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AF10 plays a key role in the survival of uncommitted hematopoietic cells.

Chamorro-Garcia R, Cervera M, Arredondo JJ - PLoS ONE (2012)

Bottom Line: The induction of hematopoietic differentiation in both human hematopoietic cell lines and murine total bone marrow cells triggers a decrease of AF10 mRNA and protein levels, particularly in stem cells and multipotent progenitors.Gain- and loss-of-function studies demonstrate that over- or under-expression of AF10 leads to apoptotic cell death in stem cells and multipotent progenitors.We conclude that AF10 plays a key role in the maintenance of multipotent hematopoietic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica & Instituto Investigaciones Biomédicas, The Autonomous University of Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain. rchamorr@uci.edu

ABSTRACT
Hematopoiesis is a complex process regulated by both cell intrinsic and cell extrinsic factors. Alterations in the expression of critical genes during hematopoiesis can modify the balance between stem cell differentiation and proliferation, and may ultimately give rise to leukemia and other diseases. AF10 is a transcription factor that has been implicated in the development of leukemia following chromosomal rearrangements between the AF10 gene and one of at least two other genes, MLL and CALM. The link between AF10 and leukemia, together with the known interactions between AF10 and hematopoietic regulators, suggests that AF10 may be important in hematopoiesis and in leukemic transformation. Here we show that AF10 is important for proper hematopoietic differentiation. The induction of hematopoietic differentiation in both human hematopoietic cell lines and murine total bone marrow cells triggers a decrease of AF10 mRNA and protein levels, particularly in stem cells and multipotent progenitors. Gain- and loss-of-function studies demonstrate that over- or under-expression of AF10 leads to apoptotic cell death in stem cells and multipotent progenitors. We conclude that AF10 plays a key role in the maintenance of multipotent hematopoietic cells.

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Related in: MedlinePlus

Effect of AF10 loss-of-function during hematopoiesis.(A) Western blot analysis of AF10 expression in HEL and K562 cell lines transfected with scrambled siControl and siAF10. (B) Analysis of annexin-V levels in cell lines transfected with scrambled siControl and siAF10 before (−TPA) and after (+TPA) the induction of megakaryocytic differentiation, in cell lines HEL, K562 and CMK, and monocytic differentiation in cell lines HEL and HL60. Dashed line indicates the annexin-V levels of the cells lines transfected with scrambled siControl plasmid normalized to 1. Paired t-test; * P<0.05; ** P<0.01 (n≥3).
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pone-0051626-g003: Effect of AF10 loss-of-function during hematopoiesis.(A) Western blot analysis of AF10 expression in HEL and K562 cell lines transfected with scrambled siControl and siAF10. (B) Analysis of annexin-V levels in cell lines transfected with scrambled siControl and siAF10 before (−TPA) and after (+TPA) the induction of megakaryocytic differentiation, in cell lines HEL, K562 and CMK, and monocytic differentiation in cell lines HEL and HL60. Dashed line indicates the annexin-V levels of the cells lines transfected with scrambled siControl plasmid normalized to 1. Paired t-test; * P<0.05; ** P<0.01 (n≥3).

Mentions: Based in our findings in the overexpression assay, we sought to determine if AF10 knockdown affected cell viability. The four cells lines were transiently transfected with siAF10 and siControl plasmids. 18 hours after transfection, cells were collected and incubated with annexin-V and apoptosis levels measured by flow cytometry. As shown in figure 3A for HEL and K562 cell lines, similar levels of knockdown were achieved in both cell lines. Since cells were transiently transfected and transfection efficiency was estimated between 40 and 80% depending on the cell line (Figure S6), we concluded that the knockdown was effective in the transfected cells. We found that AF10 knockdown in undifferentiated HEL cells led to a significant increase in the number of apoptotic cells (Figure 3B). In contrast, no significant increase was observed in any of the more committed cell lines. This result is in accordance with our hypothesis that AF10 levels must be tightly controlled throughout hematopoietic differentiation in not fully committed cells.


