Fas-mediated apoptosis is important in regulating cell replication and death in trisomy 8 hematopoietic cells but not in cells with other cytogenetic abnormalities by Elaine M. Sloand, Sonnie Kim, Monika Fuhrer, Antonio M. Risitano, Ryotaro Nakamura, Jaroslaw P. Maciejewski, A. John Barrett, and Neal S. Young Blood Volume 100(13):4427-4432 December 15, 2002 ©2002 by American Society of Hematology

Expression of active caspase in cells with trisomy 8 Expression of active caspase in cells with trisomy 8.Freshly obtained BM smears from patients with trisomy 8 were stained for activated caspase as described in “Patients, materials, and methods,” then subsequently subjected to FISH for chromosome 8. Expression of active caspase in cells with trisomy 8.Freshly obtained BM smears from patients with trisomy 8 were stained for activated caspase as described in “Patients, materials, and methods,” then subsequently subjected to FISH for chromosome 8. Cells were scored for caspase and karyotype. The histograms show increased caspase staining in cells positive for trisomy 8 when compared with cells with a normal karyotype from the same patient. Normal BM demonstrated less than 3% caspase staining. Elaine M. Sloand et al. Blood 2002;100:4427-4432 ©2002 by American Society of Hematology

Example of a blood smear of a patient with trisomy 8 stained for activated caspase-3.Freshly obtained blood smear was stained for activated caspase as described in “Patients, materials, and methods” and then subjected to FISH. A low-power view of cells stai... Example of a blood smear of a patient with trisomy 8 stained for activated caspase-3.Freshly obtained blood smear was stained for activated caspase as described in “Patients, materials, and methods” and then subjected to FISH. A low-power view of cells staining for activated caspase is seen in panel A. A close-up view of a trisomy 8 cell, also positive for caspase-3, is seen in panel B. The same cell visualized with either a green or red bandpass filter showing caspase staining (C) and trisomy 8 probe (D). Original magnification × 100. Elaine M. Sloand et al. Blood 2002;100:4427-4432 ©2002 by American Society of Hematology

Apoptotic markers on CD34 cells obtained from patients with trisomy 8 and monosomy 7.BMMNCs were obtained from patients with de novo MDS and trisomy 8 or monosomy 7 and were then stained with monoclonal antibodies: CD34-PE and annexin–fluorescein isothiocya... Apoptotic markers on CD34 cells obtained from patients with trisomy 8 and monosomy 7.BMMNCs were obtained from patients with de novo MDS and trisomy 8 or monosomy 7 and were then stained with monoclonal antibodies: CD34-PE and annexin–fluorescein isothiocyanate (annexin-FITC) or CD95-FITC. Cells were gated for CD34 cells, and the number of annexin-staining and CD95-staining cells were quantitated. CD34+, CD95+, and CD34+, PI−, annexin+ cells are shown in the histograms above. CD34 cells from patients with trisomy 8 showed increased expression of annexin and CD95 when compared with patients with monosomy 7 (n = 5) and normal cells.10 Elaine M. Sloand et al. Blood 2002;100:4427-4432 ©2002 by American Society of Hematology

Comparison of CD95 expression on cells with trisomy 8 and monosomy 7 and normal cells from the same donor.BM was obtained from 2 patients with trisomy 8 and 2 patients with monosomy 7 and stained with CD3-PE and CD95-FITC mAbs. Comparison of CD95 expression on cells with trisomy 8 and monosomy 7 and normal cells from the same donor.BM was obtained from 2 patients with trisomy 8 and 2 patients with monosomy 7 and stained with CD3-PE and CD95-FITC mAbs. CD3− cells were positively selected and sorted into CD95− and CD95+ aliquots, which were prepared for FISH with fluorescent probes for chromosome 7 or 8. The proportion of cells with each cytogenetic abnormality is shown in panel A. An example of stained cells in the Fas− and positive aliquots are seen in panel B (trisomy 8; chromosome 8 is orange; chromosome 7, a control, is green) and panel C (monosomy 7; chromosome 7). Trisomy 8 sample on the left was obtained from a patient with a history of aplastic anemia. Elaine M. Sloand et al. Blood 2002;100:4427-4432 ©2002 by American Society of Hematology

Proliferation of cells from patients with trisomy 8 and monosomy 7 in the presence of Fas agonist or Fas antagonist.BMMNCs were cultured with and without Fas agonist monoclonal antibody mAb (CH11) or Fas antagonist mAb (ZB4). Proliferation of cells from patients with trisomy 8 and monosomy 7 in the presence of Fas agonist or Fas antagonist.BMMNCs were cultured with and without Fas agonist monoclonal antibody mAb (CH11) or Fas antagonist mAb (ZB4). Slides were prepared, and FISH was performed with labeled centromeric probes for chromosomes 8 and 7 prior to culture and on days 4, 7, and 14. Days 0 and 4 are shown above (no change in expression was seen between days 4 and 14). The proportion of cells with trisomy 8 is seen in panel A; those with 5q− and monosomy 7 are seen in panel B. *Samples from patients with a history of aplastic anemia receiving CsA. Elaine M. Sloand et al. Blood 2002;100:4427-4432 ©2002 by American Society of Hematology