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. 2013 Apr;27(4):889-896.
doi: 10.1038/leu.2012.298. Epub 2012 Oct 16.

The Transporter ABCB7 Is a Mediator of the Phenotype of Acquired Refractory Anemia With Ring Sideroblasts

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The Transporter ABCB7 Is a Mediator of the Phenotype of Acquired Refractory Anemia With Ring Sideroblasts

Maryam Nikpour et al. Leukemia. .
Free PMC article

Abstract

Refractory anemia with ring sideroblasts (RARS) is characterized by mitochondrial ferritin (FTMT) accumulation and markedly suppressed expression of the iron transporter ABCB7. To test the hypothesis that ABCB7 is a key mediator of ineffective erythropoiesis of RARS, we modulated its expression in hematopoietic cells. ABCB7 up and downregulation did not influence growth and survival of K562 cells. In normal bone marrow, ABCB7 downregulation reduced erythroid differentiation, growth and colony formation, and resulted in a gene expression pattern similar to that observed in intermediate RARS erythroblasts, and in the accumulation of FTMT. Importantly, forced ABCB7 expression restored erythroid colony growth and decreased FTMT expression level in RARS CD34+ marrow cells. Mutations in the SF3B1 gene, a core component of the RNA splicing machinery, were recently identified in a high proportion of patients with RARS and 11 of the 13 RARS patients in this study carried this mutation. Interestingly, ABCB7 exon usage differed between normal bone marrow and RARS, as well as within the RARS cohort. In addition, SF3B1 silencing resulted in downregulation of ABCB7 in K562 cells undergoing erythroid differentiation. Our findings support that ABCB7 is implicated in the phenotype of acquired RARS and suggest a relation between SF3B1 mutations and ABCB7 downregulation.