AF10 plays a key role in the survival of uncommitted hematopoietic cells.

Chamorro-Garcia R, Cervera M, Arredondo JJ - PLoS ONE (2012)

Effect of AF10 loss-of-function during hematopoiesis.(A) Western blot analysis of AF10 expression in HEL and K562 cell lines transfected with scrambled siControl and siAF10. (B) Analysis of annexin-V levels in cell lines transfected with scrambled siControl and siAF10 before (−TPA) and after (+TPA) the induction of megakaryocytic differentiation, in cell lines HEL, K562 and CMK, and monocytic differentiation in cell lines HEL and HL60. Dashed line indicates the annexin-V levels of the cells lines transfected with scrambled siControl plasmid normalized to 1. Paired t-test; * P<0.05; ** P<0.01 (n≥3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526614&req=5

pone-0051626-g003: Effect of AF10 loss-of-function during hematopoiesis.(A) Western blot analysis of AF10 expression in HEL and K562 cell lines transfected with scrambled siControl and siAF10. (B) Analysis of annexin-V levels in cell lines transfected with scrambled siControl and siAF10 before (−TPA) and after (+TPA) the induction of megakaryocytic differentiation, in cell lines HEL, K562 and CMK, and monocytic differentiation in cell lines HEL and HL60. Dashed line indicates the annexin-V levels of the cells lines transfected with scrambled siControl plasmid normalized to 1. Paired t-test; * P<0.05; ** P<0.01 (n≥3).
Mentions: Based in our findings in the overexpression assay, we sought to determine if AF10 knockdown affected cell viability. The four cells lines were transiently transfected with siAF10 and siControl plasmids. 18 hours after transfection, cells were collected and incubated with annexin-V and apoptosis levels measured by flow cytometry. As shown in figure 3A for HEL and K562 cell lines, similar levels of knockdown were achieved in both cell lines. Since cells were transiently transfected and transfection efficiency was estimated between 40 and 80% depending on the cell line (Figure S6), we concluded that the knockdown was effective in the transfected cells. We found that AF10 knockdown in undifferentiated HEL cells led to a significant increase in the number of apoptotic cells (Figure 3B). In contrast, no significant increase was observed in any of the more committed cell lines. This result is in accordance with our hypothesis that AF10 levels must be tightly controlled throughout hematopoietic differentiation in not fully committed cells.

Bottom Line: The induction of hematopoietic differentiation in both human hematopoietic cell lines and murine total bone marrow cells triggers a decrease of AF10 mRNA and protein levels, particularly in stem cells and multipotent progenitors.Gain- and loss-of-function studies demonstrate that over- or under-expression of AF10 leads to apoptotic cell death in stem cells and multipotent progenitors.We conclude that AF10 plays a key role in the maintenance of multipotent hematopoietic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica & Instituto Investigaciones Biomédicas, The Autonomous University of Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain. rchamorr@uci.edu

ABSTRACT
Hematopoiesis is a complex process regulated by both cell intrinsic and cell extrinsic factors. Alterations in the expression of critical genes during hematopoiesis can modify the balance between stem cell differentiation and proliferation, and may ultimately give rise to leukemia and other diseases. AF10 is a transcription factor that has been implicated in the development of leukemia following chromosomal rearrangements between the AF10 gene and one of at least two other genes, MLL and CALM. The link between AF10 and leukemia, together with the known interactions between AF10 and hematopoietic regulators, suggests that AF10 may be important in hematopoiesis and in leukemic transformation. Here we show that AF10 is important for proper hematopoietic differentiation. The induction of hematopoietic differentiation in both human hematopoietic cell lines and murine total bone marrow cells triggers a decrease of AF10 mRNA and protein levels, particularly in stem cells and multipotent progenitors. Gain- and loss-of-function studies demonstrate that over- or under-expression of AF10 leads to apoptotic cell death in stem cells and multipotent progenitors. We conclude that AF10 plays a key role in the maintenance of multipotent hematopoietic cells.

Show MeSH
Related in: MedlinePlus