Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

Abstract

Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer.

Keywords: Cis-vaccenic acid; Erythroid progenitor stem cells; Fetal hemoglobin; Gamma globin; Mono-unsaturated fatty acid; Sickle cell anemia.

Fig. 1

CVA induces differentiation to the erythroid lineage. Differentiation was assayed as a measure of the emergence of hemoglobinized cells positive for benzidine. Analysis was carried out with K562, transgenic mice bone marrow progenitor stem cells and JK-1 cells. A. K562 cells induced with CVA for 72 h and stained with Benzidine and Giemsa stains. B. K562 cells were induced with varying concentrations of CVA and monitored for percentage of benzidine positive cells at 48 and 120 h post induction. C. Transgenic mice bone marrow progenitor stem cells depleted of plastic adherent cells in IMDM supplemented with 20% FBS, 100 U/ml penicillin, 200 μg/ml streptomycin and 2 U/ml EPO. Cells were induced with CVA for 96 h, and cytospin preparations of the cells were stained using benzidine-giemsa stain. D. Percentage of BFUe colony formation from TMbmEPSCs induced by CVA. TMbmEPSCs depleted of plastic adherent cells were seeded in plates containing semisolid IMDM supplemented with 20% FBS, 100 U/ml penicillin and 200 μg/ml streptomycin. E. JK-1 cells induced with 50 μM CVA and monitored for percentage of benzidine positive cells at 24 and 72 h post induction.

Fig. 2

CVA does not alter JK-1 Cell survival or growth. A. Cell survival was assayed as a measure of percentage positive Trypan blue stained cells. Cell viability was monitored at 24, 48 and 120 h post CVA induction. B. JK-1 cell growth was monitored for 96 h. The data represents the mean and corresponding S.D. of three independent experiments.

Fig. 3

CVA up-regulates γ and β-globin gene expression and fetal hemoglobin synthesis. CVA induced γ-globin gene expression was monitored in A. JK-1 cells using RT-PCR at 24, 48 and 72 h post CVA induction. B. qRT-PCR was used to measure JK-1 cell relative γ-globin gene expression 24, 48, 72, 96 and 120 h post CVA induction. Expression data was normalized to JK-1 GAPDH expression. C. Concentration dependent effect of CVA on JK-1 cell γ-globin gene expression was assayed using semi-quantitative RT-PCR. D. Effect of CVA induction on transgenic mice bone marrow erythroid progenitor stem cells γ-globin gene expression. Relative γ-globin gene expression was assessed using qRT-PCR at 24, 96, 168 and 240 h post CVA induction. γ-globin gene expression was normalized to transgenic mice bone marrow progenitor stem cells GAPDH gene expression. E. effect of CVA on JK-1 cells fetal hemoglobin synthesis. Fetal hemoglobin was assayed in JK-1 cell lysates using alkaline denaturation assay as previously described (Fibach et al., 1993). Hemin was used as a positive control inducer. The experiment was carried out in duplicate, and although precise globin mRNA levels appeared to fluctuate from culture to culture, but relative γ-globin levels were always higher in cells induced in transgenic mice primary bone marrow progenitor stem cells induced with 50 μM CVA. The data represents the mean and corresponding S.D. of three independent experiments. ANOVA was used to analyse the differences between groups and differences were considered significant at P<0.05.

Fig. 4

CVA lowers KLF1 gene expression in JK-1 cells. JK-1 cells were grown in RPMI 1640 medium supplemented with 20% FBS, 100U/ml penicillin and 200 μg/ml streptomycin, cells induced with CVA (50 μM). KLF1 gene expression was monitored quantitatively using qRT-PCR 24 h post induction. KLF1 gene expression was normalized to JK-1 GAPDH gene expression and compared to un-induced controls. The data represents the mean and corresponding S.D. of three independent experiments. ANOVA was used to analyse the differences between groups and differences were considered significant at P<0.05.

Fig. 5

Effects of Erythropoietin on CVA induced JK-1 cell differentiation and γ-globin gene expression. EPO has been shown to direct erythroid cell development and maturation (Orkin and Zon, 2008). JK-1 cells were induced with EPO (2 U/ml) for 48 h prior to induction with CVA; cells were then induced using 50 μM CVA, EPO (2 U/ml) and ‘CVA (50 μM)+EPO (2 U/ml)’ for 48 h. Controls were set-up for each treatment using JK-1 cells which have not been induced with EPO. JK-1 cell differentiation and γ-globin gene expression were assayed 48 h post CVA induction. A. Experimental set-up B. JK-1 relative γ-globin gene expression normalized to GAPDH C. JK-1 cell differentiation assayed by benzidine-giemsa staining. The data represents the mean and corresponding S.D. of three independent experiments. ANOVA was used to analyse the differences between groups and differences were considered significant at P<0.05.

Fig. 6

Effects of CVA on Transgenic Mice Bone Marrow Erythroid Progenitor Stem Cell Differentiation. Cells were induced with 50 μM CVA at the start of culture or 48 h after the commencement of culture. A. TMbmEPSCs were grown in IMDM supplemented with 20% FBS, 100U/ml penicillin, 200 μg/ml streptomycin and 2 U/ml EPO. γ-globin gene expression was assayed 48 h post CVA induction. B. TMbmEPSCs were grown in semisolid IMDM supplemented with 20% FBS, 100U/ml penicillin, 200 μg/ml streptomycin and 0.9% methylcellulose. BFUe colonies were enumerated 24 h post CVA induction. The data represents the mean and corresponding S.D. of three independent experiments. ANOVA was used to analyse the differences between groups and differences were considered significant at P<0.05.

Fig. 7

Inhibition of Eukaryotic Fatty acid Elongase V and Δ⁹-desaturase Modulates CVA induced differentiation and γ-globin induction capacity. JK-1 cells were grown in RPMI 1640 medium supplemented with 20% FBS, 100U/ml penicillin and 200 μg/ml streptomycin. Fatty acid elongase V (Elovl5) and Δ⁹-desaturase were inhibited at the start of culture in separate experiments using Cycloate (40 μM) or Isoxyl (10 μM) respectively or jointly. CVA was used to induce JK-1 cell γ-globin gene expression in both inhibited culture systems. A. CVA was used to induce Elovl5 and Δ⁹-desaturase inhibited JK-1 cells at the start of cultures. B. CVA (50 μg) was used to induce Elovl5 and Δ⁹-desaturase inhibited TMbmEPSCs cells at the start of cultures. C. CVA (50 μM) was added to Elovl5 and Δ⁹-desaturase inhibited JK-1 cells 48 h post inhibition, γ-globin gene expression was assayed 48 h post induction. The data represents the mean and corresponding S.D. of three independent experiments. ANOVA was used to analyse the differences between groups and differences were considered significant at P<0.05.

Fig. 8

Effect of CVA Rescue on TMbmEPSCs differentiation. CVA (50 μM) was added to Elovl5 and Δ⁹-desaturase inhibited TMbmEPSCs 48 h post inhibition on semisolid IMDM supplemented with 20% FBS, 100 U/ml penicillin and 200 μg/ml streptomycin, BFUe colonies were enumerated 24 h post induction. The data represents the mean and corresponding S.D. of three independent experiments. ANOVA was used to analyse the differences between groups and differences were considered significant at P<0.05.