Figures

Figure 1
Figure 1. Downregulation of ABCB7 in normal CD34+ marrow cells reduces viability and inhibits erythroid maturation
Bone marrow CD34+ cells from healthy individuals were transduced by lentiviral vectors expressing shRNAs either specific for ABCB7 or scrambled control. After successful transduction, aliquots were plated in CFU-assays and erythroblast cultures. A. Analysis of erythroid and myeloid colony growth from normal CD34+ cells (n=3), either untransduced (control), or transduced by scrambled-control or shABCB7, respectively. B. Expression of shABCB7-GFP (dashed black lines) or scrambled-control-GFP (grey lines) over the course of 14 days in erythroblast cultures. The percentage of GFP+ cells was analyzed by flow cytometry. Shown are two normal donors. C and D. Flow cytometric analysis of erythroid maturation during erythroblast culture on days 5, 7, 11 and 14. Shown is the degree of erythroid maturation analyzed by expression of CD36 and glycophorin A in the GFP+ fraction. Depicted are two representative examples from the same experiment as shown in A. EPO was added from day 7. E. The expression of FTMT, ALAS2, FOXO3A, and MAP3K7 was analyzed by qRT-PCR during the early phase of erythroblast culture (days 3 and 7). NBM CD34+ cells were transduced with pLKO.1-GFP-shABCB7 or pLKO.1-GFP and sorted on day 3 post transduction. Subsequently, GFP+ cells were cultured until day 7. qRT-PCR analysis was performed on cells sorted at the time of FACS (= day 3) and at day 7 of erythroblast culture. The black bar represents data of pLKO.1-GFP-shABCB7 positive cells, which was normalized to that of pLKO.1-GFP positive cells (grey bar).
Figure 1
Figure 1. Downregulation of ABCB7 in normal CD34+ marrow cells reduces viability and inhibits erythroid maturation
Bone marrow CD34+ cells from healthy individuals were transduced by lentiviral vectors expressing shRNAs either specific for ABCB7 or scrambled control. After successful transduction, aliquots were plated in CFU-assays and erythroblast cultures. A. Analysis of erythroid and myeloid colony growth from normal CD34+ cells (n=3), either untransduced (control), or transduced by scrambled-control or shABCB7, respectively. B. Expression of shABCB7-GFP (dashed black lines) or scrambled-control-GFP (grey lines) over the course of 14 days in erythroblast cultures. The percentage of GFP+ cells was analyzed by flow cytometry. Shown are two normal donors. C and D. Flow cytometric analysis of erythroid maturation during erythroblast culture on days 5, 7, 11 and 14. Shown is the degree of erythroid maturation analyzed by expression of CD36 and glycophorin A in the GFP+ fraction. Depicted are two representative examples from the same experiment as shown in A. EPO was added from day 7. E. The expression of FTMT, ALAS2, FOXO3A, and MAP3K7 was analyzed by qRT-PCR during the early phase of erythroblast culture (days 3 and 7). NBM CD34+ cells were transduced with pLKO.1-GFP-shABCB7 or pLKO.1-GFP and sorted on day 3 post transduction. Subsequently, GFP+ cells were cultured until day 7. qRT-PCR analysis was performed on cells sorted at the time of FACS (= day 3) and at day 7 of erythroblast culture. The black bar represents data of pLKO.1-GFP-shABCB7 positive cells, which was normalized to that of pLKO.1-GFP positive cells (grey bar).
Figure 1
Figure 1. Downregulation of ABCB7 in normal CD34+ marrow cells reduces viability and inhibits erythroid maturation
Bone marrow CD34+ cells from healthy individuals were transduced by lentiviral vectors expressing shRNAs either specific for ABCB7 or scrambled control. After successful transduction, aliquots were plated in CFU-assays and erythroblast cultures. A. Analysis of erythroid and myeloid colony growth from normal CD34+ cells (n=3), either untransduced (control), or transduced by scrambled-control or shABCB7, respectively. B. Expression of shABCB7-GFP (dashed black lines) or scrambled-control-GFP (grey lines) over the course of 14 days in erythroblast cultures. The percentage of GFP+ cells was analyzed by flow cytometry. Shown are two normal donors. C and D. Flow cytometric analysis of erythroid maturation during erythroblast culture on days 5, 7, 11 and 14. Shown is the degree of erythroid maturation analyzed by expression of CD36 and glycophorin A in the GFP+ fraction. Depicted are two representative examples from the same experiment as shown in A. EPO was added from day 7. E. The expression of FTMT, ALAS2, FOXO3A, and MAP3K7 was analyzed by qRT-PCR during the early phase of erythroblast culture (days 3 and 7). NBM CD34+ cells were transduced with pLKO.1-GFP-shABCB7 or pLKO.1-GFP and sorted on day 3 post transduction. Subsequently, GFP+ cells were cultured until day 7. qRT-PCR analysis was performed on cells sorted at the time of FACS (= day 3) and at day 7 of erythroblast culture. The black bar represents data of pLKO.1-GFP-shABCB7 positive cells, which was normalized to that of pLKO.1-GFP positive cells (grey bar).
Figure 1
Figure 1. Downregulation of ABCB7 in normal CD34+ marrow cells reduces viability and inhibits erythroid maturation
