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Quantification of DNase type I ends, DNase type II ends, and modified bases using fluorescently labeled ddUTP, terminal deoxynucleotidyl transferase, and formamidopyrimidine-DNA glycosylase

The role of immune cells in the cleanup of apoptotic debris is highlighted in Figure 3, I and J. We labeled the sections with ddTUNEL, Fpg-ddTUNEL, and also with FITC–anti-CD3-ε, a marker of lymphocytes and macrophages. It can be seen that the immune cells are taking up DNA fragments (into the cytosol) and are associated with, but are not part of, the apoptotic bodies.

We measured the relative levels of modified bases and AP sites in the Fpg-ddTUNEL–positive DNA present in the apoptotic bodies. Tissue sections were treated with DNP-H and then labeled using ddTUNEL, Fpg-ddTUNEL, and FITC-labeled anti-DNP antibodies (Figure 3, K and L). The levels of Fpg-ddTUNEL positive DNA are unchanged, with respect to sections that had not undergone DNP-H treatment, indicating that the DNA in the apoptotic bodies has very few AP sites, but many Fpg-labile bases. We found that there is aldehyde/ketone DNP-H–positive material in the apoptotic bodies, but it is not associated with DNA and is almost certainly oxidized protein.

Figure 4 shows the formation of DNA 3′OH ends and modified bases using ddTUNEL and Fpg-ddTUNEL; along with green DNA (YoPro) (Figure 4, A–F) or green γ-H2A.X (FITC IgG) (Figure 4, G–L). Absolute quantification of the levels of the ddTUNEL and Fpg-ddTUNEL, in their respective cellular compartments, is beyond the scope of this publication, but the data from the sample shown in Figure 4 is tabulated in Table 1 to show the approximate average concentration of the probes, assuming that each section is 6 µm thick, in the cells shown. There are few 3′OH DNA nicks/breaks in the control cells, and they appear green in Figure 4A, but in Figure 4G, the background level of ddTUNEL-positive 3′OH ends is more apparent, as is the presence of γ-H2A.X, which is mostly found in the cytosol.









Exposure of U87 cells to various modifying agents

H2O2 treatment. Hydrogen peroxide–treated cells (Figure 4, B and H) show the formation of blebs that are positively stained for both γ-H2A.X and ddTUNEL. ddTUNEL-positive DNA is found at very high levels in the cytosol, mostly within small vesicles, and 3′OH levels are low within the nucleus.

The red Fpg-ddTUNEL is also unevenly distributed; DNA that contains high levels of oxidized bases and AP sites appear to be exported from the nucleus and then concentrated up in the apoptotic vesicles. There appears to be a high degree of heterogeneity in the composition of these apoptotic vesicles, with respect to the levels of γ-H2A.X, ddTUNEL, and Fpg-ddTUNEL–positive DNA. The insert in Figure 3H is an enlargement of the indicated portion of these vesicles and conveys the degree of heterogeneity.

Paraquat treatment. Paraquat, which generates both superoxide and H2O2, has a completely different death signature to H2O2, with no evidence of blebbing. ddTUNEL correlates with γ-H2A.X and is mostly cytosolic and found in the small vesicles (Figure 4, C and I). It again appears that Fpg-ddTUNEL–positive oxidized DNA is exported out of the nucleus and concentrated into vesicles. This oxidized ddTUNEL-positive DNA is clearly associated with γ-H2A.X, as shown by the presence of the bright white spots (Figure 4I).

Carmustine and temozolomide treatment. Many chemotherapeutic agents, including temozolomide and carmustine, derive their toxicity from the acylation of the N7 position of guanine. The ability of Fpg to directly remove N7 methyl and ethyl guanine lesions is poor. However, both these modified residues can undergo slow ring opening to form formamidopyrimidines, which act as substrates for Fpg. In addition, the formation of bulky adducts in the N7 position, which are generally good Fpg substrates, occurs in vivo upon exposure of mammalian cells to carmustine (29). Bodell and coworkers (29) demonstrated that in mammalian cells, N7-(2-hydroxyethyl)guanine, N7-(2-chloroethyl) guanine, and eight other species were the major adducts following carmustine treatment. In addition, in mammalian cells, N7 methyl and ethyl guanine lesions are processed by the enzyme N-methylpurine DNA glycosylase (MPG), which removes these residues to generate AP sites. In contrast, the lesions 3-methyladenine and O6-methylguanine are primarily species responsible for temozolomide toxicity, and these lesions are also removed by MPG, generating AP sites. Therefore, treatment of cells with chemotherapeutic agents, such as carmustine and temozolomide, generates a variety of Fpg-positive species, which may be measured using Fpg-ddTUNEL.

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