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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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(A) DNA content analysis by flow cytometry. F-36P cells were synchronized by double TdR blocks. After release, the cells were cultured with rhIL-3 for the times indicated. The DNA content of cultured cells was quantitated by staining with propidium iodide and analyzing on a FACSort® with Modfit LT 2.0. The results are summarized in the table. AS, asynchronous cells. (B) Changes in expression of AIM-1, STK15, cyclin A, and cyclin B mRNA during IL-3–induced cell cycle progression. Total cellular RNA was isolated at the times indicated and subjected to Northern blot analysis. The filters were hybridized with 32P-labeled probes as indicated.
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Figure 1: (A) DNA content analysis by flow cytometry. F-36P cells were synchronized by double TdR blocks. After release, the cells were cultured with rhIL-3 for the times indicated. The DNA content of cultured cells was quantitated by staining with propidium iodide and analyzing on a FACSort® with Modfit LT 2.0. The results are summarized in the table. AS, asynchronous cells. (B) Changes in expression of AIM-1, STK15, cyclin A, and cyclin B mRNA during IL-3–induced cell cycle progression. Total cellular RNA was isolated at the times indicated and subjected to Northern blot analysis. The filters were hybridized with 32P-labeled probes as indicated.

Mentions: Since it has been shown that AIM-1 and STK15 are specifically expressed at G2/M phase of the cell cycle in nonhematopoietic cells, we initially examined expression patterns of these molecules in a human IL-3–dependent hematopoietic cell line F-36P synchronized by double TdR blocks. After the second culture with TdR, 92% of the cultured cells were synchronized at a G1/S boundary. Then, the cells were released and subjected to cell cycle (Fig. 1 A) and Northern blot analyses (Fig. 1 B), at the times indicated. After the release, 66% of the cells entered S phase at 4 h, and then progressed to G2/M phase from 12 to 20 h; the proportion of cells in G2/M phase increased maximally (37%) at 16 h. Consistent with the findings on cell cycle analysis, expression of cyclin A and cyclin B gradually increased, reached a peak at 16 h, and decreased at 24 h. The expression of AIM-1 appeared ∼4 h, peaked at 12 h, and disappeared at 20 h. Also, the expression of STK15 was observed from 12 to 20 h. Because both expression patterns were roughly coincident with those of cyclin A and cyclin B, AIM-1 and STK15 are supposed to be expressed specifically at G2/M phase in proliferating hematopoietic cells.


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

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

(A) DNA content analysis by flow cytometry. F-36P cells were synchronized by double TdR blocks. After release, the cells were cultured with rhIL-3 for the times indicated. The DNA content of cultured cells was quantitated by staining with propidium iodide and analyzing on a FACSort® with Modfit LT 2.0. The results are summarized in the table. AS, asynchronous cells. (B) Changes in expression of AIM-1, STK15, cyclin A, and cyclin B mRNA during IL-3–induced cell cycle progression. Total cellular RNA was isolated at the times indicated and subjected to Northern blot analysis. The filters were hybridized with 32P-labeled probes as indicated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: (A) DNA content analysis by flow cytometry. F-36P cells were synchronized by double TdR blocks. After release, the cells were cultured with rhIL-3 for the times indicated. The DNA content of cultured cells was quantitated by staining with propidium iodide and analyzing on a FACSort® with Modfit LT 2.0. The results are summarized in the table. AS, asynchronous cells. (B) Changes in expression of AIM-1, STK15, cyclin A, and cyclin B mRNA during IL-3–induced cell cycle progression. Total cellular RNA was isolated at the times indicated and subjected to Northern blot analysis. The filters were hybridized with 32P-labeled probes as indicated.
Mentions: Since it has been shown that AIM-1 and STK15 are specifically expressed at G2/M phase of the cell cycle in nonhematopoietic cells, we initially examined expression patterns of these molecules in a human IL-3–dependent hematopoietic cell line F-36P synchronized by double TdR blocks. After the second culture with TdR, 92% of the cultured cells were synchronized at a G1/S boundary. Then, the cells were released and subjected to cell cycle (Fig. 1 A) and Northern blot analyses (Fig. 1 B), at the times indicated. After the release, 66% of the cells entered S phase at 4 h, and then progressed to G2/M phase from 12 to 20 h; the proportion of cells in G2/M phase increased maximally (37%) at 16 h. Consistent with the findings on cell cycle analysis, expression of cyclin A and cyclin B gradually increased, reached a peak at 16 h, and decreased at 24 h. The expression of AIM-1 appeared ∼4 h, peaked at 12 h, and disappeared at 20 h. Also, the expression of STK15 was observed from 12 to 20 h. Because both expression patterns were roughly coincident with those of cyclin A and cyclin B, AIM-1 and STK15 are supposed to be expressed specifically at G2/M phase in proliferating hematopoietic cells.

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