Annexin V and DNA fragmentation

david_hedley@pmh.toronto.on.ca david_hedley at pmh.toronto.on.ca
Mon Oct 21 08:08:19 EST 1996


The question has been raised as to where annexin V labelling comes in
the apoptotic pathway, relative to the TdT assay.  The TdT assay detects
DNA fragmentation caused by activation of calcium-dependent
endonucleases.  We have used dual labelling for annexin V and calcium
(indo-1) in cells that are undergoing apoptosis.  We looked at Jurkat cells
triggered with anti-fas, and AML cells treated with cytosine arabinoside.  In
both examples we found annexin V positive cells with low calcium,
whereas as all of the high calcium cells were annexin V positive.  Hence
phosphatidylserine exposure (annexin V +ve) occurs before DNA
fragmentation.  

Using a triple labelling method that also includes mitochondrial membrane
potential, we confirmed the results of Castedo et al (J Immunol
1996;157:512-521) that loss of MMP and annexin V positivity are very
closely linked.  Again, high ionized calcium was only seen in cells with
depolarized mitochondria.  The data are consistent with the proposal that
the mitochondrial permeability transition is a key early event during
programmed cell death, and may indicate the point where cells become
irreversibly committed to death.  I guess we should get around to
publishing this some time.

My own feeling is that it is time the flow cytometry community stopped
getting hung up over the concept that measurement apoptosis is an end
in itself.  Apoptosis is a dynamic cellular process that is regulated by
genes, and increasingly can be manipulated pharmacologically.  The long
term medical significance of this is enormous.

David Hedley
Princess Margaret Hospital/Ontario Cancer Institute
Toronto




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