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MMeetthhooddss iinn MMoolleeccuullaarr BBiioollooggyy TTMM VOLUME 203 IInn SSiittuu DDeetteeccttiioonn ooff DDNNAA DDaammaaggee MMeetthhooddss aanndd PPrroottooccoollss EEddiitteedd bbyy VVllaaddiimmiirr VV.. DDiiddeennkkoo HHUUMMAANNAA PPRREESSSS Labeling DNA Damage with Terminal Transferase 1 I L DNA B U T T ABELING REAKS SING ERMINAL RANSFERASE (TUNEL A ) SSAY 2 Walker et al. Labeling DNA Damage with Terminal Transferase 3 1 Labeling DNA Damage with Terminal Transferase Applicability, Specificity, and Limitations P. Roy Walker, Christine Carson, Julie Leblanc, and Marianna Sikorska 1. Introduction Apoptotic and programmed cell death are characterized by, and indeed were first discovered from observations of, remarkable morphological changes that occur in the nucleus (see 1 for a comprehensive review of apoptosis and programmed cell death). Thus, light and electron microscopy were the first tools for the detection of apoptosis. This characteristic collapse of chromatin and ultimately the structural organization of the nucleus is triggered by the degradation of DNA, which is an active process and occurs prior to death of the cell. The degradation of DNA was subsequently found to be mediated by endonucleolytic activity that generated a specific pattern of fragments (2). The fragment sizes were multiples of approx 200 bp, the amount of DNA wound around a single nucleosome, and the pattern became known as the DNA ladder (Fig. 1A). Later it became apparent that DNA fragmentation is quite variable within cells and some cell types produce only high molecular weight (HMW) fragments (Fig. 1B, 3). The latter observations formed the basis of a convenient in vitro biochemical technique for the routine detection of apoptosis by resolving the fragmented DNA by conventional or pulsed field agarose gel electrophoresis. However, this technique requires relatively large amounts of material and DNA extraction. Subsequently, a variety of techniques have emerged to detect apoptotic DNA fragmentation in situ by exploiting the fact that the hydroxyl group at the 5' or 3' ends of the small DNA fragments becomes exposed. Nucle- otide analogues can be attached to the ends by several enzymes, with Terminal deoxynucleotide Transferase (TdT) being the most popular (4,5). The assays From: Methods in Molecular Biology, vol. 203: In Situ Detection of DNA Damage: Methods and Protocols Edited by: V. V. Didenko © Humana Press Inc., Totowa, NJ 3 4 Walker et al. Fig. 1. Patterns of DNA fragmentation in apoptosis. (A) DNA extracted from con- trol (lane 1) and glucocorticoid-treated (lane 2) thymocytes showing the DNA ladder of mono- and oligo-nucleosomes. (B) DNA extracted from untreated (lane 1), vehicle- treated (lane 2) and VM26-treated (lane 3) HL60 cells and resolved by pulsed field gel electrophoresis (see 9). Lane m is the lambda DNA ladder marker (multiples of 48.5Kb). In these cells, only high molecular weight (HMW) degradation occurs. are typically fluorescence-based, either by the direct incorporation of a nucleotide to which a fluorochrome has been conjugated, or indirectly using fluorescent dye conjugated antibodies that recognize biotin- or digoxigenin-tagged nucleotides. Radioactively labeled nucleotides can also be used. Since several million fragments are generated during complete DNA fragmentation and low levels of fluorescence can be readily detected by photo-multipliers and CCD arrays, the assays are extremely sensitive. The assays have been formatted for light and confocal microscopy as well as flow cytometry, thereby greatly facili- tating the detection and quantitation of apoptosis in situ. In addition, end- labeling techniques are employed in studies of the actual mechanism of DNA fragmentation, as well as the detection and characterization of endonucleases. Labeling DNA Damage with Terminal Transferase 5 Fig. 2. Mechanisms of endonucleolytic attack on DNA. In A the arrows indicate the site of attack by an endonuclease cleaving the phosphodiester bond at the same point on each strand of the DNA duplex to generate multiple smaller fragments each with 3'- OH and 5'-P ends. In B the endonuclease cleavage of each strand of the DNA duplex is offset generating fragments with a 3' recess. 