Bone marrow CD34+ cells from healthy individuals were transduced by lentiviral vectors expressing shRNAs either specific for ABCB7 or scrambled control. After successful transduction, aliquots were plated in CFU-assays and erythroblast cultures. A. Analysis of erythroid and myeloid colony growth from normal CD34+ cells (n=3), either untransduced (control), or transduced by scrambled-control or shABCB7, respectively. B. Expression of shABCB7-GFP (dashed black lines) or scrambled-control-GFP (grey lines) over the course of 14 days in erythroblast cultures. The percentage of GFP+ cells was analyzed by flow cytometry. Shown are two normal donors. C and D. Flow cytometric analysis of erythroid maturation during erythroblast culture on days 5, 7, 11 and 14. Shown is the degree of erythroid maturation analyzed by expression of CD36 and glycophorin A in the GFP+ fraction. Depicted are two representative examples from the same experiment as shown in A. EPO was added from day 7. E. The expression of FTMT, ALAS2, FOXO3A, and MAP3K7 was analyzed by qRT-PCR during the early phase of erythroblast culture (days 3 and 7). NBM CD34+ cells were transduced with pLKO.1-GFP-shABCB7 or pLKO.1-GFP and sorted on day 3 post transduction. Subsequently, GFP+ cells were cultured until day 7. qRT-PCR analysis was performed on cells sorted at the time of FACS (= day 3) and at day 7 of erythroblast culture. The black bar represents data of pLKO.1-GFP-shABCB7 positive cells, which was normalized to that of pLKO.1-GFP positive cells (grey bar).
Figure 1
Figure 1. Downregulation of ABCB7 in normal CD34+ marrow cells reduces viability and inhibits erythroid maturation
Bone marrow CD34+ cells from healthy individuals were transduced by lentiviral vectors expressing shRNAs either specific for ABCB7 or scrambled control. After successful transduction, aliquots were plated in CFU-assays and erythroblast cultures. A. Analysis of erythroid and myeloid colony growth from normal CD34+ cells (n=3), either untransduced (control), or transduced by scrambled-control or shABCB7, respectively. B. Expression of shABCB7-GFP (dashed black lines) or scrambled-control-GFP (grey lines) over the course of 14 days in erythroblast cultures. The percentage of GFP+ cells was analyzed by flow cytometry. Shown are two normal donors. C and D. Flow cytometric analysis of erythroid maturation during erythroblast culture on days 5, 7, 11 and 14. Shown is the degree of erythroid maturation analyzed by expression of CD36 and glycophorin A in the GFP+ fraction. Depicted are two representative examples from the same experiment as shown in A. EPO was added from day 7. E. The expression of FTMT, ALAS2, FOXO3A, and MAP3K7 was analyzed by qRT-PCR during the early phase of erythroblast culture (days 3 and 7). NBM CD34+ cells were transduced with pLKO.1-GFP-shABCB7 or pLKO.1-GFP and sorted on day 3 post transduction. Subsequently, GFP+ cells were cultured until day 7. qRT-PCR analysis was performed on cells sorted at the time of FACS (= day 3) and at day 7 of erythroblast culture. The black bar represents data of pLKO.1-GFP-shABCB7 positive cells, which was normalized to that of pLKO.1-GFP positive cells (grey bar).
Figure 2
Figure 2. Up-regulation of ABCB7 restores erythroid function in RARS progenitors
CD34+ cells from RARS patients were transduced with lentiviral vectors driving expression of ABCB7-YFP or a control vector (mock-YFP). Transduced cells were plated in CFU-assays and erythroblast cultures. A. Colony growth in RARS CD34+ cells (n=4), transduced by either mock or ABCB7-vector. Results are normalized to untransduced cells and error bars depict mean ± SE. B. Number of YFP+ colonies. Shown is the mean ± SE of YFP+ CFU-Cs. (p<0.05). C-D. Transduced cells were cultured in erythroblast cultures for 14 days and expression of ABCB7 and FTMT in RARS erythroblasts was analyzed at day 10.
Figure 2
Figure 2. Up-regulation of ABCB7 restores erythroid function in RARS progenitors
CD34+ cells from RARS patients were transduced with lentiviral vectors driving expression of ABCB7-YFP or a control vector (mock-YFP). Transduced cells were plated in CFU-assays and erythroblast cultures. A. Colony growth in RARS CD34+ cells (n=4), transduced by either mock or ABCB7-vector. Results are normalized to untransduced cells and error bars depict mean ± SE. B. Number of YFP+ colonies. Shown is the mean ± SE of YFP+ CFU-Cs. (p<0.05). C-D. Transduced cells were cultured in erythroblast cultures for 14 days and expression of ABCB7 and FTMT in RARS erythroblasts was analyzed at day 10.
Figure 2
Figure 2. Up-regulation of ABCB7 restores erythroid function in RARS progenitors
CD34+ cells from RARS patients were transduced with lentiviral vectors driving expression of ABCB7-YFP or a control vector (mock-YFP). Transduced cells were plated in CFU-assays and erythroblast cultures. A. Colony growth in RARS CD34+ cells (n=4), transduced by either mock or ABCB7-vector. Results are normalized to untransduced cells and error bars depict mean ± SE. B. Number of YFP+ colonies. Shown is the mean ± SE of YFP+ CFU-Cs. (p<0.05). C-D. Transduced cells were cultured in erythroblast cultures for 14 days and expression of ABCB7 and FTMT in RARS erythroblasts was analyzed at day 10.
Figure 2
Figure 2. Up-regulation of ABCB7 restores erythroid function in RARS progenitors
