UT, MD Anderson, Science Park - Research Division Analysis of Apoptosis

Apoptosis

Normal cells have biological programs for replication as well as suicide. Cancer cells are distinguished by their inability to die and the rate at which they proliferate. Cell death typically follows one of two paths, apoptosis (programmed cell death) or necrosis (accidental cell death). Apoptosis is different from necrosis and begins when a cell activates it's own destruction by initiating a series of complex cascading events that include; depolarization of the mitochondrial membrane potential, down regulation of Bcl-2, activation of caspases (cysteine proteases), cleavage of the DNA repair enzyme PARP, translocation of phosphatidylserine from the inner cytoplasmic membrane to the cell surface, endogenous endonuclease release promoting DNA degradation, chromatin condensation forming crescent bodies, a decrease in cell volume and an increase in cell surface side scatter properties. Necrosis by contrast is not self initiated and results from an insult or injury to the cell such as cytotoxicity. The characteristics of necrosis are a loss of membrane integrity, inflammation, and significantly less DNA degradation as compared with apoptosis.

Since a single apoptosis assay does not work in all cell models it is recommended that discrimination of apoptosis be analyzed by three methods to verify results. A good example is presented in the referenced cited below*. Microscopic analysis by confocal or electron microscopy for morphological changes such as chromatin condensation, crescent bodies and membrane blebbing is still considered the "benchmark" in identifying apoptotic cells. This combined with a flow cytometry assay and gel electrophoresis displaying the characteristic pattern of DNA 'laddering' or western analysis for reduced translation of Bcl-2 is convincing evidence of programmed cell death.

The first and simplest method involves staining cells with propidium iodide (PI) for DNA content. A highly definable sub-G₁ peak is easily quantified. However this does not work with all cells. A sub-G₁ area or trough is not generally accepted as a valid method of discriminating apoptosis. Necrotic cells, or cell debris with DNA (mitochondrial) can also stain with PI presenting an undefined sub-G₁ region. Cells should be sorted and further analyzed by microscopy to verify apoptotic cells are present. Increased fixation time is recommended (overnight) in resolving a sub-G₁ peak.

The 'TUNEL' assay is great for many cell models. Two kits supplied by PharMingen or Phoenix Flow Systems provide not only the reagents but also control cells (camptothecin treated leukemic cells providing 30-40% internucleosomal DNA cleavage in the S-phase population) for a two color profile(BrdU-FITC/PI). Excellent for developing an in-lab model system. It should be noted that in some cells types (of fibroblast or epithelial lineage) or under some conditions DNA fragmentation may stop at the initial 300 - 50kb section and fail to progress to the internucleosomal DNA section (~180bp) which is typical of extensive DNA fragmentation. If this occurs the 'TUNEL' assay may not be satisfactory since this assay relies on large numbers of DNA strand breaks.

Cells undergoing programmed cell death may translocate phosphatidylserine from their inner cytoplasmic membrane and express it on their cell surface. In the presence of Ca⁺⁺ Annexin will bind to phosphatidylserine. Propidium Iodide (PI) in un-fixed cells discriminates between apoptosis and necrosis on the basis of dye exclusion. In combination Annexin-FITC/PI is a membrane integrity assay, therefore it should be used with care in cell systems that are adherent since mechanical or enzymatic cell detachment can give false positives for necrosis if the membranes are compromised in the harvesting procedure. While necrotic cells lose membrane integrity early on in their death throws, apoptotic cells may have compromised membranes late in their programmed cell death. Therefore in advanced apoptosis cells with breached outer membranes (blebbing) may uptake PI and bind Annexin-FITC as well because the inner cytoplasmic membrane with phosphatidylserine is available for labeling. Cell sorting and further analysis by light microscopy is recommended.

In some cell model systems the nature of the cells make quantitation of apoptosis difficult if not impossible. A good example is in astrocytomas and nerve cells. Another approach in the analysis of apoptosis may be to look at other programmed cell death events such as BCL-2 down regulation, caspase activation (MCF-7 cells lack functional caspase-3), or PARP cleavage.

Inducing apoptosis can be achieved through a variety of methods depending on the cell type. The list includes but is not limited to serum deprivation; treatment with Fas ligand, campothectin, dexamethasone, or TNF-a; exposure to UV.

* Ref. Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. (1997) Cytometry in Cell Necrobiology: Analysis of Apoptosis and Accidental Cell Death (Necrosis) Cytometry 27:1 p.1-20

The above micrographs are SEM images showing the chromatin condensation, crescent bodies and membrane blebbing (morphology) characteristic in apoptotic cells. Example of induced apoptosis in established non-Hodgkins B cell lymophoma cell line generously provided by Richard J. Ford, Jr., M.D., Ph.D., Professor, Department of Hematopathology, M.D. Anderson Cancer Center.

DNA Content and Apoptosis 'TUNEL' Assay with anti-BrdU Cell Surface Labeling and 'TUNEL' Annexin-FITC/PI DNA Analysis by Gel Electrophoresis Mitochondrial Membrane Potential

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Last Updated: 10-April-03