Method of Specific Detection of Apoptosis Using Formamide-induced DNA Denaturation Assay

Abstract

S U M M A R Y We compared the reliability between apoptosis detection methods, namely, the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method and formamide-induced DNA denaturation assay using a monoclonal antibody (MAb) to single-stranded DNA (ssDNA) (formamide-MAb assay). Reaction targets in these methods are different: the TUNEL method recognizes free 39-OH DNA ends, whereas the formamide-MAb assay detects ssDNA itself (25-30 bp). We found that the formamideMAb assay immunohistochemically detected apoptotic cells, whereas the TUNEL method detected apoptotic cells as well as mitotic and necrotic cells. The TUNEL method recognized not only 39-OH DNA ends cleaved by DNase during apoptosis but also constitutive physiological nicking that occurs in DNA duplication and histone posttranslational modifications during mitosis and random DNA breaks during necrotic execution. By electron microscopy, the mean labeling density (the number of 39-OH DNA ends/nuclear area) obtained by the TUNEL method was determined to be consistently higher than that (the number of ssDNAs/ nuclear area) obtained by the formamide-MAb assay. On the basis of these findings, we conclude that the formamide-MAb assay was more specific than the TUNEL method for the detection of apoptotic cells using electron microscopy; however, the labeling intensity of the formamide-MAb assay was slightly weaker than that of the TUNEL method. (J Histochem Cytochem 54:683-692, 2006)

Molecular Imaging of Neurovascular Cell Death in Experimental Cerebral Stroke by PET

“…Because TUNEL detects internucleosomal DNA fragmentation, which is one of the hallmarks of apoptosis, this correlation supports specific binding of the tracer to target apoptotic cells. However, we cannot rule out the possibility that, in addition to uptake by apoptotic cells, 18 F-ML-10 also accumulated in cells manifesting features of necrotic cell death, because positive TUNEL staining has been reported in such cells as well (18)(19)(20). As shown in the present study, 3 H-ML-10 accumulated in apoptotic cells but not in cells undergoing necrosis in vitro ( Table 1).…”

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). For sensitive detection of druginduced DNA breaks, more intense denaturation is needed.…”

“…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).…”

Evidence of ubiquitous in vivo and in vitro expression of pro-apoptotic Smac/DIABLO protein in meningioma cell lines

“…ssDNA was stained in vivo to verify the amount of apoptosis in meningiomas, as the antibody only stains singlestranded apoptotic DNA but not necrotic cells (38)(39)(40), however no significant inverse correlation with Smac/ DIABLO or surviving expression has been seen as previously shown in glioblastomas as well (31,41 (42). Tumor cells with reduced Smac resist apoptosis leading to the suggestion for an obligatory role for Smac/DIABLO in these tumor cells during several pathways of apoptosis induction (43).…”