For proliferative responses, single-cell preparations from spleen, lymph node, and bone marrow were cultured in 96-well plates at 2 × 105 cells/ml for 24–72 h. Cells were pulsed with [3H]thymidine for the last 18 h of culture and assayed for incorporation. When you consider what’s at stake with the exam, we believe the value of our study guide gives you at least ten times your money’s worth.
For these studies, three clones of each transfectant were selected and pooled. Using the TUNEL assay for detection of DNA strand breaks, we examined apoptosis of both cell populations cultured in serum-free medium supplemented with insulin and transferrin. During the first 18 h of culture, neither U937+ nor U937− cells showed clear evidence of apoptosis, but prominent differences were seen at 38 and 68 h (Fig. 3), indicating a proapoptotic role for ICSBP in myeloid cells.
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2 E shows that Phiphilux was cleaved in T cells 4 d after anti-CD3 stimulation. Similar results were also obtained as early as 2 d of stimulation, in which a 4.7-fold increase in mean fluorescence intensity was observed (mean fluorescence intensity of 27.3 vs. 5.8 for resting cells). Interestingly, induction of apoptosis by anti-Fas antibody did not result in increased Phiphilux cleavage in activated PBMCs (Fig. 2 E), suggesting that caspase activity was already elevated in proliferating cells. Therefore, caspase-3 cleavage in stimulated lymphocytes corresponded to activation of the proenzyme. Caspase-8 is cleaved in activated lymphocytes, whereas caspase-9 remains as a proenzyme.
Three forms ranging from 17 to 21 kD were detected in lysates of activated, but not resting cells (Fig. 2 C). To verify whether these proteins originated from caspase-3, the caspase was immunoprecipitated from lysates of PHA-stimulated PBMCs using the antiserum, and whole lysates or immunoprecipitated proteins were separated on SDS-PAGE. Western blot analysis (Fig. 2 D, left) shows that the immunoprecipitate contained a doublet at 20 kD, as well as the 17-kD subunit and a 15-kD band sometimes observed in late apoptotic Jurkat cells using this antiserum (data not shown). When the membrane was probed with Extra-HRP, only the 17-kD band and the upper band from the doublet were detected (Fig. 2 D, right), showing that both caspase-3 forms were able to bind to the synthetic substrate. Another caspase subfamily including caspase-1, -5, -11, -12, and -13 is essentially involved in cytokine maturation, and does not seem to play a critical role during apoptosis. Caspase activity results in the cleavage of several cellular proteins, which leads to the apoptotic response (for a review, see reference 7).
Thus, it was of interest to determine whether caspase activation in stimulated lymphocytes resulted in cleavage of these substrates. The same extracts analyzed for PARP and Wee1 cleavage were subjected to Western blot analysis using an anti-DFF45 antiserum generated against the full-length DFF45. 6 demonstrate that DFF45 was not cleaved in these lysates, although caspase-3 was almost completely processed (Fig. 6). The cleavage of RFC140 was analyzed by Western blot, and results confirmed that this substrate was also resistant to caspase-mediated cleavage in activated lymphocytes, as we have observed previously (11; data not shown).
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After washing in PBS, cells were resuspended in saline citrate buffer containing 4× SSC, 2.5 μg/ml FITC–avidin (Boehringer Mannheim Biochemicals), 0.1% Triton X-100 (Sigma Chemical Co.), and 5% nonfat dry milk. After a 30-min incubation in the dark, cells were washed in PBS with 0.1% Triton X-100 and 0.5% BSA and resuspended in Sorter medium (Quality Biological, Inc.). DUTP incorporation by individual cells was measured by green fluorescence on a FACScan™ flow cytometer using Cellquest™ data software (Becton Dickinson).
ICSBP knockout mice exhibit dysregulated hematopoiesis, defining a role for ICSBP in the proliferation and differentiation of hematopoietic cells. Here we report that myeloid cells from ICSBP-deficient mice exhibited reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. Moreover, overexpression of ICSBP in U937 cells was sufficient to enhance their sensitivity to spontaneous and induced apoptosis. Our results thus define ICSBP as a regulator of myeloid cell survival. We also point out that ICSBP is likely to normally function in regulation of the cell cycle in hematopoietic cells, because the spontaneous proliferation of bone marrow, spleen, and lymph node cells from mutant mice was significantly increased. We next examined the role of caspases in programmed cell death of U937+ cells.
In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-XL. These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes. PBMCs were stimulated (+) or not (−) with anti-CD3 antibody and IL-2 for 4 d, and stained for 10 min with FITC-conjugated AV. Living (AV−) and dying (AV+) activated lymphocytes were purified by flow cytometric cell sorting. Proteins from sorted cells were subjected to Western blot analysis for caspase-3 and PARP. (B) Caspase-6 and -7 and the caspase substrates are processed in viable sorted cells.

