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Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation.

Bousquet M, Quelen C, Rosati R, Mansat-De Mas V, La Starza R, Bastard C, Lippert E, Talmant P, Lafage-Pochitaloff M, Leroux D, Gervais C, Viguié F, Lai JL, Terre C, Beverlo B, Sambani C, Hagemeijer A, Marynen P, Delsol G, Dastugue N, Mecucci C, Brousset P - J. Exp. Med. (2008)

Bottom Line: In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold).In vitro experiments revealed that miR-125b was able to interfere with primary human CD34(+) cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines.Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

View Article: PubMed Central - PubMed

Affiliation: Institut National de Santé et de Recherche Médicale, U563, Centre de Physiopathologie de Toulouse-Purpan, 31300 Toulouse, France.

ABSTRACT
Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34(+) cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

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Transient transfection with miR-125b blocks the differentiation of CD34+ primary cells. (A–C) Differentiation was evaluated by cell morphology with May-Grunwald-Giemsa–stained cytocentrifuge slides after 8 d of differentiation induced by IL-3 and GM-CSF treatment and transient transfection (at day 5) with the negative control (A) or miR-125b (B). Bars, 5 μm. The proportion of immature cells (B) is higher upon miR-125b transfection than in controls (A). (C) Graphic representation of morphological data. The number of differentiated cells includes myelocytes, metamyelocytes, neutrophils, monocytes, and macrophages compared with the number of undifferentiated cells (blasts, myeloblasts, and promyelocytes). The data correspond to the mean of four independent experiments (P < 0.05). (D) Corresponding CD11b expression by FACS analysis: primary blasts transfected with the negative control (blue, CD11b staining), and isotypic control [black] and primary blasts transfected with miR-125b (red, CD11b staining; green, isotypic control). A representative experiment is shown.
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fig5: Transient transfection with miR-125b blocks the differentiation of CD34+ primary cells. (A–C) Differentiation was evaluated by cell morphology with May-Grunwald-Giemsa–stained cytocentrifuge slides after 8 d of differentiation induced by IL-3 and GM-CSF treatment and transient transfection (at day 5) with the negative control (A) or miR-125b (B). Bars, 5 μm. The proportion of immature cells (B) is higher upon miR-125b transfection than in controls (A). (C) Graphic representation of morphological data. The number of differentiated cells includes myelocytes, metamyelocytes, neutrophils, monocytes, and macrophages compared with the number of undifferentiated cells (blasts, myeloblasts, and promyelocytes). The data correspond to the mean of four independent experiments (P < 0.05). (D) Corresponding CD11b expression by FACS analysis: primary blasts transfected with the negative control (blue, CD11b staining), and isotypic control [black] and primary blasts transfected with miR-125b (red, CD11b staining; green, isotypic control). A representative experiment is shown.

Mentions: Several experimental conditions were used to transfect pools (106 cells) of human CD34+ primary blasts (four experiments). The most significant effect was seen in transient transfections, as described for leukemic cells (see Materials and methods). Taking into account the criteria applied to myeloid cells in vitro, the results obtained with primary blasts revealed a blockage of differentiation that was particularly obvious with regard to morphological features. Roughly, we got half of differentiated cells upon miR-125b transfection compared with controls (Fig. 5, A–C). This was evaluated 8 d after induction of differentiation (upon GM-CSF treatment inducing both granulocytic and monocytic differentiation). Regarding the FACS results, in all experiments, there was a trend toward a delay in the acquisition of CD11b (variations of 5–10%; Fig. 5 D), but the results did not exactly fit with the morphological features seen for leukemic cells. This clearly suggests that the effect of miR-125b is greatly dependent on the stage and the pathway of differentiation of targeted cells. As seen in HL60 cells (with monocytic differentiation), miR-125b less significantly affected CD11b expression than in NB4 cells (upon granulocytic maturation).


Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation.

