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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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Changes in morphology (A) and DNA profile (B), before and after the induction of AIM-1(K/R) and STK15(K/R), in each clone of K562. The cells of each clone were cultured with or without 1 mM IPTG for 120 h. (A) Cytocentrifugation preparations were stained with May-Grunwald-Giemsa. Bar, 10 μm. (B) DNA content analyses were performed by flow cytometry.
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Figure 10: Changes in morphology (A) and DNA profile (B), before and after the induction of AIM-1(K/R) and STK15(K/R), in each clone of K562. The cells of each clone were cultured with or without 1 mM IPTG for 120 h. (A) Cytocentrifugation preparations were stained with May-Grunwald-Giemsa. Bar, 10 μm. (B) DNA content analyses were performed by flow cytometry.

Mentions: Next, we prepared stable clones that can inducibly express the Flag-tagged dn form (K/R type) of AIM-1 and STK15 from K562-LacR cells; these clones were named K562/AIM-1(K/R) and K562/STK15(K/R), respectively. The induction of AIM-1(K/R) and STK15(K/R) proteins by IPTG treatment was confirmed by immunoblot analysis using the anti-Flag antibody (data not shown). Because the data presented here suggested that AIM-1 was involved in megakaryocytic polyploidization, we initially performed immunocytochemical analyses of K562/AIM-1(K/R) cells during IPTG treatment. After the 24–36-h IPTG treatment, Flag-tagged AIM-1(K/R) was localized at the midbody of cells, with two nuclei at late mitosis in a significant proportion of the cultured cells (data not shown). After the 72-h IPTG treatment, K562/AIM-1(K/R) cells with two nuclei (4N cells) entered into mitosis and progressed to metaphase (a representative cell is shown in Fig. 9), at which point Flag-tagged AIM-1(K/R) was dispersed in the cytoplasm (Fig. 9 a). After the 96-h IPTG treatment, several cells possessed four nuclei, and expression of Flag-tagged AIM-1(K/R) was again found at the midbody of the cells at late mitosis (Fig. 9 e). These results mimic the activities of endogenous AIM-1. After the 120-h IPTG treatment, ∼30% of K562/AIM-1(K/R) cells revealed polyploid features in morphologic analysis (Fig. 10 A). In contrast, an apparent morphologic change was not induced in K562/mock or K562/STK15(K/R) cells after IPTG treatment (Fig. 10 A). Consistent with the changes in morphology, the induced expression of AIM-1(K/R) yielded 8N fraction in 21.9 ± 1.8% of the cultured cells in DNA content analysis, which was a significant difference (P < 0.001) with two-sample rank test. In contrast, STK15(K/R) hardly affected the DNA content profile of K562 cells (Fig. 10 B and Table ). Similar results were observed in several other clones of K562/AIM-1(K/R).


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

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

Changes in morphology (A) and DNA profile (B), before and after the induction of AIM-1(K/R) and STK15(K/R), in each clone of K562. The cells of each clone were cultured with or without 1 mM IPTG for 120 h. (A) Cytocentrifugation preparations were stained with May-Grunwald-Giemsa. Bar, 10 μm. (B) DNA content analyses were performed by flow cytometry.
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Related In: Results  -  Collection

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

Figure 10: Changes in morphology (A) and DNA profile (B), before and after the induction of AIM-1(K/R) and STK15(K/R), in each clone of K562. The cells of each clone were cultured with or without 1 mM IPTG for 120 h. (A) Cytocentrifugation preparations were stained with May-Grunwald-Giemsa. Bar, 10 μm. (B) DNA content analyses were performed by flow cytometry.
Mentions: Next, we prepared stable clones that can inducibly express the Flag-tagged dn form (K/R type) of AIM-1 and STK15 from K562-LacR cells; these clones were named K562/AIM-1(K/R) and K562/STK15(K/R), respectively. The induction of AIM-1(K/R) and STK15(K/R) proteins by IPTG treatment was confirmed by immunoblot analysis using the anti-Flag antibody (data not shown). Because the data presented here suggested that AIM-1 was involved in megakaryocytic polyploidization, we initially performed immunocytochemical analyses of K562/AIM-1(K/R) cells during IPTG treatment. After the 24–36-h IPTG treatment, Flag-tagged AIM-1(K/R) was localized at the midbody of cells, with two nuclei at late mitosis in a significant proportion of the cultured cells (data not shown). After the 72-h IPTG treatment, K562/AIM-1(K/R) cells with two nuclei (4N cells) entered into mitosis and progressed to metaphase (a representative cell is shown in Fig. 9), at which point Flag-tagged AIM-1(K/R) was dispersed in the cytoplasm (Fig. 9 a). After the 96-h IPTG treatment, several cells possessed four nuclei, and expression of Flag-tagged AIM-1(K/R) was again found at the midbody of the cells at late mitosis (Fig. 9 e). These results mimic the activities of endogenous AIM-1. After the 120-h IPTG treatment, ∼30% of K562/AIM-1(K/R) cells revealed polyploid features in morphologic analysis (Fig. 10 A). In contrast, an apparent morphologic change was not induced in K562/mock or K562/STK15(K/R) cells after IPTG treatment (Fig. 10 A). Consistent with the changes in morphology, the induced expression of AIM-1(K/R) yielded 8N fraction in 21.9 ± 1.8% of the cultured cells in DNA content analysis, which was a significant difference (P < 0.001) with two-sample rank test. In contrast, STK15(K/R) hardly affected the DNA content profile of K562 cells (Fig. 10 B and Table ). Similar results were observed in several other clones of K562/AIM-1(K/R).

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