Türk Biyokimya Dergisi [Turkish Journal of Biochemistry–Turk J Biochem] 2011; 36 (3) ; 261–269

While programmed cell death, apoptosis, occurs as a necessary and natural event for multicellular organisms, necrosis is a form of unplanned cell death as a result of pathological or chemical trauma. There are numerous molecular and morphological differences between these two forms of cell death, whose decision is based on the type and dose of the stress. As apoptosis is critical for homeostasis of an organism, i.e. for development to adult stage, disease progression, or response to different stimuli, it is being studied more extensively in the area of basic research and clinics, and the need for quick detection of apoptosis and well established criteria for the discrimination of apoptosis have also gained more popularity. Here, we review our knowledge on the most commonly used methods for both qualitatively and quantitatively measuring apoptosis, including morphological imaging (i.e. through light, fluorescence, phase contrast or electron microscopy), immunohistochemical (i.e. Annexin V-FITC, TUNEL, M30 antigen or caspase 3 detection), biochemical (i.e. DNA- or protein-based electrophoresis or flow cytometry-based methods), immunological (i.e. ELISA), and molecular biology techniques (i.e. array-based techniques) while focusing on the differences for distinction between the two forms of cell death. Indeed, one has to confirm that cell death occurs through apoptosis based on more than one of these protocols depending on the specific
purpose of the user.

Rather than providing a specific protocol, the following can serve as a general guide to the most frequently utilized options for apoptosis assessment. This is by no means a comprehensive list of options, as the field continues to evolve. For each assay listed, the cellular events and accompanying detection method are described. It may be useful to consider taking advantage of the multiparametric capability of flow cytometry by combining two or more of these assays of apoptosis in a given cell system. For example, Annexin V can be combined with a mitochondrial membrane potential probe rather than a viability dye. Or, multiple regulatory proteins can be combined to evaluate changes in expression with apoptosis induction. In addition to the items below, several review articles are listed as information sources.

Assay Options:
Membrane Phosphotidyl Serine Exposure: In early apoptosis, the cell membrane loses the asymmetry of its membrane phospholipids and phosphotidylserine, a negatively charged phospholipid normally found in the inner leaflet of the plasma membrane becomes exposed on the cell surface. Exposed PS can be detected by binding with Annexin V (a calcium and phospholipid binding protein) conjugated with a fluorochrome. Combining detection using directly-conjugated Annexin V with viability exclusion using Propidium Iodide or 7-AAD (7-aminoactinomycin D) allows for assessment of non-apoptotic vs. early and late apoptotic cells. Numerous assay kits are commercially available.

Mitochondrial Membrane Permeability Transition: A frequently used apoptotic assay, mitochondrial membrane potential assays use permeant live cell probes. The dyes are retained in the mitochondria and thereby fluorescent while the transmembrane potential is intact and released when potential is disrupted (with corresponding loss in fluorescence). Among the probes available for MMP assessment are Rhodamine 123, CMX Rosamine, and TMRE. Choice of these or other similar dyes will depend upon experiment plan.

DNA Strand Breaks (TUNEL): Toward end-stage apoptosis, the final event of DNA strand breaks can be visualized using a TUNEL assay. Fragmented ends of DNA are accessible to terminal deoxynucleotidyl transferase (Tdt), which facilitates the incorporation of DNA precursors onto the fragmented ends. These DNA precursors are detected via antibody or are directly labeled with fluorochrome. There are several commercially available kits which utilize this principle.

Detection of Apoptotic Regulatory Proteins: Commercially available antibodies to some of the apoptotic regulatory proteins can be used in flow cytometry. Among these are the Bcl-2 family (Bcl-2, Bax, Bcl-XL) and the CD95 family of proteins. Antibodies are also available for the active subunit of Caspase 3 and cleaved poly ADP ribose polymerase (PARP). Utilizing more than one of these markers can help delineate subpopulations and apoptotic-stage related expression of both pro- and anti-apoptotic proteins.

Generation of Reactive Oxygen Intermediates: Reactive oxygen intermediates (ROI) play a host of biological roles in the cell-signaling, oxidative burst, etc. In apoptosis, the accumulation of ROI can induce stress leading to cell death. Detection of ROI involves the use of cell probes which are non-fluorescent in their inert state but are converted to fluorescent products upon oxidation by ROI present in cell. Examples of cell probes typically utilized in this procedure include Dichlorofluorescein diacetate, dihydroethidium and dihydrorhodamine 123.

Glutathione (GSH): A complement to the ROI assay, this is a measure of glutathione (antioxidant) levels decreased as a result of increased production of ROI. Assessment of glutathione levels can be assessed by cell probes which react with cellular GSH and become highly fluorescent.

