Characterizing functional changes that occur in different cellular compartments during programmed cell death/apoptosis are of universal interest. Many techniques have been developed to analyze this process, and indeed, the quantification of cells undergoing apoptosis is also important to determine the efficacy of new drugs, such as anticancer agents or antiviral compounds. Mitochondria represent key organelles for the cell survival, and their role in programmed cell death is known since several years (1–3). Mitochondrial alterations during cell death have been widely described (4, 5); even if cytochrome c release or caspase activation, and consequent DNA degradation can be independent of loss of mitochondrial membrane potential (ΔΨ) (6), we accept that this event is a hallmark of apoptosis (7).
As far as ΔΨ is concerned, taking advantage of the possibility to use different sensitive dyes, it is nowadays simple to identify cells with high or low ΔΨ. The dye JC-1, excited by the argon laser present in all flow cytometers emits in FL-1 (monomers, typical of low ΔΨ) and FL-2 (aggregates, whose formation is due to a high ΔΨ) (8, 9). However, its spectral characteristics can create some difficulties when used simultaneously with other dyes excitable by such a laser. Thus, until now, the study of changes in DNA content and ΔΨ in the same cell during apoptosis have been difficult if not impossible, and all data, including ours (10), have been obtained by separate measures.
We describe here a 5-color staining that allows the simultaneous determination of four independent parameters that can change during apoptosis: ΔΨ, exposure of phosphatidilserine (PS) on plasma membrane's external leaflet, cell viability (in terms of lack of permeability to propidium iodide, PI), and DNA content. In addition, we used a 4-color staining where detection of PS exposure was replaced by analysis of mitochondrial mass. We took advantage of a flow cytometer equipped with three lasers and a UV lamp, which can collect up to 12 fluorescences. By inducing apoptosis in the myeloblastic cell line U937 with flavonoid quercetin (Qu) (11), we found that apoptotic populations with different ΔΨ were also discordant for other parameters, such as DNA content and PI permeability, but not for PS exposure. This was well distinguishable after short time of Qu incubation, while, at prolonged times, cells with different ΔΨ were mostly similar. This indicates that apoptosis proceeds independently from loss of ΔΨ, which is however, the first, irreversible event detectable by the methodology used in this article. Moreover, by the simultaneous analysis of several parameters in the same cell, it is possible to characterize the kinetics of programmed cell death and to determine early and late alterations of various relevant phenomena.
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In this article, we show that, in some models of apoptosis, a population of cells with intermediate ΔΨ can appear. Multiparametric analysis of this population during Qu-induced apoptosis revealed that they have different characteristics from those with high or low ΔΨ. This was well detectable at short times after apoptotic engagement, when Qu induces a relevant oxidative stress (17).
We took advantage of a multilaser flow cytometer that allowed us to detect several independent apoptotic parameters in the same cell: cells with different ΔΨ were investigated for Annexin V positivity, plasma membrane permeability to PI and DNA content. Following JC-1 staining, we identified three populations with different ΔΨ that belong to different stages of the apoptotic process, because they are discordant for other parameters associated to apoptosis. Short times after Qu treatment (6 h), cells with high ΔΨ did not display alterations in physical parameters, exposure of PS, cell viability, and DNA fragmentation/condensation. On the contrary, cells with intermediate ΔΨ were Annexin V+ and had low FSC, but other parameters were still similar to those of the control; this suggests that they could be early apoptotic. Moreover, in these cells, loss of ΔΨ was not accompanied by changes in DNA content (measured by Hoechst fluorescence); thus, changes in ΔΨ seem to precede DNA fragmentation. Hypodiploid DNA content and PI permeability, in addition to PS exposure, were found in cells with fully collapsed ΔΨ. Inside these cells, we also detected a subpopulation of cells that, despite the presence of fragmented DNA, was PI negative. These could be either cellular fragments that are not able to retain PI, or apoptotic bodies that have a very low DNA content, and thus are not fluorescent.
Prolonging the time of treatment with Qu resulted in an increase in cells with intermediate or collapsed ΔΨ. However, despite differences in ΔΨ, cells with intermediate ΔΨ became permeable to PI and they were mostly similar to those with collapsed ΔΨ, except for DNA content. This indicates that cells with intermediate ΔΨ switched to a more advanced apoptotic stage and it suggests that, in this model of apoptosis, loss of ΔΨ seems to be independent from other events associated to apoptosis, because PI permeability and, in part, DNA degradation occurred also in cells with incomplete collapse in ΔΨ. This raises many questions about the role of ΔΨ during apoptosis. It is known that maintaining of ΔΨ is indispensable for the production of ATP which is also required for cell death induction. Recent data suggest that a tight link between glycolysis and apoptosis exists because glucokinase and BAD reside in a mitochondrial macromolecular complex (18). Thus, alterations, but not collapse, of ΔΨ could be important to maintain, at least in part, ATP production that is necessary for the formation of the apoptotsome and for the activation of caspases (19, 20). Nevertheless, more studies are needed to characterize this population with intermediate ΔΨ.
The simultaneous analysis of several parameters has a relevant interest for a better understanding of complex phenomena such as apoptosis. In particular, the role of changes in ΔΨ are under investigations for several years (3, 21, 22). An impairment of mitochondrial function leading to decrease in ΔΨ and the consequent permeability transition are considered hallmarks of apoptosis (7), even if it has been demonstrated that the release of cytochrome c can be independent of mitochondrial depolarization both in a cell free system (23), and in intact cells (24). More recently, it has been shown that decrease in ΔΨ or permeability transition phenomena are per se incapable of triggering caspase activation and nuclear apoptosis (25, 26). Thus, it is possible to conceive that, even in the models in which mitochondrial function is involved, decrease in ΔΨ would be an ancillary event of the apoptotic process. Furthermore, it is not known if all mitochondria are impaired in a similar manner and to what extent, nor if alteration of all mitochondria is required to trigger cell death.
Recently, we found that certain cells do not dissipate completely their ΔΨ after staurosporine treatment (10). We observed the same phenomenon in Qu treated cells. The presence of the intermediate population could be explained by the fact that, during mitochondrial depolarization, the simultaneous presence of a low number of aggregates (orange) and a high number of monomers (green) can be present at the same time. Another possibility exists, which does not contradict what has been previously discussed, i.e. that cells do not depolarize all organelles simultaneously. In any case, further characterization of these cells is of interest for understanding their possible fate.
On the whole, our data indicate that, in this model of apoptosis, (i) loss of ΔΨ, PS exposure, and decrease of mitochondrial mass are early events and they precede permeability to PI and DNA condensation/fragmentation; (ii) populations with different ΔΨ, as revealed by flow cytometry after JC-1 staining, also differ for other parameters associated to apoptosis; (iii) the simultaneous analysis of multiple parameters with polychromatic flow cytometry allows the identification of many stages of the same phenomenon, i.e. apoptosis, and the consequent different sensibility of populations under study with the drug tested. This polychromatic approach thus allows more sophisticated analyses that can reveal the heterogeneity of the response to a given compound or stimulus.