caspase-dependent permeabilization of mitochondria: evidence for a cytochrome c feedback loop

Abstract

Previous studies introduced cytochrome c into intact cells via the disruptive techniques of microinjection or electroporation to provide support for the hypothesis that, in whole cells, cytochrome c release from mitochondria triggers caspase activation and other degradative changes. However, the types of measurements that could be undertaken with these techniques was limited. We used the simple and relatively gentle technique of pinocytic loading to demonstrate that, in intact cells, cytosolic cytochrome c specifically induced activation of caspase-3-and -9-like enzymes, and a loss of mitochondrial polarization coincident with an increase in mitochondrial permeability. Our results support the prediction from in vitro studies that activation of caspases-3 and -9 is downstream of cytochrome c release and provide the first direct evidence that, in whole cells, cytochrome c-dependent caspase-activation can exert a feedback effect to elicit mitochondrial permeabilization and collapse of the mitochondrial trans-membrane potential. Cell Death and Differentiation (2001) 8, 631 ± 639.

Comparative analysis of proapoptotic activity of cytochrome c mutants in living cells

“…Caspases or some cytosolic Human T-cell lymphoblastoid Jurkat yes [11][12][13] Human myelo-monocytic U-937 yes 13…”

“…A statement that pinocytic loading of cytochrome c in cells is a "relatively gentle technique" as compared with "the disruptive techniques of microinjection or electroporation" 13 should be considered as a literary hyperbole (exaggeration), since this method is based on the enhancement of pinocytosis at the hyper-osmolar conditions followed by lysis (disruption) of intracellular vesicles, pinosomes, induced by hypo-osmolar shock. As for the viability of cells, here we report on an optimized electroporation procedure that results in a specific activation of apoptosis in up to 80% of cells after cytochrome c electroinjection and in a very low level of dead and apoptotic cells in control experiments.…”

“…13 Keeping in mind that the analyzed lysates were prepared from different (living, apoptotic and, probably, dead) cells, these values can be considered as a very approximate low estimation of the apoptosis-activating amount of cytochrome c in the above cell types. Microinjection of kidney 293 cells with 120 fg/cell cytochrome c corresponded to a threshold of apoptosis activation, 8 but, in fact, the delivered amount of cytochrome c was not exactly known.…”

“…[8][9][10][11][12][13] Currently, three techniques are exploited to load cytochrome c into the cytoplasm of cells: microinjection technique, 8,14-18 electroporation procedure [9][10][11][12] and the method of pinocytic loading of macromolecules. 13 Independently of the way of cytochrome c loading, the investigators agree that exogeneous cytochrome c induces activation of the caspase cascade in living cells and, finally, produces other apoptotic effects such as cell shrinkage, chromatin condensation, oligonucleosomal fragmentation of DNA, fragmentation of nuclei and phosphatidylserine externalization.…”

Mitochondrial Regulation of Caspase Activation by Cytochrome Oxidase and Tetramethylphenylenediamine via Cytosolic Cytochrome c Redox State

“…Previous studies (14) have shown that pinocytic loading of cytochrome c into the cells induced caspase activation and apoptosis. In agreement with this, we found that when macrophage cells were scraped-loaded with oxidized cytochrome c, this resulted in about 2-fold increase in caspase activity measured 2 h after the treatment (Fig.…”