Treating cells with carmustine (Figure 4, D and J) induced 6-fold increases in ddTUNEL and Fpg-ddTUNEL. In carmustine-treated cells, ddTUNEL ends are found in a 2:1 ratio in the cytosol compared with the nucleus. The levels of γ-H2A.X, mostly cytoplasmic, are elevated more than 30-fold with respect to the controls.
Temozolomide-treated cells (Figure 4, E and K) gave the highest Fpg-ddTUNEL–positive result, and it is obvious that this methylated DNA is exported into and concentrated in cytosolic vesicles; these vesicles are also γ-H2A.X– and ddTUNEL-rich. In comparing the ethylation agent, carmustine, to the methylation agent, temozolomide, the striking difference is the 10-fold difference in cytosolic γ-H2A.X levels in carmustine-treated cells and that these histones are not packaged into inclusion bodies. We can only conclude that methylated DNA is treated in a completely different fashion than ethylated/acylated DNA.
Irinotecan treatment. The final pairing (Figure 4, F and L) shows the effect of the topoisomerase I inhibitor, irinotecan, on cell death. ddTUNEL-rich DNA is intimately associated with both γ-H2A.X and Fpg-ddTUNEL, but strikingly, the apoptotic vesicles are heterogeneous, containing either ddTUNEL with γ-H2 A. X or ddTUNEL with Fpg-ddTUNEL.
We differentiate between DNA base modification and the presence of AP sites using NaBH4 reduction. We expect that H2O2 would increase the levels of oxidized DNA and that carmustine would increase ethylation. We prepared slides that had irinotecan-, H2O2-, and carmustine-treated cells and incubated half of a slide in methanolic NaBH4 to reduce all the AP sites and then ran Fpg-ddTUNEL,
In irinotecan- and carmustine-treated cells, the levels of Fpg-ddTUNEL–sensitive generated 3′OH ends were insensitive to NaBH4 treatment. In striking contrast, in H2O2-treated cells, the Fpg-ddTUNEL signal was abolished by NaBH4 treatment (Figure 5). Irinotecan increases DNA breaks and does not directly modify DNA bases, carmustine ethylates DNA bases, and H2O2 is a DNA base oxidant.
We found low levels of DNase type II 3′PO4 ends in the both rat mammary tissue and also in the various U87 cell samples. To demonstrate that CIAP-ddTUNEL was able to measure the levels of DNase type II 3′PO4 ends, positive controls were generated by incubating U87 cells with DNase type II. DNase type II–treated cells underwent a first round of biotin-ddUTP-ddTUNEL and were labeled with avidin-FITC. Half the samples were then incubated with CIAP, half were in buffer with the enzyme omitted, and then all samples underwent a second round of ddTUNEL using biotin-ddUTP/avidin–Texas Red. Supplementary Figure S3 shows the visualization of this type of labeling. There is very little (<5%) labeling in the second round of ddTUNEL in the absence of CIAP, demonstrating that it is the presence of CIAP, with its 3′PO4→3′OH activity, that allows labeling in the second round of ddTUNEL.
We have modified the traditional TUNEL assay (1) to allow the absolute levels of TdT-accessible 3′OH to be measured. We have shown that the substitution of ddUTP for dUTP allows the levels of both 3′OH and 3′PO4 DNA ends to be measured in the same sample, using ddTUNEL and CIAP-ddTUNEL, respectively. We have also demonstrated that E. coli enzyme Fpg can be used to excise oxidized/acylated DNA bases and AP sites in vitro using U87 cells in combination with oxidative and acetylating agents.
Fpg-ddTUNEL is very useful for converting AP sites and modified bases into 3′OH-labeled ends. In addition to 8-oxo-7,8-dihydroguanine, a wide range of modified pyrimidines are recognized and excised by Fpg (30). In addition, some modified purines are also substrates for this enzyme (31,32).
The broad substrate specificity of Fpg can also be discerned by the protection it affords mammalian cells that have been transformed with E. coli Fpg. Fpg-transfected cells are much less sensitive to DNA acylating agents [ThioTEPA (14,33), bis(2-chloroethyl)-N-nitrosourea (20), and carmustine (19)], DNA oxidants [potassium bromate, H2O2, and γ-rays (34)], and hyperoxia (18).
We have shown that it is possible to derivativize AP sites with dinitrophenyl hydrazine, which allows one to interrogate the localization of ROS damage within a cell using an anti-DNP antibody. Derivatization of AP sites by fluorophores linked to an aminooxy group (24,35) or using a commercially available biotinylated hydroxylamine (Aldehyde Reactive Probe; Invitrogen, Carlsbad, CA, USA) are neutral pH reactions and may be less harsh alternatives to DNP-H/antibody labeling.
Treatment of DNA with methanolic NaBH4 reduces AP sites, and then Fpg-ddTUNEL allows the discrimination between the levels of modified bases and AP sites. We find that AP sites are the principle product of oxidative stressors, such as H2O2 and paraquat (Figure 5 and unpublished data). Fenton chemistry is a major cause of oxidative cellular damage (36), and it has previously been postulated that DNA damage caused by Fenton chemistry results in much higher levels of AP sites than oxidized bases, such as oxyguanine (37).
In breast tissue, the Fpg-ddTUNEL– positive DNA concentrated in apoptotic bodies had few AP sites, since Fpg-ddTUNEL was insensitive to either DNP-H or NaBH4; this would suggest that the DNA in the apoptotic bodies is highly methylated.
In vitro, both methyl and ethyl guanine can be converted into formamidopyrimidines, by incubation at high pH (38), and the Fpg-ddTUNEL assay could be modified to include such an alkyl-hydrolysis step. However, similar to Speit and coworkers (17), we find that the products of DNA methylation/ethylation generated within mammalian cells in vivo are Fpg-labile without prealkalinization. Moreover, they found that the changes in pH imposed by an alkalinization/neutralization protocol resulted in a considerable loss in sensitivity in their combined Fpg-Comet assay.
The three techniques—ddTUNEL, CIAP-ddTUNEL, and Fpg-ddTUNEL, introduced and validated herein—can be combined with the use of tissue phantoms for signal calibration, allowing the absolute levels of three different types of DNA damage to be quantified within individual cells for the first time.
We thank our research assistant, Sophie Lopez, for all her assistance in cell growth, sample preparation, and many other duties. We also thank Kalika M. Landua of Nikon Instruments for her aid in the calibration of our microscope and camera. Funding for this research was provided by The Henry J.N. Taub Fund for Neurological Research, The Pauline Sterne Wolff Memorial Foundation, Golfers Against Cancer, and the Methodist Hospital Foundation.
The authors declare no competing interests.
Correspondence Address correspondence to Martyn A. Sharpe, Department of Neurosurgery, The Methodist Hospital, 6565 Fannin Street, Houston, TX 77030, USA. e-mail: [email protected]References
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