Cleavage of hRad21 in caspase-3-deficient MCF-7 breast cancer cells. Apoptosis was induced by treatment of MCF-7 cells with 60 μM etoposide for 6 h. Cells treated with DMSO (vehicle) served as a control. Whole-cell lysates or lysates from the cytoplasmic and nuclear fractions were electrophoresed on an SDS-6% PAGE gel and subjected to Western blot analysis using hRad21 mAb. Arrows indicate the hRad21 products.

IP and Western blot (WB) analyses of the hRad21 cleavage products. Apoptosis was induced in Molt4 T cells by treatment with 10 μM etoposide (Etop) for 8 h. Protein lysates were subjected to IP using hRad21 mAb or a control bacterial trpE mAb. Both monoclonal and polyclonal C-terminal antibodies detected 64- and 60-kDa bands (closed arrows), indicating that these bands are from the C-terminal portion of the cleaved protein.

Caspase peptide inhibitors inhibit etoposide-induced cleavage of hRad21. Molt4 cells were treated with 20 μM z-VAD-FMK, a broad-spectrum caspase inhibitor, 1 h prior to etoposide (10 μM) treatment for 6 h. At the end of the incubation period, protein lysates were analyzed on an SDS-6% PAGE gel followed by Western blot analysis using hRad21 C-terminal pAb.

Effect of p53 status on the cleavage of hRad21. Apoptosis was induced in the myeloid leukemia cell lines ML-1 and HL-60, with WT and null p53 genotypes, respectively, by treatment of the cells with UV light (20 J/m²) or ionizing radiation (IR) (20 Gy). Six hours after treatment, protein lysates were made and resolved by SDS-6% PAGE followed by Western blot analysis using hRad21 mAb. Arrows indicate hRad21 cleavage products. C, control without UV or IR treatment.

Apoptosis-induced cleavage of hRad21 in Molt4 T-cell leukemia and JEG3 choriocarcinoma cells treated with etoposide, prostaglandin, and proteasome inhibitors. (A) Dose-related cleavage of hRad21 in Molt4 T-cell leukemia cells treated with increasing concentrations of etoposide (10 to 50 μM) for 6 h. (B) JEG3 cells treated with 15DPGJ₂ (2.4 to 8 μM) for 16 h. (C) Molt4 cells were also treated with a 0.025 mM concentration of proteasome inhibitors, MG115, and MG132 for 8 h. Lysates of these samples were resolved on an SDS-PAGE (4 to 20% acrylamide) gel, transferred to a nitrocellulose membrane, and analyzed by Western blot using a monoclonal hRad21 antibody. Induction of apoptosis resulted in the generation of approximately 64- and 60-kDa hRad21 cleavage products (shown by the closed arrows). Full-length hRad21 (122 kDa) is indicated by the open arrow.

C-terminal Rad21 cleavage product translocates to the cytoplasm after induction of apoptosis. Apoptosis was induced by treatment of EL-12 mammary epithelial cells with UV light (100 J/m²). UV-treated (bottom panel) and untreated control (middle panel) cells were subjected to immunofluorescence staining and microscopy using a C-terminal hRad21 pAb (green fluorescence) and hRad21 mAb (red fluorescence), respectively. The signals of mAb and pAb were visualized by the addition of rhodamine-labeled goat anti-mouse IgG and fluorescein-labeled goat anti-rabbit IgG, respectively. The upper panel shows the background staining (negative control) from the fluorescein-labeled secondary antibody in the presence of normal mouse and rabbit IgGs. The nuclear material is visualized by DAPI staining (blue fluorescence). Panels at right show merged images of red, blue, and green fluorescence.

Effect of the cytoplasmic expression of hRad21 on apoptosis and cleavage of hRad21 protein. Molt4 cells were transiently transfected with the constructs pCMV/myc/cyto-hRad21 and pCMV/myc/nuc-hRad21, which direct the expression of the recombinant protein to the cytoplasm and nucleus, respectively. Empty PCMV/myc/cyto vector served as a control. At 48 h posttransfection, protein lysates were made and subjected to Western blot analysis using c-myc epitope (9E10) and hRad21 mAb to detect the fusion and/or native hRad21 proteins, respectively. Lysates were also used for the assaying of caspase-3 activity as described in Materials and Methods. The data for caspase-3 activity shown at right are the averages and standard errors of the mean from two experiments. Individual values were compared using Student's t test, and values labeled “a” are significantly different (P < 0.05) from that labeled “b.”

Analysis of the proapoptotic activity of C-terminal hRad21 in 293T cells by examination of cellular morphology (A) and staining with TUNEL (B), Annexin V (C), and DAPI (D). In panels A to C, 293T cells were transiently transfected with CMV promoter-driven myc-tagged mammalian expression plasmids (pCS2MT) encoding the full-length hRad21 or cDNAs encoding the two cleavage products, hRad21 N-terminal (aa 1 to 279) and hRad21 C-terminal (aa 280 to 631) proteins. In panel D, in addition to the above constructs, cells were also cotransfected with a red fluorescence plasmid (pDsRed1-Mito) to account for the hRad21-transfected cells. Note that C-terminal-hRad21-transfected cells have round morphology, as shown in light micrographs (A) and are positive for TUNEL (B) and Annexin V (C) staining (arrows). PI was used to detect the integrity of cell membranes in Annexin V-FITC staining; hence, the nuclei of some apoptotic cells were red (arrowhead). In panel D, more than 50% of the C-terminal-hRad21-transfected cells with red fluorescence have fragmented nuclei and condensed chromatin, typical of apoptotic cells.

