Do TUNEL and Other Apoptosis Assays Detect Cell Death in Preclinical Studies?

Razmik Mirzayans,David Murray

doi:10.3390/ijms21239090

Abstract

The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay detects DNA breakage by labeling the free 3ʹ-hydroxyl termini. Given that genomic DNA breaks arise during early and late stages of apoptosis, TUNEL staining continues to be widely used as a measure of apoptotic cell death. The advantages of the assay include its relative ease of performance and the broad availability of TUNEL assay kits for various applications, such as single-cell analysis of apoptosis in cell cultures and tissue samples. However, as briefly discussed herein, aside from some concerns relating to the specificity of the TUNEL assay itself, it was demonstrated some twenty years ago that the early stages of apoptosis, detected by TUNEL, can be reversed. More recently, compelling evidence from different biological systems has revealed that cells can recover from even late stage apoptosis through a process called anastasis. Specifically, such recovery has been observed in cells exhibiting caspase activation, genomic DNA breakage, phosphatidylserine externalization, and formation of apoptotic bodies. Furthermore, there is solid evidence demonstrating that apoptotic cells can promote neighboring tumor cell repopulation (e.g., through caspase-3-mediated secretion of prostaglandin E2) and confer resistance to anticancer therapy. Accordingly, caution should be exercised in the interpretation of results obtained by the TUNEL and other apoptosis assays (e.g., caspase activation) in terms of apoptotic cell demise.

Highlights

The Nomenclature Committee on Cell Death (NCCD) has formulated several caveats concerning the misuse of words and concepts that have slowed down progress in the area of cell death research
We briefly review: (i) stress-induced responses that are associated with persistent genomic DNA breakage and might be detected by the transferase-mediated dUTP nick end labeling (TUNEL) assay (Section 2); (ii) reversal of p53-induced apoptosis in the absence of exogenous stress (Section 3); (iii) recovery from the brink of apoptotic death following treatment with anticancer agents (Section 4); and (iv) the generation of growth-stimulating signals by apoptotic cells (Section 5)
There is an urgent need for developing robust biomarkers using in vitro models that will enable the discrimination of cells that exhibit manifestations of apoptosis with respect to whether an individual cell is likely to survive through recovery/anastasis or to undergo terminal apoptotic death [27]

Summary

Introduction

The Nomenclature Committee on Cell Death (NCCD) has formulated several caveats concerning the misuse of words and concepts that have slowed down progress in the area of cell death research. We briefly review: (i) stress-induced responses that are associated with persistent genomic DNA breakage and might be detected by the TUNEL assay (Section 2); (ii) reversal of p53-induced apoptosis in the absence of exogenous stress (Section 3); (iii) recovery from the brink of apoptotic death following treatment with anticancer agents (Section 4); and (iv) the generation of growth-stimulating signals by apoptotic cells (Section 5). Based on the evidence provided, we conclude that caution should be exercised in the interpretation of results obtained by TUNEL and other apoptosis assays in terms of cell death This cautionary note is relevant to preclinical studies that are performed under conditions that permit cancer cell recovery from the brink of apoptotic death. We will refer to such giant cells collectively as polyploid giant cancer cells (PGCCs) All of these outlier responses are associated with genomic instability, such as DNA strand breakage and nuclear fragmentation.

Reversal of p53-Induced Apoptosis in the Absence of Exogenous Stress

Apoptotic Cells Generate Growth-Stimulating Signals

The Principle of the TUNEL Assay

Advantages and Disadvantages of TUNEL in Preclinical Anticancer Studies

Concluding Remarks