The biology of cell death

Particular interest in biology of cell death comes from the discovery of multitude complex regulatory circuits that control the cellular demise.

Historically cells were known die in two distinct morphologically and biochemically processes such as apoptosis and necrosis often referred to as programmed and accidental cell death (necrosis), respectively.

Both were initially identified based on characteristic changes in cell morphology. Figures below outline major morphological and molecular changes occurring during apoptosis versus accidental cell death (herein termed necrosis).

Alterations in those complex cellular parameters become a basis for development of a variety of markers for fluorescence microscopy and flow cytometry.

Despite subsequent introduction of numerous molecular assays, the morphological changes, detected by light and electron microscopy, still remain the ‘‘gold standard’’ to differentiate these two distinct modes of cell death.

Mitochondria as sentinels of xenobiotic-induced cell damage

Apart from functions in bioenergetics the mitochondrial networks stand at the nexus of sensing and integrating diverse incoming stress signals, and mitochondrial disturbances often occur long before any marked morphological symptoms of cell death.

In recent years multiple mechanisms have been revealed that explain mitochondrial function in apoptosis, including release of apoptogenic proteins into the cytosol upon mitochondrial outer membrane permeabilization (MOMP), loss of mitochondrial physiological processes indispensable for cell survival and generation of reactive oxygen species (ROS).

The MOMP is a fundamental event leading to a release of holocytochrome c (cyt c) and an array of cell death modulating small proteins such as AIF, EndoG, Omi/HtrA2, Smac/DIABLO, Smac b, normally enclosed in the intermembrane space of the organell. Dissipation of mitochondrial inner transmembrane potential is frequently associated with MOMP.

Interestingly, as described by our laboratory and others, dissipation of mitochondrial inner transmembrane potential may not be an ultimate point of no return for cell commitment to die.

The detection of mitochondrial inner transmembrane potential loss is a sensitive marker of early apoptotic events. Procedures are based on lipofilic cationic probes that are readily taken up by live cells and accumulate in mitochondria according to the Nernst equation. The extent of their uptake, as measured by intensity of cellular fluorescence, is proportional to mitochondrial inner transmembrane potential.

Majority of sensitive probes are easily applicable for multiparameter detection with other apoptotic markers including caspase activation by fluorescently labelled inhibitors of caspases (FLICA), phosphatidylserine (PS) exposure by Annexin V and plasma membrane permeabilization by propidium iodide (PI) or YO-PRO 1.

Our laboratory has pioneered a plethora of cytometric and imaging teniques and bioassays for the detection of apoptosis as well as mitochondrial function.

Analysis of programmed cell death

In view of recent discoveries of alternative cell death pathways it has been, postulated to restrict the term apoptosis to only the traditional cascade featuring all canonical ‘‘hallmarks of apoptotic cell death,’’ such as (i) activation of caspases as an absolute marker of cell death ; (ii) high degree of compaction of chromatin; (iii) activation of endonucleases(s) causing internucleosomal DNA cleavage and extensive DNA fragmentation; (iv) appearance of distinctive cellular morphology with preservation of organelles, (v) cell shrinkage, (vi) plasma membrane blebbing, and (vii) nuclear fragmentation followed by formation of apoptotic bodies..

Markers for many diverse modes of caspase-dependent cell death have been particularly well developed in aquatic vertebrate models and can be analysed using techniques ranging from molecular techniques, microscopy and flow cytometry as well as imaging cytometry.

Our laboratory employs diverse methods that allow for implementation of apoptotic assays on live suspension, adherent cells, in situ using transgenic models and innovative fluorescent probes as well as fixed specimens such as cell and tissue samples.

We specialise in multi parameter flow and imaging bioassays to detect programmed cell death, cell proliferation and analyse changes in cell cycle such as for instance cell cycle arrest. The major advantages of cytometry include the possibility of multiparameter measurements (correlation of different cellular events at a time), single cell analysis (avoidance of bulk analysis), and rapid analysis of cell populations. Cytometry overcomes a frequent problem of traditional bulk techniques such as fluorimetry, spectrophotometry, or gel techniques (e.g., Western blot, WB). These are based on analysis of a total cell population that averages the results from every given cell.

Moreover, by virtue of multiparameter analysis, cytometry allows correlative studies between many cell attributes based on both light scatter and fluorescence measurements. For example, when cellular DNA content, the parameter that reports the cell cycle position, is one of the measured attributes, an expression of other measured attribute(s) can be then directly related to the cell cycle position.

This advantageous when investigating mechanisms of toxicant action because change in expression of particular cell constituents, or co-expression of different events, if correlated within the same cell, may yield clues regarding a possible cause–effect relationship between the detected events.

Novel technologies such as cell imaging in flow and laser scanning cytometry (LSC) deliver even more sophisticated features that combine superior statistical power of cytometric analysis coupled with in situ and in vivo imaging capabilities.