Figure 1.
Direct comparison of DNase γ-dependence of DNA fragmentation in apoptosis and necrosis.
Ramos (R) and γRamos-25 (25, a stable transformant over-expressing human DNase γ) cells were induced to undergo apoptosis with staurosporine (lanes 3 and 4) or necrosis by a freeze-thaw procedure (lanes 5 and 6) or saponin (lanes 7 and 8). DNA fragmentation analyzed by agarose-gel electrophoresis and PARP cleavage (from 116 kDa to 85 kDa) by western blot are shown at the top and bottom panels, respectively. Control DNA (top panel) and protein (bottom panel) from untreated cells are in lanes 1 and 2. M, 1-kb ladder DNA marker.
Figure 2.
DNase γ-dependence of DNA fragmentation in TNF-receptor mediated necrosis.
(A, B) Expression of DNase γ and ICAD in U937 and its derivatives (UI, UG, and UIG). (A) RT-PCR analysis of DNase γ and GAPDH expression. (B) Western blot detection of mouse ICAD-CR (mICAD), endogenous human ICAD (hICAD) and β-Actin. (C) Morphological changes in U937 cells and its derivatives undergoing apoptosis or necrosis. Apoptosis was induced with TNF-α and CHX (TNF/CHX, middle panels) and necrosis by additional pretreatment with Z-VAD-fmk (TNF/CHX/ZVAD, right panels). Cells were examined by phase contrast microscopy after 3 h incubation. Control untreated cells are shown in the left panels. Arrowheads in the middle and right panels indicate examples of cells with apoptotic bodies or swelling cells, respectively. (D) Western blot analysis showing PARP cleavage (from 116 kDa to 85 kDa). Lanes 1–4: untreated cells; Lanes 5, 7, 9, and 11: cells treated with TNF/CHX for 6 h; Lanes 6, 8, 10, and 12: cells treated with TNF/CHX/ZVAD for 6 h. (E) U937 and its derivatives were induced as in (C) and incubated for the indicated time periods (0–48 h). DNA samples were then examined by agarose gel electrophoresis. (F, G) Ca2+ is required for necrotic but not apoptotic DNA fragmentation. Necrosis and apoptosis were induced in UIG with TNF/CHX/ZVAD for 6 h (F) and U937 with TNF/CHX for 6 h (G), respectively, in the presence of various concentrations of EGTA (lanes 1–5; 0 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM). After the incubation, DNA from each sample was analyzed by agarose gel electrophoresis. Data shown are representative of two to three separate experiments.
Figure 3.
DNase γ is responsible for generation of TUNEL-positive DNA ends in necrotic cells.
(A) HepG2 and G2G cells were treated with digitonin for 1 h, and genomic DNAs from these cells were electrophoresed on agarose gels (top panel): M, 1-kb ladder DNA marker. DNase γ and GAPDH expression levels in the same cells were examined by RT-PCR assay (middle and bottom panels). (B) HepG2 and G2G cells were treated with digitonin for 30 m and subjected to TUNEL assay (right panels) and stained with propidium iodide (PI, left panels). (C) Fixed mouse liver sections were treated with (+, bottom panels) or without (−, top panels) recombinant DNase γ (2.5 ng/ml) at 37°C for 30 min and subjected to TUNEL assay (right panels) and stained with PI (left panels). PI stained both DNA and RNA. Scale bar, 200 µm.
Figure 4.
Necrosis-associated DNA fragmentation is abrogated in DNase γ-deficient mice.
(A–C, E–G) Fragmentation of genomic DNA was analyzed by agarose gel electrophoresis. (A) Apoptosis was induced in DNase γ +/+ (+/+) and DNase γ −/− (−/−) mice by γ-irradiation. Genomic DNA from spleen (S) or thymus (T) was analyzed (lanes 5–8). Lanes 1–4, DNA from non-irradiated mice. Lane 9, 1-kb ladder. (B) Primary hepatocytes from mice of the indicated genotypes were incubated at 37°C with anti-Fas antibody for the indicated time periods (0–12 h) and DNA fragmentation was analyzed. (C) Hepatocyte nuclei were incubated at 37°C for 2 h in buffer containing 5 mM Mg2+ and various concentrations of Ca2+ (lanes 1 and 6, 0 mM; lanes 2 and 7, 0.3 mM; lanes 3 and 8, 1 mM; lanes 4 and 9, 5 mM; lanes 5 and 10, 10 mM), and DNA was analyzed. Lane 11, 1-kb ladder. (D) The same nuclei as in (C) were incubated for 2 h in buffer with 5 mM Mg2+ and with or without 5 mM Ca2+ (bottom and top panels, respectively), stained with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) and examined by confocal microscopy. Magnification, ×600. A high power image of chromatin condensation is shown in the inset. (E) Splenocytes from mice of the indicated genotypes were incubated with saponin in DMEM at 37°C for the indicated time periods (0–2 h) to induce necrosis, and DNA from these cells was analyzed. Lane 10, 1-kb ladder. (F) Frozen tissues (L: liver; K: kidney; S: spleen; T: thymus) from mice of the indicated genotypes were smashed to a homogenate, incubated in DMEM at 37°C for 1 h and then used for DNA purification. (G) DNA fragmentation in liver after ischemia-induced necrosis. Ischemia was induced in one of the hepatic lobes of two mice from each genotype and DNA was prepared from the ischemic (+) and nonischemic lobes (−). Arrowhead, 180 bp DNA fragments corresponding to a single nucleosome.