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Downregulation of an AIM-1 kinase couples with megakaryocytic polyploidization of human hematopoietic cells.

Kawasaki A, Matsumura I - J. Cell Biol. (2001)

Bottom Line: In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester.Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not.Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology/Oncology, Osaka University Medical School, Osaka 565-0871, Japan.

ABSTRACT
During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

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Immunocytochemical analysis of murine proliferating cells and polyploid megakaryocytes. Murine bone marrow mononuclear cells were cultured with rmIL-3 (10 ng/ml) and rmSCF (100 ng/ml) (a–d) or with rhTPO (100 ng/ml) (e–h) for 3–5 d. Cytocentrifugation preparations were stained with murine anti–AIM-1 antibody and Alexa-conjugated goat anti–mouse IgG antibody (a and e), FITC-conjugated anti-CD41 mAb (b and f), and DAPI (c and g). The triple staining of the same cell is shown in d and h. Bar, 10 μm. a–d show a CD41-negative (nonmegakaryocytic) proliferating cell, and e–h show a CD41-positive megakaryocyte and a CD41-negative cell also stained for AIM-1.
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Figure 5: Immunocytochemical analysis of murine proliferating cells and polyploid megakaryocytes. Murine bone marrow mononuclear cells were cultured with rmIL-3 (10 ng/ml) and rmSCF (100 ng/ml) (a–d) or with rhTPO (100 ng/ml) (e–h) for 3–5 d. Cytocentrifugation preparations were stained with murine anti–AIM-1 antibody and Alexa-conjugated goat anti–mouse IgG antibody (a and e), FITC-conjugated anti-CD41 mAb (b and f), and DAPI (c and g). The triple staining of the same cell is shown in d and h. Bar, 10 μm. a–d show a CD41-negative (nonmegakaryocytic) proliferating cell, and e–h show a CD41-positive megakaryocyte and a CD41-negative cell also stained for AIM-1.

Mentions: To examine the expression of AIM-1 in polyploidizing megakaryocytes by immunocytochemical analysis, we prepared murine bone marrow mononuclear cells and cultured them with rhTPO. Also, to obtain proliferating cells as a positive control for the AIM-1 staining, the murine bone marrow mononuclear cells were cultured with rmSCF and rmIL-3. The cells obtained from both culture conditions were subjected to the double staining with an anti-CD41 antibody and an anti–AIM-1 antibody in batched and parallel reactions. As a positive control, we could find several CD41-negative dividing cells after the 5-d culture with rmIL-3 and rmSCF, in which AIM-1 expression was condensed at their midbody (Fig. 5, a–d). In contrast, all CD41-positive polyploid megakaryocytes that developed after the culture with TPO were essentially negative for anti–AIM-1 staining, even in metaphase or anaphase, whereas some CD41-negative small cells were stained with an anti–AIM-1 antibody on the same glass slide (Fig. 5, e–h). These results again suggested that AIM-1 expression was continuously downregulated at the protein level in normal megakaryocytes during polyploidization.


Downregulation of an AIM-1 kinase couples with megakaryocytic polyploidization of human hematopoietic cells.

Kawasaki A, Matsumura I - J. Cell Biol. (2001)

Immunocytochemical analysis of murine proliferating cells and polyploid megakaryocytes. Murine bone marrow mononuclear cells were cultured with rmIL-3 (10 ng/ml) and rmSCF (100 ng/ml) (a–d) or with rhTPO (100 ng/ml) (e–h) for 3–5 d. Cytocentrifugation preparations were stained with murine anti–AIM-1 antibody and Alexa-conjugated goat anti–mouse IgG antibody (a and e), FITC-conjugated anti-CD41 mAb (b and f), and DAPI (c and g). The triple staining of the same cell is shown in d and h. Bar, 10 μm. a–d show a CD41-negative (nonmegakaryocytic) proliferating cell, and e–h show a CD41-positive megakaryocyte and a CD41-negative cell also stained for AIM-1.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199624&req=5

Figure 5: Immunocytochemical analysis of murine proliferating cells and polyploid megakaryocytes. Murine bone marrow mononuclear cells were cultured with rmIL-3 (10 ng/ml) and rmSCF (100 ng/ml) (a–d) or with rhTPO (100 ng/ml) (e–h) for 3–5 d. Cytocentrifugation preparations were stained with murine anti–AIM-1 antibody and Alexa-conjugated goat anti–mouse IgG antibody (a and e), FITC-conjugated anti-CD41 mAb (b and f), and DAPI (c and g). The triple staining of the same cell is shown in d and h. Bar, 10 μm. a–d show a CD41-negative (nonmegakaryocytic) proliferating cell, and e–h show a CD41-positive megakaryocyte and a CD41-negative cell also stained for AIM-1.
Mentions: To examine the expression of AIM-1 in polyploidizing megakaryocytes by immunocytochemical analysis, we prepared murine bone marrow mononuclear cells and cultured them with rhTPO. Also, to obtain proliferating cells as a positive control for the AIM-1 staining, the murine bone marrow mononuclear cells were cultured with rmSCF and rmIL-3. The cells obtained from both culture conditions were subjected to the double staining with an anti-CD41 antibody and an anti–AIM-1 antibody in batched and parallel reactions. As a positive control, we could find several CD41-negative dividing cells after the 5-d culture with rmIL-3 and rmSCF, in which AIM-1 expression was condensed at their midbody (Fig. 5, a–d). In contrast, all CD41-positive polyploid megakaryocytes that developed after the culture with TPO were essentially negative for anti–AIM-1 staining, even in metaphase or anaphase, whereas some CD41-negative small cells were stained with an anti–AIM-1 antibody on the same glass slide (Fig. 5, e–h). These results again suggested that AIM-1 expression was continuously downregulated at the protein level in normal megakaryocytes during polyploidization.

Bottom Line: In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester.Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not.Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology/Oncology, Osaka University Medical School, Osaka 565-0871, Japan.

ABSTRACT
During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

Show MeSH
Related in: MedlinePlus