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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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The expressions of AIM-1 and STK15 during the developmental processes of normal megakaryocytes. CD34+ cells were purified from normal human bone marrow cells, and then cultured with TPO (20 ng/ml) and PPP (20%) for the indicated times. (A) Phase–contrast micrographs of cells before and after the culture. Bar, 20 μm. (B) Light micrographs of cells. Cytocentrifugation preparations of the cells before and after the culture were stained with May-Grunwald-Giemsa. Bar, 10 μm. (C) DNA content of cultured cells was analyzed by flow cytometry before (dashed line) and after (solid line) culture. (D) Semiquantitative RT-PCR analysis on the expression of AIM-1 and STK15 mRNA. Total cellular RNA was isolated at the times indicated and cDNA was synthesized. The amounts of cDNA products were normalized according to the amounts of PCR products of GAPDH. The adjusted amounts of cDNA products from each sample were subjected to the PCR reaction for AIM-1, STK15, GPIIb, and GAPDH. The PCR products were size fractionated on 3.5% polyacrylamide gels, dried, and autoradiographed. NTC, no template control.
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Figure 4: The expressions of AIM-1 and STK15 during the developmental processes of normal megakaryocytes. CD34+ cells were purified from normal human bone marrow cells, and then cultured with TPO (20 ng/ml) and PPP (20%) for the indicated times. (A) Phase–contrast micrographs of cells before and after the culture. Bar, 20 μm. (B) Light micrographs of cells. Cytocentrifugation preparations of the cells before and after the culture were stained with May-Grunwald-Giemsa. Bar, 10 μm. (C) DNA content of cultured cells was analyzed by flow cytometry before (dashed line) and after (solid line) culture. (D) Semiquantitative RT-PCR analysis on the expression of AIM-1 and STK15 mRNA. Total cellular RNA was isolated at the times indicated and cDNA was synthesized. The amounts of cDNA products were normalized according to the amounts of PCR products of GAPDH. The adjusted amounts of cDNA products from each sample were subjected to the PCR reaction for AIM-1, STK15, GPIIb, and GAPDH. The PCR products were size fractionated on 3.5% polyacrylamide gels, dried, and autoradiographed. NTC, no template control.

Mentions: Next, we examined changes in expression levels of AIM-1 and STK15 during the developmental processes of normal megakaryocytes. CD34+ cells were purified from human bone marrow cells and cultured with rhTPO and PPP for the indicated times. After the 6-d culture, we found that most of the surviving cells became large in size by phase–contrast microscopic analysis (Fig. 4 A). Also, cytospin preparations of the cultured cells revealed that ∼95% of the cells surviving and developing after the 6-d culture of CD34+ cells with rhTPO and PPP were megakaryocytes (a typical cell is shown in Fig. 4 B). In agreement with morphologic changes, DNA content analysis revealed that most of the cultured cells possessed polyploid nuclei (solid line), whereas almost all of the cells were diploid before the culture (dashed line) (Fig. 4 C). Consistent with these findings, the expression level of megakaryocyte-specific surface antigen GPIIb was upregulated gradually until day 6 by semiquantitative RT-PCR analysis (Fig. 4 D). The expressions of AIM-1 and STK15 were induced at day 2, and then decreased to an undetectable level from days 4 to 6 (Fig. 4 D). In this culture condition, the purified CD34+ cells temporarily proliferated in response to TPO around day 2 (data not shown), and then underwent polyploidization until day 6. Thus, it was speculated that the expressions of AIM-1 and STK15 were transiently induced at the proliferative process (day 2) and were continuously suppressed during the subsequent polyploidizing process (days 4–6).


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

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

The expressions of AIM-1 and STK15 during the developmental processes of normal megakaryocytes. CD34+ cells were purified from normal human bone marrow cells, and then cultured with TPO (20 ng/ml) and PPP (20%) for the indicated times. (A) Phase–contrast micrographs of cells before and after the culture. Bar, 20 μm. (B) Light micrographs of cells. Cytocentrifugation preparations of the cells before and after the culture were stained with May-Grunwald-Giemsa. Bar, 10 μm. (C) DNA content of cultured cells was analyzed by flow cytometry before (dashed line) and after (solid line) culture. (D) Semiquantitative RT-PCR analysis on the expression of AIM-1 and STK15 mRNA. Total cellular RNA was isolated at the times indicated and cDNA was synthesized. The amounts of cDNA products were normalized according to the amounts of PCR products of GAPDH. The adjusted amounts of cDNA products from each sample were subjected to the PCR reaction for AIM-1, STK15, GPIIb, and GAPDH. The PCR products were size fractionated on 3.5% polyacrylamide gels, dried, and autoradiographed. NTC, no template control.
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Related In: Results  -  Collection

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Figure 4: The expressions of AIM-1 and STK15 during the developmental processes of normal megakaryocytes. CD34+ cells were purified from normal human bone marrow cells, and then cultured with TPO (20 ng/ml) and PPP (20%) for the indicated times. (A) Phase–contrast micrographs of cells before and after the culture. Bar, 20 μm. (B) Light micrographs of cells. Cytocentrifugation preparations of the cells before and after the culture were stained with May-Grunwald-Giemsa. Bar, 10 μm. (C) DNA content of cultured cells was analyzed by flow cytometry before (dashed line) and after (solid line) culture. (D) Semiquantitative RT-PCR analysis on the expression of AIM-1 and STK15 mRNA. Total cellular RNA was isolated at the times indicated and cDNA was synthesized. The amounts of cDNA products were normalized according to the amounts of PCR products of GAPDH. The adjusted amounts of cDNA products from each sample were subjected to the PCR reaction for AIM-1, STK15, GPIIb, and GAPDH. The PCR products were size fractionated on 3.5% polyacrylamide gels, dried, and autoradiographed. NTC, no template control.
Mentions: Next, we examined changes in expression levels of AIM-1 and STK15 during the developmental processes of normal megakaryocytes. CD34+ cells were purified from human bone marrow cells and cultured with rhTPO and PPP for the indicated times. After the 6-d culture, we found that most of the surviving cells became large in size by phase–contrast microscopic analysis (Fig. 4 A). Also, cytospin preparations of the cultured cells revealed that ∼95% of the cells surviving and developing after the 6-d culture of CD34+ cells with rhTPO and PPP were megakaryocytes (a typical cell is shown in Fig. 4 B). In agreement with morphologic changes, DNA content analysis revealed that most of the cultured cells possessed polyploid nuclei (solid line), whereas almost all of the cells were diploid before the culture (dashed line) (Fig. 4 C). Consistent with these findings, the expression level of megakaryocyte-specific surface antigen GPIIb was upregulated gradually until day 6 by semiquantitative RT-PCR analysis (Fig. 4 D). The expressions of AIM-1 and STK15 were induced at day 2, and then decreased to an undetectable level from days 4 to 6 (Fig. 4 D). In this culture condition, the purified CD34+ cells temporarily proliferated in response to TPO around day 2 (data not shown), and then underwent polyploidization until day 6. Thus, it was speculated that the expressions of AIM-1 and STK15 were transiently induced at the proliferative process (day 2) and were continuously suppressed during the subsequent polyploidizing process (days 4–6).

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