CD34+ cells from RARS patients were transduced with lentiviral vectors driving expression of ABCB7-YFP or a control vector (mock-YFP). Transduced cells were plated in CFU-assays and erythroblast cultures. A. Colony growth in RARS CD34+ cells (n=4), transduced by either mock or ABCB7-vector. Results are normalized to untransduced cells and error bars depict mean ± SE. B. Number of YFP+ colonies. Shown is the mean ± SE of YFP+ CFU-Cs. (p<0.05). C-D. Transduced cells were cultured in erythroblast cultures for 14 days and expression of ABCB7 and FTMT in RARS erythroblasts was analyzed at day 10.
Figure 3
Figure 3. ABCB7 exon usage in RARS and NBM progenitors
ABCB7 exons expression was quantified by q-PCR at day 7 (Figure 3A) and day 14 (Figure 3B) in RARS and NBM controls. Graph shows marked difference exon usage within RARS samples at both time points. C-F. ABCB7 Exon usage analysis at day 7. C: Exon 2-4, D: Exon 6-8; E: 7J8-9; F: Exon 11-13.
Figure 3
Figure 3. ABCB7 exon usage in RARS and NBM progenitors
ABCB7 exons expression was quantified by q-PCR at day 7 (Figure 3A) and day 14 (Figure 3B) in RARS and NBM controls. Graph shows marked difference exon usage within RARS samples at both time points. C-F. ABCB7 Exon usage analysis at day 7. C: Exon 2-4, D: Exon 6-8; E: 7J8-9; F: Exon 11-13.
Figure 3
Figure 3. ABCB7 exon usage in RARS and NBM progenitors
ABCB7 exons expression was quantified by q-PCR at day 7 (Figure 3A) and day 14 (Figure 3B) in RARS and NBM controls. Graph shows marked difference exon usage within RARS samples at both time points. C-F. ABCB7 Exon usage analysis at day 7. C: Exon 2-4, D: Exon 6-8; E: 7J8-9; F: Exon 11-13.
Figure 3
Figure 3. ABCB7 exon usage in RARS and NBM progenitors
ABCB7 exons expression was quantified by q-PCR at day 7 (Figure 3A) and day 14 (Figure 3B) in RARS and NBM controls. Graph shows marked difference exon usage within RARS samples at both time points. C-F. ABCB7 Exon usage analysis at day 7. C: Exon 2-4, D: Exon 6-8; E: 7J8-9; F: Exon 11-13.
Figure 3
Figure 3. ABCB7 exon usage in RARS and NBM progenitors
ABCB7 exons expression was quantified by q-PCR at day 7 (Figure 3A) and day 14 (Figure 3B) in RARS and NBM controls. Graph shows marked difference exon usage within RARS samples at both time points. C-F. ABCB7 Exon usage analysis at day 7. C: Exon 2-4, D: Exon 6-8; E: 7J8-9; F: Exon 11-13.
Figure 3
Figure 3. ABCB7 exon usage in RARS and NBM progenitors
ABCB7 exons expression was quantified by q-PCR at day 7 (Figure 3A) and day 14 (Figure 3B) in RARS and NBM controls. Graph shows marked difference exon usage within RARS samples at both time points. C-F. ABCB7 Exon usage analysis at day 7. C: Exon 2-4, D: Exon 6-8; E: 7J8-9; F: Exon 11-13.
Figure 4
Figure 4. Relative expression levels of SF3B1 and ABCB7 post SF3B1 silencing
K562 cells were transfected by using three non-overlapping siRNAs (#13-15) targeting SF3B1 (A and B). Identical experiments were also performed using hemin to induce erythroid differentiation in K562 cells (C, D and E). Depicted is the relative gene expression of SF3B1, ABCB7 and γ-globin. Percentages are calculated with respect to expression levels of both genes in cells transfected with two scramble sequences with GC content similar to the one of siRNA sequences.
Figure 4
Figure 4. Relative expression levels of SF3B1 and ABCB7 post SF3B1 silencing
K562 cells were transfected by using three non-overlapping siRNAs (#13-15) targeting SF3B1 (A and B). Identical experiments were also performed using hemin to induce erythroid differentiation in K562 cells (C, D and E). Depicted is the relative gene expression of SF3B1, ABCB7 and γ-globin. Percentages are calculated with respect to expression levels of both genes in cells transfected with two scramble sequences with GC content similar to the one of siRNA sequences.
Figure 4
Figure 4. Relative expression levels of SF3B1 and ABCB7 post SF3B1 silencing
K562 cells were transfected by using three non-overlapping siRNAs (#13-15) targeting SF3B1 (A and B). Identical experiments were also performed using hemin to induce erythroid differentiation in K562 cells (C, D and E). Depicted is the relative gene expression of SF3B1, ABCB7 and γ-globin. Percentages are calculated with respect to expression levels of both genes in cells transfected with two scramble sequences with GC content similar to the one of siRNA sequences.
Figure 4
Figure 4. Relative expression levels of SF3B1 and ABCB7 post SF3B1 silencing
K562 cells were transfected by using three non-overlapping siRNAs (#13-15) targeting SF3B1 (A and B). Identical experiments were also performed using hemin to induce erythroid differentiation in K562 cells (C, D and E). Depicted is the relative gene expression of SF3B1, ABCB7 and γ-globin. Percentages are calculated with respect to expression levels of both genes in cells transfected with two scramble sequences with GC content similar to the one of siRNA sequences.
Figure 4
Figure 4. Relative expression levels of SF3B1 and ABCB7 post SF3B1 silencing
K562 cells were transfected by using three non-overlapping siRNAs (#13-15) targeting SF3B1 (A and B). Identical experiments were also performed using hemin to induce erythroid differentiation in K562 cells (C, D and E). Depicted is the relative gene expression of SF3B1, ABCB7 and γ-globin. Percentages are calculated with respect to expression levels of both genes in cells transfected with two scramble sequences with GC content similar to the one of siRNA sequences.

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