Figure 6.
SCF up-regulates Bcl-2 and Bcl-XL, which are able to protect erythroblasts from apoptosis induced by chemotherapeutic drugs. (A) Western blot analysis of Bcl-2 and Bcl-XL levels in day-7 erythroblasts cultured in standard erythroid medium (- SCF) or preincubated from day 5 to day 7 in erythroid medium supplemented with 100 ng/mL SCF (+ SCF).(B) Western bolt analysis of erythroblasts transduced with empty vector, Bcl-2, or Bcl-XL. Cycling CD34+ cells were transduced with a retroviral vector containing cDNAs for Bcl-2 or Bcl-XL and GFP as a reporter gene. Cells were sorted for GFP expression, placed in standard erythroid medium, and analyzed at day 4 of differentiation. One representative experiment of 5 performed with cells from different donors is shown. (C) Comparison of Bcl-2 or Bcl-XL expression in SCF-treated erythroblasts (Epo + SCF) versus erythroblasts grown in standard medium and transduced with Bcl-2 or Bcl-XL. The analysis was performed most cells reached the basophilic stage of differentiation, which corresponded to day 7 of erythroid unilineage culture for untransduced erythroblasts (Epo + SCF vs Epo) and to day 4 of erythroid unilineage culture for transduced cells (Bcl-2 vs empty vector and Bcl-XL vs empty vector). The latter were previously cultivated with cycling growth factors for 5 days (including the transduction and sorting procedures) as described in “Materials and methods.” Counting of unilineage erythroid culture days started when sorted GFP-positive cells were placed in erythroid specific medium. Data show the increase in gene-transduced erythroblasts as compared to empty vector-transduced erythroblasts (▪) or in SCF-treated versus untreated erythroblasts (□). (D) Cells at day 4 of erythroid unilineage culture, previously transduced with empty vector (▪), Bcl-2 (□), or Bcl-XL (▦) as in C, were incubated with medium alone (Con), 50 ng/mL camptothecin (CPT), 2 μM etoposide (VP-16), or 3 μg/mL cisplatin (CDDP). The percentage of cell death was evaluated after 24 hours by staining with ethidium bromide—acridine orange. The results shown are the mean ± SD of 3 independent experiments performed with cells from different donors.