Surface staining was performed for CD69, HLA-DR, and CD25 on PBMCs activated for 4 d with anti-CD3/IL-2, in the presence or absence of 100 μM of zVAD. Purified T lymphocytes isolated from PBMCs by negative selection were preincubated for 1 h in medium alone or with 100 μM zVAD, and cultured for 4 d without stimulation (NS) or in the presence of IL-2, anti-CD3, or both. Proliferation was assessed by [3H]thymidine incorporation as described in A. Although it was initially assumed that CML is caused by uncontrolled cell proliferation resulting in the clonal expansion evident in this disease, some studies have indicated that the rates of proliferation are not significantly increased 20,21. Other studies have shown that myeloid cells of CML patients have increased resistance to apoptosis, indicating that this disease may result from suppression of cell death rather than ungoverned proliferation 22,23. To investigate the possible relatedness of the disease of ICSBP−/− mice to CML, we first studied the levels of spontaneous proliferation and apoptosis of cells from normal and mutant mice.

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Caspase-3 is cleaved into multiple fragments in stimulated lymphocytes. Purified PBMCs were obtained through Ficoll gradient separation and stimulated with 1 μg/ml of anti-CD3 and 20 U/ml of IL-2 (Anti-CD3) or not (Medium). After 1–4 d at 37°C, thymidine incorporation was determined as in the legend to Fig. Proteins were extracted from the same PBMCs treated in A by cell lysis using a denaturing Laemmli buffer containing 8 M urea. Total proteins extracted from 106 fresh (d0), unstimulated (−), or stimulated cells (+) were separated on SDS-PAGE, and Western blot analysis was performed using a polyclonal rabbit anti–caspase-3 (reference 5).
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Substrate processing during T cell activation is caspase dependent and is different than after apoptosis. Fresh PBMCs were stimulated for 48 h with anti-CD3 and IL-2 in the presence or absence of 100 μM of zVAD. An aliquot was conserved for quantitation of DNA synthesis by thymidine incorporation, and cells were lysed in denaturing buffer for Western blot analysis of PARP, Wee1, lamin B, and caspase-3 cleavage. Black arrows indicate the full-length proteins, and white arrows designate the cleaved forms. PBMCs activated for 4 d with anti-CD3 and IL-2 were placed in anti-Fas–coated plates and cultured for 6 h at 37°C. Apoptosis was assessed by AV/PI staining, and the cells were subjected to Western blot analysis using the DFF45 antiserum.

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Wee1 is a critical component of the G2/M cell cycle checkpoint machinery, and mediates cell cycle arrest after DNA damage by phosphorylation of Cdc2 45. Therefore, cleavage of Wee1 in proliferating lymphocytes could lead to its inactivation, thus allowing cell cycle progression. Of note, Wee1 processing by caspases during apoptosis in Jurkat cells correlates with a 20-fold decrease in Wee1 activity and an increase in Cdc2 activity 13. The nuclear protein lamin B is considered as a caspase-6 substrate 7. Our results show that in fresh PBMCs, two forms of 66 and 45 kD can be identified by Western blot using an anti–lamin B antibody, and at least two fragments of 35 and 28 kD are produced after T cell activation.
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(A) Cells were cultured for 24–72 h and pulsed with [3H]thymidine for the last 18 h of culture. (B) Cells from −/− mice and +/+ littermates were cultured for 1 and 2 d and analyzed for apoptosis by TUNEL assay. Whole cell extracts were prepared by lysing cells for 30 min at 4°C with lysis buffer (50 mM Tris, pH 7.5, 2 mM EDTA, 100 mM NaCl, 1 mM Na3VO4, 1% NP-40, 10 μg/ml leupeptin, 5 μg/ml aprotinin, and 10 μg/ml PMSF) at a concentration of 106 cells/50 μl.