Caspases 3 and 7: key mediators of mitochondrial events of apoptosis

Abstract

The current model of apoptosis holds that upstream signals lead to activation of downstream effector caspases. We generated mice deficient in the two effectors, caspase 3 and caspase 7, which died immediately after birth with defects in cardiac development. Fibroblasts lacking both enzymes were highly resistant to both mitochondrial and death receptor-mediated apoptosis, displayed preservation of mitochondrial membrane potential, and had defective nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, the early apoptotic events of Bax translocation and cytochrome c release were also delayed. We conclude that caspases 3 and 7 are critical mediators of mitochondrial events of apoptosis.

Fig. 1

(A to F) Defective cardiac development in DKO embryos. Hematoxylin-and-eosin staining of transverse sections of caspase 3^+/−/caspase 7^+/− and DKO E20 embryo hearts. Higher magnification of the right ventricular free wall (E and F). Scale bars: (A and B), 500 μm; (C and D), 200 μm; and (E and F), 100 μm. Abbreviations: A, aorta; PT, pulmonary trunk; RA, right atrium; LA, left atrium; RSVC, right superior vena cava; LSVC, left superior vena cava; LV, left ventricle; RV, right ventricle; S, septum; TV, tricuspid valve; RVFW, right ventricular free wall.

Fig. 2

Apoptosis resistance in caspase 3– and caspase 7–deficient MEFs. (A) Caspase 3^+/−/caspase 7^+/−, (B) caspase 3^+/−/caspase 7^−/−, (C) caspase 3^−/−/caspase 7^+/− and (D) DKO MEFs were treated with UV irradiation at 30 J/m², fixed after 6 hours, then stained with DAPI to visualize chromatin condensation (arrows) and nuclear fragmentation (arrowhead). Partial chromatin condensation was seen in caspase 3^−/−/caspase 7^+/− MEFs (asterisks). (E) Caspase 3^+/−/caspase 7^+/− and (F) DKO MEFs, 24 hours after UV irradiation. Light microscopy at 24 hours after UV in (G) caspase 3^+/−/caspase 7^+/−, (H) caspase 3^+/−/caspase 7^−/−, (I) caspase 3^−/−/caspase 7^+/− (some with partial preservation of morphology, arrows), and (J) DKO MEFs. (K) Improved survival in DKO MEFs. MEFs were treated as indicated, FasL and TNF-α each supplemented with cyclohexamide (10 μg/ml). After 24 hours, survival was determined using a live/dead cytotoxicity/viability assay. Data are means ± SD from a single experiment in triplicate, representative of at least three separate experiments. P < 0.01 for pairwise comparison of differences between DKO and each of the other six genotypes. (L) DNA cleavage is associated with caspase 3 alone. Cells were treated with UV and harvested after 3 hours, then nucleosome ELISA was done to measure DNA fragmentation. Data are means ± SD from a single experiment in duplicate, representative of at least three separate experiments. (M) PARP cleavage is a function of caspase 3. Cells were treated with UV (20 J/m²) and harvested at indicated time points, then Western blots were performed on whole-cell lysates.

Fig. 3

Thymocyte apoptosis. (A) Reduced subG₀ population in caspase 3^−/−/caspase 7^+/− and DKO thymocytes. Thymocytes from RAG^−/− chimeras were plated in media alone or treated with plate-bound antibodies against CD3 and CD28 (20 μg/ml each), the Fas-specific antibody Jo2 (1 μg/ml) + cyclohexamide (10 μg/ml), staurosporine (0.1 μM), or etoposide (25 μM). Cells were fixed at 24 hours and stained with propidium iodide. SubG₀ population was identified by using flow cytometry in the FL-2 channel and was expressed as a percentage of total cells. Data are means ± SD from a single experiment in triplicate, representative of at least three separate experiments. For each treatment, P < 0.001 for the difference between DKO and C3KO. (B) Reduced mitochondrially mediated cell death in DKO thymocytes. Thymocytes with above treatments were analyzed at 24 hours using a live-dead cytotoxicity-viability assay, and viable cells were expressed as a percentage of total cells analyzed. Data are means ± SD from a single experiment in triplicate, representative of at least three separate experiments.

Fig. 4

(A) Preservation of Δψ_m in DKO MEFs. MEFs were treated with UV irradiation at 30 J/m² and harvested at indicated time points. Δψ_m was assessed by incubating with JC-1 reagent and by fluorescence-activated cell sorting (FACS). JC-1 enters cells and gives a green fluorescent signal (FL-1, x axis), and is processed to give an additional red fluorescent signal (FL-2, y axis) only in mitochondria with preserved Δψ_m. Cells with preservation of Δψ_m are both FL-1 high, FL-2 high (top right in each plot); cells with loss of Δψ_m are FL-1 high, FL-2 low (bottom right in each plot). FACS data plots are shown, with percentage of total events indicated. Data are from a single experiment, representative of at least three independent experiments. (B and C) Control of release of mitochondrial apoptotic factors by caspases 3 and 7. (B) Delayed Bax translocation and cytochrome c release in DKO MEFs. MEFs treated with UV irradiation at 30 J/m², mitochondrial (top) and cytoplasmic (bottom) fractions were separated at indicated time points. Western blots were probed with indicated antibodies. (C) Absent AIF nuclear translocation in DKO MEFs. MEFs were treated with UV irradiation at 30 J/m² and fixed after 6 hours. Cells with AIF nuclear translocation were counted in five random fields and expressed as a percentage of total cells (minimum 120 total cells counted per genotype). Data are means ± SD from a single experiment, representative of two separate experiments.