1.1. The Nature of the DNA Fragments Endonucleases cleave DNA by attacking the phosphodiester bonds of the sugar-phosphate backbone of each strand (Fig. 2A). The phosphodiester bond can be cleaved in two ways such that the phosphate is left on either the 3' end of the DNA strand or the 5' end, the opposite end being a hydroxyl group in each case. In addition, the distance between the point at which the bond is broken on opposite strands of the DNA duplex also varies. If the breaks are exactly opposite, the fragment is considered blunt-ended. If they are offset, they generate either 3' or 5' overhangs (Fig. 2B). Thus, DNA can be cleaved by a variety of nucleases, operating by different mechanisms, and each type of nuclease gen- erates a characteristic “signature” in terms of nature of the ends it creates. The DNA fragments that are produced during apoptosis are usually, but not always, created by an endonuclease that cleaves the DNA strand at the 6 Walker et al. phosphodiester bond such that the 5' end of the DNA retains the phosphate group and the 3' end is an hydroxyl group (Fig. 2A). Generally there is little or no overhang (6,7). The terminal transferase assays take advantage of this observation, and add nucleotide analogs to the 3'-hydroxyl of the DNA fragment. However, numerous exceptions to this observation have been docu- mented. In some cells, the DNA is believed to be cleaved by DNAse II, an enzyme that produces 5'-OH (8). Such ends would not be labeled with terminal transferase. In addition, the cleavage of each strand is sometimes offset, leav- ing a variety of sizes of overhang (9). The reasons for this are not clear, but probably relate to the fact that different endonucleases cleave the DNA in different cell types or under different physiological conditions. Terminal transferase can still add nucleotides to the 3'-OH of many of these fragments and a variety of other enzymes can also be used to add fluorescent or radioactive nucelotides to those DNA strands, as discussed below. In some cells undergoing apoptosis, the DNA is not cleaved into small fragments at all. Instead, larger fragments of about 50 Kb are produced (Fig. 1B)(3). These fragments appear to have 3'-OH groups, but since the number of fragments per cell is orders of magnitude lower, their detection becomes more difficult. 1.2. Terminal Deoxynucleotidyl Transferase DNA nucleotidylexotransferase. (E.C. 2.7.7.31, common name: Terminal deoxynucleotidyl Transferase, TdT) is a DNA polymerase that catalyzes the addition of deoxyribonucleotides to the 3'-OH end of DNA strands without the need for a template or a primer. This is in contrast to most enzymes that incorporate nucleotides into duplex DNA, since they require a string of nucleotides on the opposite strand to create a template so that the enzyme recognizes which nucleotides to select. A reaction mixture containing all four nucleotides is required by these enzymes. DNA polymerases and the smaller Klenow fragment are typical examples and these enzymes are ideally suited to incorporate nucleotides into DNA fragments that possess overhangs. On the other hand, TdT requires only 1 nucleotide type (typically, deoxyuridine triphosphate, dUTP) in end-labeling assays and will continue to add it to generate a homo- polymer. TdT also has other advantages such as the ability to add nucleotides to very small fragments of DNA making it ideal for labeling fragments in apoptotic cells. The enzyme will also label single stranded DNA molecules containing a 3'-OH and will attach nucleotides to a single-strand nick in DNA. This is particularly useful since many single-strand breaks are also introduced into DNA during fragmentation in apoptotic cells (10). 1.3. Nucleotides Used in Labeling Assays Initially, radioactively-labeled nucleotides were used in DNA-labeling experiments, but more recently, fluorescent nucleotide analogs have been Labeling DNA Damage with Terminal Transferase 7 Fig. 3. Nucleotide analogs used in end-labeling assays. The three compounds com- monly conjugated to dUTP are Fluoroscein, biotin and digoxigenin. The compounds are linked via a spacer to the C-5 of the nucleotide. Radioactively labeled dUTP is com- monly on the alpha-Phosphate which becomes incorporated into the sugar phosphate backbone of DNA. Also shown is the substitution that terminates polymerization by removing the hydroxyl that interacts with the phosphate of the next nucleotide. developed. The fluorochrome, usually fluorescein isothiocyanate (FITC), can be directly conjugated to the nucleotide and its green fluorescence readily detected using standard filter sets (Fig. 3). The nucleotide of choice is deoxyuridine triphosphate (dUTP) and conjugation is usually to the C-5 posi- tion of uridine which does not participate in hydrogen bonding. A spacer is used to decrease steric hindrance, with the length of the spacer dependent upon manufacturer and the nature of the molecule being conjugated. In other for- mats, the nucleotide is conjugated with biotin or digoxigenin derivatives and these molecules are detected with fluorescently-tagged proteins. This is fluo- 8 Walker et al. rochrome-conjugated streptavidin for biotin detection and anti-digoxigenin antibodies for digoxigenin detection. Because more than one molecule of FITC can be conjugated to the protein, the fluorescence signal is amplified. Thus, the indirect assays are more sensitive than direct FITC conjugation to the nucleotide (11). Moreover, since digoxigenin is found only in plants, its antibody does not recognize any mammalian proteins, thereby reducing the background that is usually caused by non-specific binding. Other non-fluorescent detection systems have been used, particularly for tissue sections and blots, including alkaline phosphate-colorimetry, peroxidase, chemi-luminescence and colloidal gold. Under optimal conditions, the sensitivity of fluorescence detection approaches that of radioactivity and the small number of fragments that occur during high molecular weight DNA fragmentation are readily detectable (12). In addition, fluorescence affords the opportunity for multicolor counterstaining and labeling protocols, which are particularly useful for flow cytometry and confocal microscopy. 