Bousquet M, Quelen C, Rosati R, Mansat-De Mas V, La Starza R, Bastard C, Lippert E, Talmant P, Lafage-Pochitaloff M, Leroux D, Gervais C, Viguié F, Lai JL, Terre C, Beverlo B, Sambani C, Hagemeijer A, Marynen P, Delsol G, Dastugue N, Mecucci C, Brousset P - J. Exp. Med. (2008)

Transient transfection with miR-125b blocks the differentiation of CD34+ primary cells. (A–C) Differentiation was evaluated by cell morphology with May-Grunwald-Giemsa–stained cytocentrifuge slides after 8 d of differentiation induced by IL-3 and GM-CSF treatment and transient transfection (at day 5) with the negative control (A) or miR-125b (B). Bars, 5 μm. The proportion of immature cells (B) is higher upon miR-125b transfection than in controls (A). (C) Graphic representation of morphological data. The number of differentiated cells includes myelocytes, metamyelocytes, neutrophils, monocytes, and macrophages compared with the number of undifferentiated cells (blasts, myeloblasts, and promyelocytes). The data correspond to the mean of four independent experiments (P < 0.05). (D) Corresponding CD11b expression by FACS analysis: primary blasts transfected with the negative control (blue, CD11b staining), and isotypic control [black] and primary blasts transfected with miR-125b (red, CD11b staining; green, isotypic control). A representative experiment is shown.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571925&req=5

fig5: Transient transfection with miR-125b blocks the differentiation of CD34+ primary cells. (A–C) Differentiation was evaluated by cell morphology with May-Grunwald-Giemsa–stained cytocentrifuge slides after 8 d of differentiation induced by IL-3 and GM-CSF treatment and transient transfection (at day 5) with the negative control (A) or miR-125b (B). Bars, 5 μm. The proportion of immature cells (B) is higher upon miR-125b transfection than in controls (A). (C) Graphic representation of morphological data. The number of differentiated cells includes myelocytes, metamyelocytes, neutrophils, monocytes, and macrophages compared with the number of undifferentiated cells (blasts, myeloblasts, and promyelocytes). The data correspond to the mean of four independent experiments (P < 0.05). (D) Corresponding CD11b expression by FACS analysis: primary blasts transfected with the negative control (blue, CD11b staining), and isotypic control [black] and primary blasts transfected with miR-125b (red, CD11b staining; green, isotypic control). A representative experiment is shown.
Mentions: Several experimental conditions were used to transfect pools (106 cells) of human CD34+ primary blasts (four experiments). The most significant effect was seen in transient transfections, as described for leukemic cells (see Materials and methods). Taking into account the criteria applied to myeloid cells in vitro, the results obtained with primary blasts revealed a blockage of differentiation that was particularly obvious with regard to morphological features. Roughly, we got half of differentiated cells upon miR-125b transfection compared with controls (Fig. 5, A–C). This was evaluated 8 d after induction of differentiation (upon GM-CSF treatment inducing both granulocytic and monocytic differentiation). Regarding the FACS results, in all experiments, there was a trend toward a delay in the acquisition of CD11b (variations of 5–10%; Fig. 5 D), but the results did not exactly fit with the morphological features seen for leukemic cells. This clearly suggests that the effect of miR-125b is greatly dependent on the stage and the pathway of differentiation of targeted cells. As seen in HL60 cells (with monocytic differentiation), miR-125b less significantly affected CD11b expression than in NB4 cells (upon granulocytic maturation).

Bottom Line: In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold).In vitro experiments revealed that miR-125b was able to interfere with primary human CD34(+) cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines.Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

View Article: PubMed Central - PubMed

Affiliation: Institut National de Santé et de Recherche Médicale, U563, Centre de Physiopathologie de Toulouse-Purpan, 31300 Toulouse, France.

ABSTRACT
Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34(+) cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

Show MeSH
Related in: MedlinePlus