Technical Considerations:
Assay controls: In order to assess that the kit/method you choose is working, it is recommended that known biological positive and negative controls are employed, i.e. a known apoptotic-inducing system and corresponding negative control. Also, independent assessment of test cell condition is useful in correlating the status of cells at the time of assay.

Monolayer cell removal: Scraping can physically disrupt cells and cause artifact in data interpretation, so it is best to use a cell removal method that involves a gentler method of cell removal. This is especially a problem in assays such as Annexin V which usually also contain a membrane impermeant cell viability dye like propidium iodide.

Monolayer cell removal, revisited: The dead/dying cells are usually loose and/or floating, therefore it is essential to retain these cells for analysis purposes.

Assay Timing: For any given apoptosis-inducing system, obtaining optimal flow cytometry data will depend on the timing of the assay; therefore performing a treatment time course is recommended for any unknown system.

Apoptosis Regulatory Protein Detection: As these intracellular proteins are all detected using antibodies following some type of fixation/permeabilization, all of the technical considerations listed under Intracellular Antigen Detection apply.

References:

Steemsa DP, Timm M, Witzig TE. Flow cytometric methods for detection and quantification of apoptosis. Methods in Molecular Medicine, 85:323-32, 2003.

Omerod MG. Using flow cytometry to follow the apoptotic cascade. Redox Report, 6 (5):275-87, 2001.

Vermes I, Haanen C, Reutelingsperger C. Flow cytometry of apoptotic cell death. Journal of Immunological Methods, 243(1-2):167-90, 2000 Sep 21.

Gorczyca W, Melamed MR, Darzynkiewicz, Z. Analysis of apoptosis by flow cytometry. Methods in Molecular Biology, 91:217-38, 1998.

Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1-20, 1997.

Kaynak: http://www.basic.northwestern.edu/sharedresources/flowcytometry/Protocols-appoptosis%20assesment.htm

Suzanne M, Steller H.

J Biol. 2009;8(5):49. Epub 2009 May 28. Review.

Apoptosis, a morphologically and mechanistically distinct form of programmed cell death, is essential for normal animal development and tissue homeostasis. The key executioners in apoptosis are caspases (cysteine aspartases), a family of proteases that have been conserved through much of animal evolution. Caspases are present as inactive precursor proteins in virtually all cells and are specifically activated by proteolytic cleavage. Their activation is regulated by both activators, which promote the conversion of the weakly active precursor caspase to the mature protease, and inhibitors, which prevent unwanted caspase activity and cell death [1]. One important family of caspase inhibitors comprises the inhibitor of apoptosis proteins (IAPs), which can directly bind to and inhibit caspases. In Drosophila, Diap1 is required to prevent inappropriate caspase activation and ubiquitous apoptosis. In response to death-inducing stimuli, antagonists of IAPs such as Reaper, Hid and Grim are produced to inactivate Diap1 and thereby remove the ‘brakes on death’. Although caspases are often viewed as general destroyers of cellular components during apoptosis, there are now many studies showing that they can act with a great degree of local specificity to remove unwanted cellular compartments [2-4].

Cleavage by caspases can either activate or inactivate their substrates; for example, cleavage activates the Rho-associated kinase ROCK1, which promotes membrane blebbing [5,6], whereas proteolysis by a caspase inhibits the DNase inhibitor iCAD and unleashes DNA fragmentation by the CAD nuclease [7,8]. Among the very large number of caspase substrates identified so far, only a few have been linked to a specific apoptotic function. In a recent paper in BMC Developmental Biology, Kessler and Muller [9] describe one such example. They show that cleavage of the &豪-catenin homolog Armadillo (Arm) by the effector caspase DrICE in Drosophila is essential to regulate the adhesive properties of apoptotic cells.

Caspase-mediated cleavage of &豪-catenin promotes changes in cell adhesion and cell shape (a) Drosophila; (b) mammals. Adherens junctions are composed of adhesion complexes of E-cadherin (gray bars), &豪-catenin (Armadillo (Arm); green ovals) and &豩-catenin (&豩-cat; blue circles), which link to the actin cytoskeleton. When apoptosis is induced, DrICE in Drosophila or its homolog caspase-3 in mammals are activated in the apoptotic cell (dark gray). DrICE cleaves Armadillo near the amino terminus (Arm&豌N), whereas mammalian capsase-3 cleaves &豪-catenin near both the amino and carboxyl termini. In Drosophila, an early stage of apoptosis has been described in which the cleaved form of Armadillo remains at the membrane linked to &豩-catenin, whereas E-cadherin is removed from the membrane by an unknown mechanism. In mammals, nothing is known so far about an intermediate step in adherens junction degradation in response to induction of apoptosis. At a later stage of apoptosis, all adherens junction components are removed from the membrane and the actin cytoskeleton retracts. Meanwhile, neighboring cells form new adherens junctions with each other and close the gap created by the retraction of the dying cell.

