Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays.

TLDR

The authors labeled DNA strand breaks in individual fixed HL‑60 cells by incorporating biotinylated dUTP with terminal deoxynucleotidyl transferase or nick‑translation, then quantified the labeling by flow cytometry to relate break kinetics to cell‑cycle position. They found that early apoptotic breaks localize to the nuclear periphery, later become uniformly distributed, TdT labeling is faster than nick‑translation, camptothecin, teniposide, and 4′‑(9‑acridinylamino)-3‑methanesulfon‑m‑anisidide preferentially induce breaks in S‑phase cells while fostriecin acts indiscriminately, and the method can serve as a prognostic marker in tumor biopsies.

Abstract

DNA strand breaks which occur in HL-60 cells as a result of activation of endonuclease during apoptosis induced by cell treatment with the DNA topoisomerase I inhibitor camptothecin and topoisomerase II inhibitors teniposide, 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide, and fostriecin were labeled in situ, in individual fixed and permeabilized cells, with biotinylated dUTP (detected by fluoresceinated avidin), using the terminal deoxynucleotidyl transferase or nick translation assays. During the early stage of apoptosis, prior to nuclear fragmentation, the breaks were predominantly localized at the nuclear periphery, close to the nuclear envelope. In more advanced stages, all cellular DNA, then localized within the cell as dense, homogeneous granules of a variety of sizes, was strongly labeled, indicating extensive and more uniform distribution of breaks throughout genomic DNA. Bivariate analysis of the incorporated biotinylated dUTP and cellular DNA content by flow cytometry made it possible to estimate the kinetics of the labeling reaction and relate DNA breaks to cell position in the cycle. The kinetics of biotinylated dUTP incorporation was faster, and the distinction of cells with DNA breaks was more pronounced, using the terminal transferase rather than the nick translation assay. Camptothecin, teniposide, and 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide induced DNA breaks preferentially in S-phase cells, having little effect on cells in the G1 phase of the cycle. In contrast, fostriecin affected cells indiscriminately, in all phases of the cell cycle. The method of detection of DNA strand breaks (3'-hydroxyl termini) in individual cells offers several advantages and can be applied to clinical material (tumor biopsies) to study the induction of apoptosis in tumors during treatment, as a possible prognostic marker. The protein-associated DNA breaks in the "cleavable" DNA-topoisomerase complexes, which are the primary lesions induced by the inhibitors and precede apoptosis, were not detectable by the present methods.