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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 K562/AIM-1(WT) cells during IPTG and TPA treatment. The cells were pretreated with 1 mM of IPTG for 24 h, and then TPA was added to the culture medium (1 nM, final concentration). 48 h after adding TPA, the cultured cells were stained with murine anti-Flag antibody, M2, and Alexa-conjugated goat anti–mouse IgG antibody (a), FITC-conjugated murine anti–α-tubulin antibody (b), and DAPI (c). The triple staining of the same cell is shown in d. Bar, 5 μm.
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Figure 8: Immunocytochemical analysis of K562/AIM-1(WT) cells during IPTG and TPA treatment. The cells were pretreated with 1 mM of IPTG for 24 h, and then TPA was added to the culture medium (1 nM, final concentration). 48 h after adding TPA, the cultured cells were stained with murine anti-Flag antibody, M2, and Alexa-conjugated goat anti–mouse IgG antibody (a), FITC-conjugated murine anti–α-tubulin antibody (b), and DAPI (c). The triple staining of the same cell is shown in d. Bar, 5 μm.

Mentions: To further clarify the mechanisms by which the induced AIM-1(WT) abrogated TPA-induced polyploidization of K562 cells, TPA- and IPTG-treated K562/AIM-1(WT) cells were subjected to immunocytochemical analyses with anti-Flag (Fig. 8 a) and anti–α-tubulin antibodies (Fig. 8 b). Also, the nuclei were visualized with DAPI staining (Fig. 8 c). During interphase, Flag-tagged AIM-1(WT) was dispersed in the cytoplasm, and then visualized by staining with anti-Flag mAb (data not shown). After 36–48 h, some of the cultured cells were supposed to enter mitosis, even in the presence of TPA, because separation of sister chromatids was observed in these cells (a representative cell is shown in Fig. 8). In addition, it was noted that Flag-tagged AIM-1(WT) was localized at the midzone of the central spindle in the cell around anaphase B (Fig. 8), telophase, and cytokinesis (data not shown). These results mimic the activities of endogenous AIM-1.


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 K562/AIM-1(WT) cells during IPTG and TPA treatment. The cells were pretreated with 1 mM of IPTG for 24 h, and then TPA was added to the culture medium (1 nM, final concentration). 48 h after adding TPA, the cultured cells were stained with murine anti-Flag antibody, M2, and Alexa-conjugated goat anti–mouse IgG antibody (a), FITC-conjugated murine anti–α-tubulin antibody (b), and DAPI (c). The triple staining of the same cell is shown in d. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Immunocytochemical analysis of K562/AIM-1(WT) cells during IPTG and TPA treatment. The cells were pretreated with 1 mM of IPTG for 24 h, and then TPA was added to the culture medium (1 nM, final concentration). 48 h after adding TPA, the cultured cells were stained with murine anti-Flag antibody, M2, and Alexa-conjugated goat anti–mouse IgG antibody (a), FITC-conjugated murine anti–α-tubulin antibody (b), and DAPI (c). The triple staining of the same cell is shown in d. Bar, 5 μm.
Mentions: To further clarify the mechanisms by which the induced AIM-1(WT) abrogated TPA-induced polyploidization of K562 cells, TPA- and IPTG-treated K562/AIM-1(WT) cells were subjected to immunocytochemical analyses with anti-Flag (Fig. 8 a) and anti–α-tubulin antibodies (Fig. 8 b). Also, the nuclei were visualized with DAPI staining (Fig. 8 c). During interphase, Flag-tagged AIM-1(WT) was dispersed in the cytoplasm, and then visualized by staining with anti-Flag mAb (data not shown). After 36–48 h, some of the cultured cells were supposed to enter mitosis, even in the presence of TPA, because separation of sister chromatids was observed in these cells (a representative cell is shown in Fig. 8). In addition, it was noted that Flag-tagged AIM-1(WT) was localized at the midzone of the central spindle in the cell around anaphase B (Fig. 8), telophase, and cytokinesis (data not shown). These results mimic the activities of endogenous AIM-1.

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