Nucleus counterstaining by Hoescht showed clearly that mitochondrial depolarization occurred in non apoptotic cells (Fig. of specific products. HSP90 is usually shown as a loading control. (D) CD36 cells (2 105 cells/ml) were cultured in the presence of Epo with (+) or without (?) 150 M DEVD-cmk (DEVD). Every 2 or 3 3 d, cells were diluted to 2 105 cells/ml, and cytokines and DEVD-cmk were added. Cell morphology was examined after May-Grnwald-Giemsa staining on day 8 of the culture. (E) The hydrolysis of IETD-AFC (caspase-8), LEHD-AFC (caspase-9), and VDVAD-AFC (caspase-2) peptide substrates was monitored in whole cell lysates from 0.5 106 CD36+ cells cultured for indicated times in the presence of Epo or Epo + TGF-1. For caspase-3, -9, and -2 activation; results are expressed as percentage of the activity measured in 12-h growth factorCdeprived cells (Deprived), and results for caspase-8 activity (A) are expressed as percentage of the activity measured in lysates of cells cultured with Epo + 500 ng/mL of an agonist anti-CD95 (Fas) mAb (Fas-L) for 12 h. For detection of caspase-3 (BD PharMingen), poly(ADP-ribose) polymerase (PARP; Boehringer Mannheim), ICAD (inhibitor of caspase-activated DNase [CAD]) (DFF45; Euromedex-Upstate Biotechnology), lamin B (provided by J. Courvalin, Centre National de la Recherche Scientifique, Paris, France), acinus (provided by Y. Tsujimoto, Osaka University Medical School, Suita, Japan), HSP 90 (StressGen), and GATA-1 (provided by I. Dussanter, INSERM U363, Hopital Cochin, Paris, France), cells were lysed in Laemmli buffer or in 1% SDS, 10 mM Tris, pH 7.4, 1 mM vanadate, 1 mM PMSF, 1 mM dithiothreitol, and 10 g/ml leupeptin, aprotinin, and pepstatin. Protein concentration was measured in the supernatant by the use of micro BCA protein assay (Pierce Chemical Co.). Proteins were analyzed by standard immunoblot procedure as described earlier 13. Results and Discussion To determine whether caspases could play a role in terminal erythroid differentiation, we used a ID1 two-step amplification culture system in which normal human CD34+ progenitor cells were amplified during 7 d in the presence of SCF, IL-3, and Olcegepant IL-6. Then, CD36+ erythroid progenitors were isolated and cultured in the presence of SCF, IL-3, and Epo. After 7 d of this second step of culture (day 7), all stages of erythroblast differentiation were observed (Fig. 1 A), though the percentage of enucleated erythrocytes remained limited to 1C5%. To increase the percentage of mature erythroblasts and erythrocytes, TGF-1 (2.5 Olcegepant ng/ml) was added to the culture medium of CD36+ cells. While inhibiting cell proliferation, TGF-1 accelerates and synchronizes erythroid differentiation with production at day 7 of 75 and 25% of mature polychromatic or orthochromatic erythroblasts and enucleated red cells, respectively 12. Open in a separate window Open in a separate window Physique 1 The caspase inhibitor z-VAD-fmk arrests erythroid maturation at the basophilic stage. CD36 cells (2 105 cells/ml) were cultured for 8 d in the presence of Epo or Epo + TGF-1 with (+) or without (?) 150 M z-VAD-fmk (ZVAD). Every 2 or 3 3 d, cells were diluted to 2 105 cells/ml, and cytokines and z-VAD-fmk were added. (A) Cell morphology was examined after May-Grnwald-Giemsa staining on day Olcegepant 8 of the culture. Absolute numbers of each type of erythroblasts (immature cells, mature cells, and erythrocytes) in the presence or absence of z-VAD-fmk are indicated above percentages. (B) Percentage of cells made up of hemoglobin was estimated after benzidine staining. All results are the Olcegepant mean of four impartial experiments. Addition to culture.