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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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Morphologic and DNA content analyses of various types of leukemia cell lines before and after the induction of megakaryocytic differentiation. The cells were cultured with TPA (1 nM), IPTG (1 mM), and TPO (30 ng/ml) for 120 h. (A) Light micrographs of cells. A cytocentrifugation preparation from each culture was stained with May-Grunwald-Giemsa. Bar, 10 μm. (B) DNA content of the cultured cells was analyzed at the times indicated by flow cytometry.
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Figure 2: Morphologic and DNA content analyses of various types of leukemia cell lines before and after the induction of megakaryocytic differentiation. The cells were cultured with TPA (1 nM), IPTG (1 mM), and TPO (30 ng/ml) for 120 h. (A) Light micrographs of cells. A cytocentrifugation preparation from each culture was stained with May-Grunwald-Giemsa. Bar, 10 μm. (B) DNA content of the cultured cells was analyzed at the times indicated by flow cytometry.

Mentions: We previously found that expression of AIM-1 was downregulated during TPA-induced polyploidization of several megakaryocytic cell lines (Katayama et al. 1998). Here, we investigated the changes in expression of AIM-1 and STK15 during various types of megakaryocytic differentiation. Human erythro/megakaryocytic leukemia cell lines K562 and CMK are known to undergo polyploidization in response to TPA (Tetteroo et al. 1984; Matsumura et al. 1997). In addition, we previously reported that TPO treatment of F-36P-mpl yielded highly differentiated polyploid megakaryocytes, and that the induced expression of activated ras (H-rasG12V) provoked polyploidization of F-36P/ras and K562/ras cells (Matsumura et al. 1998, Matsumura et al. 2000b). Before each treatment, K562, CMK, F-36P-mpl, K562/ras, and F-36P/ras cells were exclusively composed of undifferentiated blastoid cells (Fig. 2 A). In contrast, the 120-h treatment with TPA, TPO, or IPTG individually gave rise to mature megakaryocytes with polyploid nucleus (several typical cells are shown in Fig. 2 A). In accordance with the morphologic data, DNA content analyses revealed that polyploid formation was induced by each treatment in all of the clones (Fig. 2 B). When expression levels of AIM-1 and STK15 were examined by Northern blot analysis, both expressions gradually decreased and were retained at a hardly detectable level until 120 h in all of the culture conditions (Fig. 3 A). Because cyclin B is a crucial regulator of mitosis, we also monitored expression levels of cyclin B during these cultures by Northern blot analysis. As shown in Fig. 3 A, the expressions of cyclin B were downregulated in all of the cultures. Consistent with the Northern blot analysis results, immunoblotting showed that the expression levels of cyclin B1 were reduced during polyploidization in all cultures (Fig. 3 B). These findings were largely consistent with previous reports that the expression of cyclin B was downregulated during polyploidization of several megakaryocytic cell lines (Zhang et al. 1996, Zhang et al. 1998; Matsumura et al. 2000a), but at variance with the finding that the cyclin B expression was retained in normal polyploidizing megakaryocytes (Vitrat et al. 1998). As reviewed by Zimmet and Ravid 2000, it was speculated that the reduction of cyclin B expression might be necessary for rapid reentry into S phase during polyploidization in highly proliferative megakaryocytic cell lines, which possess higher levels of cyclin B and cdc2 activity than normal megakaryocytes.


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

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

Morphologic and DNA content analyses of various types of leukemia cell lines before and after the induction of megakaryocytic differentiation. The cells were cultured with TPA (1 nM), IPTG (1 mM), and TPO (30 ng/ml) for 120 h. (A) Light micrographs of cells. A cytocentrifugation preparation from each culture was stained with May-Grunwald-Giemsa. Bar, 10 μm. (B) DNA content of the cultured cells was analyzed at the times indicated by flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Morphologic and DNA content analyses of various types of leukemia cell lines before and after the induction of megakaryocytic differentiation. The cells were cultured with TPA (1 nM), IPTG (1 mM), and TPO (30 ng/ml) for 120 h. (A) Light micrographs of cells. A cytocentrifugation preparation from each culture was stained with May-Grunwald-Giemsa. Bar, 10 μm. (B) DNA content of the cultured cells was analyzed at the times indicated by flow cytometry.
Mentions: We previously found that expression of AIM-1 was downregulated during TPA-induced polyploidization of several megakaryocytic cell lines (Katayama et al. 1998). Here, we investigated the changes in expression of AIM-1 and STK15 during various types of megakaryocytic differentiation. Human erythro/megakaryocytic leukemia cell lines K562 and CMK are known to undergo polyploidization in response to TPA (Tetteroo et al. 1984; Matsumura et al. 1997). In addition, we previously reported that TPO treatment of F-36P-mpl yielded highly differentiated polyploid megakaryocytes, and that the induced expression of activated ras (H-rasG12V) provoked polyploidization of F-36P/ras and K562/ras cells (Matsumura et al. 1998, Matsumura et al. 2000b). Before each treatment, K562, CMK, F-36P-mpl, K562/ras, and F-36P/ras cells were exclusively composed of undifferentiated blastoid cells (Fig. 2 A). In contrast, the 120-h treatment with TPA, TPO, or IPTG individually gave rise to mature megakaryocytes with polyploid nucleus (several typical cells are shown in Fig. 2 A). In accordance with the morphologic data, DNA content analyses revealed that polyploid formation was induced by each treatment in all of the clones (Fig. 2 B). When expression levels of AIM-1 and STK15 were examined by Northern blot analysis, both expressions gradually decreased and were retained at a hardly detectable level until 120 h in all of the culture conditions (Fig. 3 A). Because cyclin B is a crucial regulator of mitosis, we also monitored expression levels of cyclin B during these cultures by Northern blot analysis. As shown in Fig. 3 A, the expressions of cyclin B were downregulated in all of the cultures. Consistent with the Northern blot analysis results, immunoblotting showed that the expression levels of cyclin B1 were reduced during polyploidization in all cultures (Fig. 3 B). These findings were largely consistent with previous reports that the expression of cyclin B was downregulated during polyploidization of several megakaryocytic cell lines (Zhang et al. 1996, Zhang et al. 1998; Matsumura et al. 2000a), but at variance with the finding that the cyclin B expression was retained in normal polyploidizing megakaryocytes (Vitrat et al. 1998). As reviewed by Zimmet and Ravid 2000, it was speculated that the reduction of cyclin B expression might be necessary for rapid reentry into S phase during polyploidization in highly proliferative megakaryocytic cell lines, which possess higher levels of cyclin B and cdc2 activity than normal megakaryocytes.

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