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ARTICLE |
Identification of Apoptotic Cells by Formamide-induced DNA Denaturation in Condensed Chromatin
Oskar S. Frankfurta,b and Awtar Krishanaa Experimental Therapeutics Division, Radiation Oncology Department, University of Miami Medical School, Miami, Florida
b Apostain, Inc., Miami, Florida
Correspondence to: Oskar S. Frankfurt, Experimental Therapeutics Div., U. of Miami Medical School (R-71), PO Box 016960, Miami, FL 33101. E-mail: apostain@bellsouth.net
In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells based on this effect. Our observations show that formamide induces DNA denaturation in apoptotic nuclei but has no such effect on DNA of non-apoptotic cells. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it possible to specifically identify the apoptotic cells. This procedure produced intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity of the formamide–MAb assay for the apoptotic cells. However, TUNEL stained 90–100% of necrotic cells in all three models of necrosis. Because the MAb did not stain cells with single- or double-stranded DNA breaks in the absence of apoptosis, we conclude that staining of the apoptotic nuclei is not influenced by DNA breaks and is induced by specific changes in condensed chromatin, such as damage to the DNA–histone interactions. Importantly, the formamide–MAb technique identified apoptotic cells in frozen sections and in histological sections of formalin-fixed, paraffin-embedded tissues. (J Histochem Cytochem 49:369–378, 2001)
Key Words: apoptosis, single-stranded DNA, chromatin, formamide, DNA denaturation, monoclonal antibody, immunohistochemistry, TUNEL
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