Quantitative Differentiation of Both Free 3′ OH and 5′ OH DNA Ends Using Terminal Transferase-Based Labeling Combined with Transmission Electron Microscopy

Abstract

The initial concept of apoptosis was proposed by Kerr et al. (1972) (1) and was based on characteristic morphological criteria. Transmission electron microscopy (TEM) allows visualization of fine details of cellular morphology and is one of the most reliable methods for detecting of apoptotic cells. However, TEM has a number of limitations as an apoptosis detection method. First, apoptotic cells detected by TEM are those at the terminal stage of apoptosis. Therefore, TEM cannot identify apoptotic cells entering early stages of apoptotic process. For this purpose, investigators prefer to use fluorescent probes such as a combination of Hoechst 33342 and propidiumiodide (2). Second, chromatin condensation detected by TEM does not necessarily indicate extensive DNA fragmentation. In the thymus, thymocytes with chromatin condensation do not always exhibit extensive nuclear DNA fragmentation (3,4). Recently, Sahara et al. reported the discovery of a caspase-3-activated protein that is required for apoptotic chromatin condensation without DNA fragmentation (5). Therefore, chromatin condensation and DNA fragmentation are independent events of apoptosis. Both agarose gel electrophoresis using extracted DNA and in situ end labeling of DNA strand breaks (ISEL) technique using light microscopy (ISEL/LM) have been widely used for the detecting of DNA fragmentation. To overcome the above-mentioned limitations of TEM, we applied the ISEL technique to TEM (ISEL/TEM technique) (Fig. 1). The procedure of the ISEL/TEM technique is fundamentally the same as that of the ISEL/LM technique, except for the incorporation of immunogold particles, as a substitution for the immunoperoxidase in the ISEL/LM technique (6,7,8). Briefly, dideoxynucleotides labeled with digoxigenin are bound to the 3′ OH DNA ends by reacting with the TdT enzyme in ultrathin sections on a nickel grid. After incubation with an anti-digoxigenin antibody conjugated with 10-nm colloidal gold, the sections are then stained with uranyl acetate and lead citrate and are examined by TEM. The ISEL/TEM technique using immunogold staining has some advantages in terms of detection of apoptotic cells and semiquantification of free 3/t’ OH DNA ends. First, the ISEL/TEM technique enables determination of apoptotic cells entering early stages of apoptosis. It is well known that the increase in the number of sites of newly formed free 3′ OH DNA ends precedes morphological changes associated with apoptosis (6,9). The ISEL/TEM study clearly shows apoptotic cells in the early stage of apoptosis: the moderately increased number of immunogold particles on the initial chromatin condensation but otherwise normal ultrastructural appearance, and in the late stage, the typical chromatin condensation with a numerous immunogold particles in the nuclei (6,8). Second, the ISEL/TEM technique provides information on the mode of DNA cleavage for both apoptosis and necrosis. More than 10 endonucleases have been identified and classified into the following two DNA cleavage types: the 3′ OH/5′ P type and the 3′ P/5′ OH type (8,10). There are only few studies on the mode of DNA cleavage, particularly the 3/t’ P/5/t’ OH type, for both apoptosis and necrosis (8,11,12), although several studies have reported that apoptotic cells possess abundant DNA termini of the 3′ OH/5′ P type (13,14,15). In order to detect the free 5′ OH DNA ends using a nonradioactive method, the 3′ P ends located on the opposite side which hold the 5′ OH ends that were generated by cleavage, were dephosphorylated into 3′ OH ends using alkaline phosphatase (ALP). In this chapter, we describe two techniques for detecting both 3′ OH and 5′ OH ends in a nucleus (see Subheadings 3.3.1, 3.3.2). As described in Subheading 3.3.1, the exact number of 5′ OH ends in a nucleus was determined by calculating the difference between the number of immunogold particles labeled by ISEL/TEM using ALP (ALP/ISEL/TEM) and that by ISEL/TEM, because the ALP/ISEL/TEM technique recognizes both inherent 3′ OH ends and 3′ OH ends newly formed by dephosphorylation. As shown in 3.3.2. (Fig. 1), we can simultaneously detect 3′ OH ends labeled with 5-nm immunogold particles and 3′ P ends labeled with 10-nm immunogold particles in a nucleus, although the number of labeled immunogold particles may decrease due to double staining using different sizes of immunogold particles. Third, the ISEL/TEM technique provides objective data due to the image analysis of quantitative changes for both the 3′ OH and 5′ OH DNA ends in each apoptotic or necrotic cell, when images are processed by employing NIH Image and Adobe Photoshop programs (6,8) (Figs. 2, 3).