Effects of CrmA and antisense cDNA constructs complementary to single ICE proteases on apoptosis. (A) Inhibition of apoptosis in MG63 cells by cDNA microinjection. Cells (8 × 105) were seeded in 60-mm tissue dishes. After overnight incubation, cell nuclei were microinjected with a CrmA expression plasmid or antisense constructs targeting ICH-1, ICE, or CPP32. Cell injections were performed with an automatic microinjection system equipped with glass micropipettes loaded with DNA and Texas red-labeled dextran. After 20 hours after microinjection, cells were treated with anti–APO-1, CR, DX, or MT as described in Fig 1 and incubated for a further 48 hours. Anti–APO-1 treatment was applied in the presence of 5 μg/mL cycloheximide, which was not toxic by itself. Cells were fixed with 2.5% glutaraldehyde and inspected microscopically. Microinjected cells were identified by red fluorescent staining. Cells were regarded as apoptotic when they showed membrane blebbing and/or a condensed cell nucleus. At least 180 cells were analyzed for each condition. The values represent the mean ± SD from three independent experiments. (B) Effect of cDNA expression after electroporation of CEM cells. Cells (2 × 108) were transfected the antisense constructs using an electroporator (975 μF, 230 V). After electroporation, cells were seeded at 1 × 106 cells/well in 6-well plates. Eight hours later, cells were treated with G-418 to deplete untransfected cells. Dead cells were removed after a further 36 hours by a washing step in a Ficoll-Paque gradient. The enriched population containing 65% to 88% of transfected cells was then treated with anti–APO-1 for 4 hours or with CR, DX, or MT at the concentrations indicated in Fig 1 for 20 hours. Apoptosis was measured by propidium iodide/Hoechst 33342 uptake using flow cytometry. Data are given as the mean percentage of cell death from four experiments with duplicate samples.
(C) Morphologic analysis of doxorubicin-induced apoptosis after microinjection of CrmA cDNA and antisense constructs complementary to ICE family members in SHEP cells. Cells were microinjected as described in (A) using 0.2% Texas red-dextran together with cDNA constructs encoding (a) empty pCDNA-3 vector, (b) pSV25S-CrmA, (c) antisense ICE, and (d) antisense CPP32. Eight hours after transfection, cells were treated with anti–APO-1, CR, DX, and MT and evaluated 30 hours later by two independent scientists. Microinjected cells were identified by red fluorescent staining (left panel) and are indicated by arrows (right panel). The scale bar represents 20 μm. Quantitative values of specific inhibition after scoring of about 300 cells were 58% for CrmA, 62% for pCDNA-3-a300h.ICE, 61% for pCDNA-3-a300h.CPP32, and 64% for the mixture of ICE and CPP32 antisense constructs microinjected together.
(D) Long-term protection from drug-induced apoptosis by antisense constructs. CEM cells were transfected with empty vector, antisense ICE, or CPP32 constructs and selected in G418-containing medium for 36 hours. Cells were then treated with the indicated cytotoxic drugs. After 30 hours, cells were harvested and limiting dilutions (1:3) were performed, starting from 3 × 104 cells per well. Cell viability was assayed by trypan blue exclusion after 14 days. The highest dilutions still containing living cells are indicated. Samples of cells transfected with control vector did not contain living cells after 14 days of drug treatment; therefore, the vector alone bar is not included. Control indicates the clonogenicity of vector-transfected cells without drug treatment. The data represent experiments with quadruplicated samples. Standard deviations were in the range of one dilution step.
