Identification of Apoptotic Cells by Formamide-induced DNA Denaturation in Condensed Chromatin

Abstract

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.

Application of anti‐ssDNA monoclonal antibody to study exogenous and apoptosis‐associated DNA damage

“…This conclusion is in disagreement with the data reported in several earlier studies (2)(3)(4)(5)(6)(7).…”

“…Under these conditions, the MAb detects DNA damage associated with apoptotic chromatin condensation per se, but does not detect presence of DNA breaks that require more intense denaturation (2,14). Electron microscopic studies confirmed this conclusion (6).…”

“…nal antibody (MAb) method originally developed for the measurement of apoptosis (2) and concluded that this procedure detects drug-induced exogenous but not apoptosis-associated DNA damage. This conclusion is in disagreement with the data reported in several earlier studies (2)(3)(4)(5)(6)(7).…”

“…The addition of a high concentration of MgCl 2 to the denaturation solution suppressed exogenous but not apoptosis-associated DNA damage, thus providing a specific test for the apoptotic cells (14). A practical and reliable apoptosis assay was developed when the denaturation was performed in formamide at or below 75°C (2). Under these conditions only apoptosisassociated but not necrosis-associated or exogenous DNA breaks were detected (2,6).…”

“…A practical and reliable apoptosis assay was developed when the denaturation was performed in formamide at or below 75°C (2). Under these conditions only apoptosisassociated but not necrosis-associated or exogenous DNA breaks were detected (2,6). Heating of cells suspended in formamide made it possible to develop an apoptosis ELISA assay for high-throughput drug screening and for detection of selective toxicity in cancer cells (15,16).…”