The effect of amifostine on differentiation of the human megakaryoblastic Dami cell line

Abstract

Amifostine is a cytoprotective drug that was initially used to control and treat nuclear radiation injury and is currently used to provide organ protection in cancer patients receiving chemotherapy. Clinical studies have also found that amifostine has some efficacy in the treatment of cytopenia caused by conditions such as myelodysplastic syndrome and immune thrombocytopenia, both of which involve megakaryocyte maturation defects. We hypothesized that amifostine induced the differentiation of megakaryocytes and investigated this by exposing the human Dami megakaryocyte leukemia cell line to amifostine (1 mmol/L). After 12 days of amifostine exposure, optical microscopy showed that the proportion of Dami cells with diameters >20 μm had increased to 24.63%. Transmission electron microscopy identified the development of a platelet demarcation membrane system, while flow cytometry detected increased CD41a expression and decreased CD33 expression on the Dami cell surface. Ploidy analysis found that the number of polyploid cells with >4N DNA content increased to 27.96%. We did not detect any elevation in the mRNA or protein levels of megakaryocytic differentiation-associated transcription factors GATA-binding factor 1 (GATA-1) and nuclear factor, erythroid 2 (NF-E2), but nuclear import assay revealed an increased nuclear translocation of these proteins. These findings indicate that amifostine induced the differentiation of Dami cells into mature megakaryocytes via a mechanism involving increased nuclear translocation of the transcription factors, NF-E2 and GATA-1.

Keywords: Amifostine; CD41a; DNA ploidy; Dami cells; transcription factor.

Figure 1

Dami cell growth curves in the presence of the indicated concentrations of amifostine. Amifostine (0.1 – 5 mmol/L) inhibited the proliferation of Dami cells. The number of Dami cells remained constant during exposure to 1 mmol/L amifostine, indicating cell differentiation.

Figure 2

The morphology of Dami cells exposed to 1 mmol/L amifostine for the indicated number of days (d). The Dami cell volume and cytoplasm increased over time (A) and the proportion of cells with diameter >20 μm was counted in three randomly selected fields (B). The differences between the control cells and cells treated with amifostine for 4 and 8 days were statistically significant (P < 0.05). Giemsa staining (C) confirmed that the differentiated Dami cells had increased cytoplasmic volume and DNA ploidy. Cells with 32N ploidy were also observed.

Figure 3

Transmission electron microscopy image of Dami cells treated with 1 mmol/L amifostine for 12 days. Mature megakaryocyte features are visible, including well‐developed ribosomes and endoplasmic reticulum, and sponge‐like edges indicating the platelet demarcation membrane system.

Figure 4

Flow cytometric analysis of Dami cell surface CD41a, CD33, and CD34 expression during amifostine treatment (1 mmol/L) for the indicated times. Increased expression of CD41a was observed (A). Dami cells are megakaryoblasts, and did not express CD34 (B). After 12‐day exposure to amifostine induction, cell surface CD33 expression was reduced (C).

Figure 5

DNA ploidy in Dami cells exposed to 1 mmol/L amifostine for the indicated number of days (d). The proportion of 2N cells decreased, while the proportion of 4N and 8N cells increased, as the treatment duration increased; 16N cells were also observed. The differences between the control cells (A) and those treated with amifostine were statistically significant (B). *P < 0.05; **P < 0.01.

Figure 6

The effects of amifostine exposure (1 mmol/L) for the indicated numbers of days (d) on the differentiation‐related transcription factors, GATA‐binding factor 1 (GATA‐1) and nuclear factor, erythroid 2 (NF‐E2), in Dami cells. Amifostine did not alter the mRNA (A) or protein (B) expression levels of GATA‐1 and NF‐E2. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase. After 12 days of treatment with amifostine, the nuclear translocation of GATA‐1 and NF‐E2 were enhanced in Dami cells (C).