Quantification of DNase type I ends, DNase type II ends, and Modified Bases using Fluorescently Labeled ddUTP, Terminal Deoxynucleotidyl Transferase, and Formamidopyrimidine-DNA Glycosylase

Abstract

Here we describe the substitution of fluorescently labeled ddUTP for dUTP in the TUNEL assay to allow quantification of generated fluorescence signals by epifluorescence microscopy. The capping of DNase type I 3′OH DNA ends using ddTUNEL was further combined with phosphatase treatment for detection of DNase type II 3′PO₄ ends in the same sample using a second round of ddTUNEL. Levels of modified DNA bases in tissues and fixed cultured cells could be interrogated in the ddTUNEL assay with the base modification repair enzyme formamidopyrimidine DNA glycosylase. Using rat mammary gland, from days 1 and 7 of involution, we validate the methodology's ability to label apoptotic nuclei and apoptotic inclusion bodies. In addition, we examined the types of DNA damage and modification that occur in human glioblastoma, U87 cells, following exposure to reactive oxygen stressing agents, chemotherapeutic alkylating agents, and a topoisomerase I inhibitor, irinotecan.

Digging a New TUNEL

The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay is a standard method for detecting DNA fragmentation associated with apoptosis. Labeled dUTP is added by terminal deoxynucleotidyl transferase to the 3′OH ends of DNA fragments that are generated by type I DNases during apoptosis. However, this assay is inherently nonquantitative since the added dUTP itself has a 3′OH end, resulting in the formation of deoxyuridine polymers of indeterminate length at each primary 3′OH site. In this issue, Baskin et al. from the Methodist Hospital (Houston, T X) present a modified TUNEL assay they call ddTUNEL, which uses ddUTP instead of dUTP to stoichiometrically label each DNA3′OH end with a sing le dideoxyuridine base that cannot be further extended, thereby permitting quantitation of the number of 3′OH ends by fluorescence microscopy. This capping of the DNase type I–induced 3′OH ends has the added benefit of enabling additional rounds of ddTUNEL to examine 3′OH ends subsequently generated from other forms of DNA damage in the same sample. For instance, the 3′ phosphate ends of DNA fragments generated by type II DNases associated with caspase independent apoptosis can be detected by treatment of the sample from a first round of ddTUNEL with calf intestinal alkaline phosphatase (CIAP). This creates additional 3′OH ends that can be differentially labeled in a second round of ddTUNEL. Modified bases and apurinic/apyrimidinic (AP) sites in DNA can also be independently assayed by using the bacterially derived enzyme formamidopyrimidine-DNA glycosylase, which excises many modified bases to form an AP site that is then hydrolyzed to a nick containing a 3′ phosphate end that can then be converted to a 3′OH end with CIAP. These new ddTUNEL assays were validated in a study of mammary involution, which involves large-scale apoptosis, and by an examination of DNA damage in U87 cells upon exposure to various DNA-modifying agents.

See “Quantification of DNase type I ends, DNase type II ends, and modified bases using fluorescently labeled ddUTP, terminal deoxynucleotidyl transferase, and formamidopyrimidine-DNA glycosylase” on page 505.

Keywords: :

• TUNEL
• ddUTP
• formamidopyrimidine-DNA glycosylase
• DNase
• apoptosis
• mammary gland
• U87
• chemotherapy

Acknowledgments

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.

Competing interests

The authors declare no competing interests.

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.tandfonline.com/doi/suppl/10.2144/000113439

Additional information

Funding

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