1.4. Assays Based on Terminal Transferase The first end-labeling protocol developed for the detection of DNA fragmentation in apoptosis was the Terminal Uridine Nucleotide End Labeling (TUNEL) technique of Gavrielli et al (4). This method exploited the ability of the enzyme, terminal transferase, to add biotin-conjugated nucleotides onto the 3' OH of a DNA strand. By using either a fluorescently tagged or radioactively labeled nucleotide analog, the DNA fragments become detectable. Formula- tion of the reaction buffer with cobalt ensures that the enzyme can add multiple bases to the 3'-end of each strand. As mentioned above, all types of 3'-end can be labeled, including those of single and double stranded DNA as well as recessed, protruding and blunt ends. The enzyme appears to have a preference for single stranded and 3'-protruding ends. The method can be used on cell suspensions and monolayers as well as frozen or paraffin tissue sections. If only one nucleotide is to be incorporated onto each end of the double- stranded DNA fragment in order, for example, to accurately quantitate the number of fragments, then dideoxynucleotides can be used to create strand termination (Fig. 3). Usually, however, the objective is to increase sensitivity by incorporating multiple nucleotides and under optimal conditions as many as 50–100 monomers may be incorporated (13). Unmodified nucleotides are included in the reaction mixture to “space out” the modified nucelotides in order to increase the ability of the binding protein to recognize its target. Typi- cally, the methods use either digoxigenin-conjugated dUTP detected by staining with a FITC-conjugated anti-digoxigenin antibody or biotin-conjugated dUTP detected by staining with FITC-conjugated streptavidin. Labeling DNA Damage with Terminal Transferase 9 If radioactivity is needed, the alpha-phosphate of dUTP is substituted with 32P, since this is the phosphate that becomes incorporated into the sugar phosphate backbone of the DNA (Fig. 3). 1.5. Other Enzymes That Can Label DNA Since some endonucleases also leave an overhang (i.e. a run of nucleotides on one strand only, Fig. 2B) the other strand can be extended or “filled in” by the Klenow fragment of DNA polymerase. The Klenow fragment of DNA polymerase I is used since it retains the ability to create a polymer, but does not possess the 5'–3' exonuclease activity which would degrade the fragment. In other situations, it is necessary to examine the 5' end of the DNA fragments. To confirm that the 5' end is indeed phosphorylated, the fragments can be incu- bated in the presence of the enzyme T4 kinase and 32P-labeled inorganic phosphate. T4 kinase phosphorylates any 5'-OH. Thus, if the phosphate group is already present no radioactivity can be incorporated. However, if the phosphate is absent, or has been removed by incubation with alkaline phosphatase, the radioactively-labeled phosphate becomes attached to the fragment and this can be detected by autoradiography. Since T4 kinase can add only one phosphate, whereas terminal transferase or the Klenow fragment can add multiple nucleotides, the 5' labeling technique is much less sensitive than the 3' labeling techniques and is not generally used in routine assays. However, it is very useful for determining the nature of the ends of DNA from apoptotic cells. 1.6. Limitations DNA fragments with 3'-OH ends can be produced in a number of situations where apoptosis is not occurring. For example, some forms of DNA damage produce DNA breaks or nicks with 3'-OHs. Moreover, the DNA degradation that occurs during necrosis also produces fragments with 3'-OH that would be labeled by TUNEL or ISEL (InsituEndLabeling,14). Over-reliance on these techniques has led to considerable controversy in studies in brain where, following some insults, both apoptosis and necrosis occur simultaneously making it very difficult to establish and quantitate true apoptotic cell death (15–17). It is evident, therefore, that TdT-based labeling techniques should not be used as the sole criterion for establishing the nature of the cell death mechanism. In order to establish that apoptosis is occurring, other criteria must also be used. Since it is possible to use multiple fluorochromes in the same experiments, another marker such as the appearance of annexin on the cell surface, can be used simultaneously. Once it has been established that the cell death is indeed apoptotic, then the TdT-based assays can be used for routine quantitation by microscopy or flow cytometry (3,18).