Cleavage of in vitro translated hRad21 by recombinant caspase-3 and caspase-7. (A) In a cleavage reaction, in vitro translated unlabeled (nonisotopic) WT hRad21 or ACS mutant hRad21 (ACSmut-I or ACSMut-II) in the rabbit reticulocyte lysate were incubated with saline (vehicle) (lanes a), caspase-3 (lanes b), or caspase-7 (lanes c). TNT reactions in the absence of plasmid DNA served as a negative control. Samples were analyzed on an SDS-6% PAGE gel followed by Western blotting with hRad21 C-terminal pAb. Molecular weight markers are shown at left. (B) ³⁵S-hRad21 was incubated in the absence of extract (left lane) or in the presence of Molt4 cell lysates treated with DMSO (middle lane) or 10 μM etoposide (right lane) for 6 h. Samples were resolved on a 4 to 20% gradient SDS-PAGE gel, fixed with methanol and acetic acid for 30 min, dried on a gel dryer, and exposed to a Storm imager.

Characterization of the apoptotic cleavage site of hRad21. The hRad21 cleavage site was mapped biochemically, as described in Materials and Methods, through N-terminal sequencing of a Coomassie-stained PVDF membrane that was electroblotted with immunoprecipitated hRad21 cleavage products. (A) Comparison of the apoptotic cleavage recognition site and the adjoining sequence of hRad21 with those of other vertebrates (MouseRad21 and Xenopus Rad21) and simpler eukaryotes. C.elegans, Caenorhabditis elegans. The arrow indicates the peptide bond cleaved during apoptosis. (B) Construction of the apoptotic cleavage site mutants, ACS-mut-I and ACS-mut-II, to verify whether specific cleavage occurs at D²⁷⁹ in hRad21 after induction of apoptosis, by introduction of a point mutation to substitute an alanine (A) for aspartate (D) (mut-I) or alanine (A) for aspartate (D) and serine (S) (mut-II). (C) Molt4 cells were transiently transfected with blank vector (pCS2MT), WT hRad21 (pCS2MT-hRAD21), or ACS mutants tagged with myc epitope at their N termini (pCS2MT-ACSmut-I or pCS2MT-ACSmut-II) and treated with etoposide as indicated. Lysates were analyzed with SDS-6% PAGE followed by Western blot analysis using antibody against myc tag (9E10) to distinguish the cleavage products from the native forms of transfected hRad21 WT and hRad21 ACS-mut-I and ACS-mut-II proteins.

Quantitative analysis of the proapoptotic activity of C-terminal hRad21 in 293T and Molt4 T-cell leukemia cells. Cells were transiently transfected with CMV promoter-driven myc-tagged mammalian expression plasmids encoding the full-length hRad21 or cDNAs encoding the two cleavage products, hRad21 N-terminal (aa 1 to 279) and hRad21 C-terminal (aa 280 to 631) proteins. 293T cells were also cotransfected with the red fluorescence plasmid pDSRed1-mito to account for the hRad21-transfected cells. Nuclear degradation of 293T cells (A) and caspase-3 activity in Molt4 cells (B) were measured as described in Materials and Methods. For the nuclear degradation assay, cells were collected and fixed 24 and 48 h after transfection. The samples were stained with DAPI and examined with fluorescence microscopy. Cells with fragmented nuclei or condensed chromatin were counted as apoptotic. For each sample, a total of 50 intact and degraded nuclei of cells coexpressing pDsRed1-mito (with red fluorescence) was counted. Each data point represents the average and standard errors of the mean from three experiments. The data for caspase-3 activity shown in panel B are the averages and standard errors of the mean from two experiments. The results of the apoptosis assay were analyzed using a binomial test of proportions and those for caspase-3 activity were analyzed using Student's t test. Data labeled “a” are significantly different (P < 0.05) from data labeled “b.”

Time course of etoposide-induced hRad21 cleavage. Molt4 cells were incubated in the absence (0 h) or presence of 10 μM etoposide for 1, 2, 3, 4, 6, and 12 h. At the end of the incubation period, lysates from cytoplasmic and nuclear fractions were made. (A) Induction of apoptosis was verified by determination of caspase-3 activity in a Western blot analysis using anti-caspase-3 mAb. Pro-caspase-3 and active caspase-3 are indicated. (B and C) The time course of cleavage of hRad21 protein in etoposide-induced cytoplasmic and nuclear fractions was examined using C-terminal (B) and N-terminal (C) Rad21 pAbs. Full-length hRad21 (122 kDa; open arrow) and C-terminal cleaved 60- and 64-kDa fragments and N-terminal 50- and 55-kDa fragments (closed arrows) are indicated. (D and E) The purities of the cytoplasmic (D) and nuclear (E) fractions were verified with antibodies to tubulin and nuclear lamin, respectively. (F) Quantitative measurement of Annexin V staining showing early and late events of apoptosis. Cells stained with Annexin V (green fluorescence in the outer cell membrane) represent early stages of apoptosis, while cells with both PI-stained nuclei (red fluorescence) and Annexin V staining (green fluorescence in the outer cell membrane) represent late stages of apoptosis. Cells with PI-stained red fluorescence represent necrotic cells only. Normal cells lack any staining. Examples from each category are shown at right. Values represent the percentage of green (early stage of apoptosis), green plus red (late stage of apoptosis), or red (necrotic) fluorescent cells, with 120 cells being used at each time point.