Apoptosis Related Gene Expression in Marrow Cells from Aplastic Anemia Patients.

Increased apoptosis is a possible pathogenetic mechanism for the damage of hematopoietic stem cells in aplastic anemia (AA), but the regulation of apoptotic machinery remains unclear. We investigated the parallel mRNA expression of specific pro-apoptotic and anti-apoptotic genes in AA patients and controls. We also examined the presence of apoptosis in the same populations with flow cytometry (FC) analysis. Bone marrow was obtained from 11 aplastic anemia patients (6 male, 5 female) of median age 38 years (range 21–77) and 8 healthy volunteers of similar age. Seven AA patients had active disease (untreated or refractory) and eight patients had long term complete or partial hematological remission after immunosuppressive therapy. BMMNC from three patients and CD34⁺ cells from two patients were examined in active disease and then in remission. We investigated the apoptosis-related gene mRNA expression (Fas, FasL, TRAIL, bcl-xl, bcl-2, bax, caspase 3, caspase 8 and GAPDH as an internal control) by RT-Multiplex PCR in total bone marrow mononuclear cells (BMMNC) or via MACS (magnetic cell sorting) selected CD34⁺ cells, when possible, from aplastic anemia patients and controls. Two-or three-color FC analysis was used for quantitative measurement of cell surface expression of Apo-1/Fas receptor in CD34⁺ cells and Annexin⁺/PI⁻ total BMMNC and CD34⁺ cells. We also estimated the CFCs and LTC-IC derived CFCs values from five AA patients, which were very low, even in two patients who were in hematological remission, compared to controls. Two-color FC analysis revealed a significant difference in Fas expression on BMMNC and on CD34⁺ in active disease patients compared to controls, whereas in patients in remission this difference remains on CD34⁺ cells only. CD34⁺/Annexin⁺/PI⁻ cells are significantly increased in both active disease (p=0.02) and in remission patients (p=0.05) compared to controls. The patients in remission had significantly higher proportion of early apoptotic total BMMNC than the patients with active disease. The results from patients’ BMMNC and CD34⁺ cells’ mRNA gene expression are presented in the table. No mRNA expression of any of the above apoptosis-related genes was detected in CD34⁺ cells from normal controls whereas their expression in normal total BMMNC was variable. There is a variability of gene expression, especially in patients’ BMMNC. Apart from Fas mRNA expression in BMMNC and/or CD34⁺ cells, which is a characteristic finding during active disease, TRAIL mRNA expression was also found in almost all samples. The BMMNC’s TRAIL mRNA expression remains a constant finding even in patients in remission. The expression of anti-apoptotic bcl-xl and/or bcl-2 in BMMNC and CD34⁺ cells from patients with active disease seems unable to inhibit the TRAIL or Fas induced apoptosis, pointing to the possibility that the ratio of pro-apoptotic and anti-apoptotic signals is of importance. In conjunction with our in vitro data (Ref: abstract 1293, ASH 2003), along with Fas, TRAIL expression may be of importance in the pathogenesis of AA.

Expression of apoptosis related genes (I)

Expression of apoptosis related genes (II)