Biochem J. 1997 Aug 15;326 ( Pt 1):1-16.

Apoptosis is a major form of cell death, characterized initially by a series of stereotypic morphological changes. In the nematode Caenorhabditis elegans, the gene ced-3 encodes a protein required for developmental cell death. Since the recognition that CED-3 has sequence identity with the mammalian cysteine protease interleukin-1 beta-converting enzyme (ICE), a family of at least 10 related cysteine proteases has been identified. These proteins are characterized by almost absolute specificity for aspartic acid in the P1 position. All the caspases (ICE-like proteases) contain a conserved QACXG (where X is R, Q or G) pentapeptide active-site motif. Capases are synthesized as inactive proenzymes comprising an N-terminal peptide (prodomain) together with one large and one small subunit. The crystal structures of both caspase-1 and caspase-3 show that the active enzyme is a heterotetramer, containing two small and two large subunits. Activation of caspases during apoptosis results in the cleavage of critical cellular substrates, including poly(ADP-ribose) polymerase and lamins, so precipitating the dramatic morphological changes of apoptosis. Apoptosis induced by CD95 (Fas/APO-1) and tumour necrosis factor activates caspase-8 (MACH/FLICE/Mch5), which contains an N-terminus with FADD (Fas-associating protein with death domain)-like death effector domains, so providing a direct link between cell death receptors and the caspases. The importance of caspase prodomains in the regulation of apoptosis is further highlighted by the recognition of adapter molecules, such as RAIDD [receptor-interacting protein (RIP)-associated ICH-1/CED-3-homologous protein with a death domain]/CRADD (caspase and RIP adapter with death domain), which binds to the prodomain of caspase-2 and recruits it to the signalling complex. Cells undergoing apoptosis following triggering of death receptors execute the death programme by activating a hierarchy of caspases, with caspase-8 and possibly caspase-10 being at or near the apex of this apoptotic cascade.

Link için tıklayınız

Apoptosis is a naturally occurring process by which a cell is directed to Programmed Cell Death. Apoptosis is based on a genetic program that is an indispensable part of the development and function of an organism. In this process, cells that are no longer needed or that will be detrimental to an organism or tissue are disposed of in a neat and orderly manner; this prevents the development of an inflammatory response, which is often associated with Necrotic cell death. There are at least two broad pathways that lead to Apoptosis, an “Extrinsic” and an “Intrinsic” Pathway. In both pathways, signaling results in the activation of a family of Cys (Cysteine) Proteases, named Caspases that act in a proteolytic cascade to dismantle and remove the dying cell

Tam metin için tıklayınız

“Apoptozis klasik hücre ölüm şekli olarak bilinen nekrozisden birçok özelliği açısından oldukça farklı olan bir hücre ölüm mekanizmasıdır. Biyolojik bilimler literatüründe apoptozis terimi, ilk defa İskoçyalı araştırmacılar olan Kerr, Wyllie ve Currie tarafından 1972 yılında kullanılmış ve canlı dokulardaki hücre azalmalarından sorumlu olan, yapısal olarak özgün bir hücre ölüm tipi olarak tanımlanmıştır. Apoptozis veya, daha genel anlamda söylemek gerekirse, hücre ölümü uzun süre araştırmacıların çok ilgilenmedikleri bir alan olarak kalmıştır. Fakat apoptozisin gelişim biyolojisinde, normal doku “turnover”ında ve immun sistem hücrelerinin sitotoksik fonksiyonları gibi bazı önemli fizyolojik süreçlerdeki rolü ortaya çıktıkça önemi de hızla artmıştır.
Her saniye yaklaşık bir milyon hücremiz apoptozisle vücuttan uzaklaştırılmaktadır. Bunların yerine yenileri yapılmaktadır. Yapım (mitozis) ile yıkım (apoptozis) arasında kontrollu bir denge vardır. İşte bu dengenin apoptozisin lehine veya aleyhine bozulması birçok önemli hastalığın patogenezine katkıda bulunur. Görüldüğü gibi apoptozis organizmada doğru bir şekilde işlemelidir. Olmaması gerekirken gerçekleşen apoptozis veya hızlanmış ya da tam tersine yavaşlamış apoptozis organizma için tehlikelidir. Apoptozisin gereksiz yere oluştuğu veya hızlandığı hastalıklara örnek olarak AIDS, nörodejeneratif hastalıklar, insüline bağımlı tip diyabet, hepatit C infeksiyonu, miyokard enfarktüsü, ateroskleroz gibi hastalıklar verilebilirken; apoptozisin yavaşladığı hastalıklara örnek olarak ise otoimmün hastalıklar ve kanser verilebilir.”