Induction of human fetal hemoglobin expression by adenosine-2',3'-dialdehyde

Abstract

Background: Pharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and β-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2',3'-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells.

Methods: Expression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP).

Results: Adox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5).

Conclusions: The ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and β-thalassemia.

Figure 1

Adox induced γ-globin gene expression in K562 cells. (A) Northern blot analysis of total RNA from K562 cells treated with indicated concentrations of Adox for 2 days. (B) Northern blot analysis of total RNA from K562 cells treated with Adox (2.5 μM) for the indicated number of days. (C) Effects of Adox on in vitro growth of K562 cells. K562 cells were treated with the indicated concentrations of Adox or PBS control (Ctrl) and cell number/ml was determined at the indicated time. (D) Benzidine staining of K562 cells treated with Adox (2.5 μM) or PBS control (Ctrl) for 2 days. (E) Q-RT-PCR quantification of γ-globin from K562 cells treated with PBS control (Ctrl), Adox (2.5 μM) for 2 days, or decitabine (Deci, 4 μM) for one week (refreshed once after 3 days treatment). Graphs show mean ± SD, n = 3.

Figure 2

Modulation of DNA methylation and histone methylation by Adox in K562 cells. (A) Histone H4R3me2s ChIP analysis of γ-globin promoter from K562 cells treated with Adox or PBS control. Graphs show mean ± SD, n = 3. (B) Western blot analysis of proteins with histone H4R3me2s and anti-tubulin antibodies from K562 cells treated with indicated concentrations of Adox for 2 days. (C) Quantification of DNA methylation at the human γ-globin gene in K562 cells treated with Adox, decitabine (Deci), or PBS control as in Figure 1E. Numbers on the bottom represent the positions of the CpG dinucleotides relative to the transcriptional start site of the γ-globin gene. Ave indicates the average methylation of the 4 total CpGs. **P < 0.01, Chi-squared test.

Figure 3

The effects of Adox to induce fetal globin gene expression in human bone marrow erythroid progenitor cells. (A) Q-RT-PCR quantification of γ-globin from human BM treated with indicated concentrations of Adox or decitabine (Deci, 4 μM) with refreshment twice a week compared to PBS control (Ctrl) after 21 days of culture. Graphs show mean ± SD, n = 3. (B) Wright-Giemsa-stained adult bone marrow erythroid progenitor cells at day 21 of differentiation. (C) HPLC analysis of globin in human BM cells treated with Adox (20 μM) or decitabine (4 μM). Peaks for HbF are indicated. Data are representative of three independent experiments. (D) Q-RT-PCR quantification of mRNA levels of β-globin as percentage of (β-globin + γ-globin) from human BM treated with Adox (20 μM) or decitabine (Deci, 4 μM) with refreshment twice a week compared to PBS control (Ctrl) after 21 days of culture. Graphs show mean ± SD, n = 3. (E) Q-RT-PCR quantification of mRNA levels of β-globin as percentage of (β-globin + γ-globin) from human BM treated with Adox (20 μM) or decitabine (Deci, 4 μM) with refreshment twice a week compared to PBS control (Ctrl) after 21 days of culture. Graphs show mean ± SD, n = 3. (F) Q-RT-PCR quantification of α-globin and β-globin from BM treated with Adox (20 μM) with refreshment twice a week compared to PBS control (Ctrl) after 21 days of culture. Graphs show mean ± SD, n = 3.

Figure 4

Modulation of DNA methylation and histone methylation by Adox in human adult erythroid progenitor cells. (A) Histone H4R3me2s ChIP analysis of γ-gene promoter from human BM cells treated with either PBS control, Adox or decitabine as in Figure 3C. Graphs show mean ± SD, n = 3. (B) Histone H4R3me2s ChIP analysis of CDH1 promoter from human BM cells treated with either PBS control or Adox as in Figure 3C. Graphs show mean ± SD, n = 3. (C) Histone H4 acetylation ChIP analysis of γ-promoter from human BM cells treated with either PBS control or Adox as in Figure 3C. Graphs show mean ± SD, n = 3. (D) Quantitation of DNA methylation at the human γ-globin gene in human BM cells treated as in (A). The numbers on the bottom represent the positions of the CpG dinucleotides relative to the transcriptional start site of the γ-gene. Ave indicates average methylation of the 4 total CpGs. *P < 0.05, #P > 0.05 using Chi-squared test.