Detection of apoptosis

DNA cleavage has been one of the main biochemical criteria used to distinguish apoptosis from necrosis.However,it should not be the sole factor used.Both morphological and biochemical characteristics need to be exemined collectively to determine that cell death hasoccured via apoptosis.The main methods methods of apoptosis detection are summarized below.

Microscopy

Histological sections can be treated with reagents(e.g.haematoxylin and eosin)that have an affinity for negatively charged molecules and stain the nuclear material,revealing its distribution.When there sections are examined under a light microscope,apoptosis is shown by shrinkage of the nuclei of the cells and apoptotic bodies show up as small darkly stained structures.

By counting apoptic cells before and a given treatment,it is possible to asses the efficacy of that treatment in inducing apoptosis and how readily a given tissue or tumour undergoes the process.Alternatively, cells grown in culture can be examined by scanning microscopy,which shows changes in the shape of individual cells undergoing apoptosis and the characteristic blebing on the cell's surface.

Flow cytometry

Cells in suspension are stained with fluorescent antibodies by attachment of these antibodies to the cell surface antigens.A laser beam is directed at the cell,causing the antibody to fluoresce.The intensity of the fluorescense reveals the number of cells and the amount of scattering of the beam depends on the size of the cells.When cells undergo apoptosis they shrink and break into apoptotic bodies.This is revealed as a reduction in light scattering by the cell sample.

DNA Electrophoresis

DNA is isolated from cells,placed on a gel and an electric current is passed through the gel.Since DNA has a negative charge.the electric current will cause the DNA to migrate depending on its net charge,size and shape. The DNA is placed on a solution with a powerful negatively charged detergent.The DNA then migrates towards a positive electrode when a voltage is applied.The different fragments of DNA migrate across the gel according to their size,with small fragments moving much more readily than large ones.The result is a series of descrete bands arranged in order of molecular weight,which can be detected by photographing the gel under ultraviolet light.The sizes of the fragments of DNA are calculatde with refrence to markers of known size run along the sample.

Apoptosis is revealed by the breakdown of the DNA into fragments.This hallmark of apoptosis proceeds in two steps:

In vitro experiments suggest that the initial breakage of DNA into large fragments is of crucial importance to apoptosis,whereas the DNA cleavage into smaller fragments now appears to be a by-product of the apoptotic process.These experiments have demonstrated that cells undergo the morphological changes characteristic of apoptosis despite selective inhibition of internucleosomal DNA fragmentation by intracellular Ca+2 chelation or by treatment with phorbol ester.However,in contrast,neither apoptopic morphology more internucleosomal DNA fragmentation was blocked by inhibition of nucleoside analogue incorporation into DNA.(Huang et al.,in press).

Detection of apoptosis in patients

Most work on apoptosis has been done in animals,cell samples or cell cultures.If apoptosis could be detected in tissue samples from cancer patients,this would support the clinical significance of this process, particularly if a treatment colud be shown to induce apoptosis in tumor cells.To date it has not been possible to isolate apoptotic cells in blood or tissue taken from patients after treatment in vivo,probably because apoptotic cells are rapidly ingested and destroyed by phagocytes and neutrophils.Recently,however,large nuclesomal fragmentation has been detected in leukaemic lymphocytes from patients after chemotheraphy fpr CLL (Huang et al.,submitted).