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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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Effects of the induced expression of Flag-AIM-1(K/R) on CMK cells. (A) Western blot analysis on the induction of Flag-AIM-1(K/R) before and after IPTG treatment. CMK/AIM-1(K/R) cells were cultured with 1 mM of IPTG for the times indicated. Total cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the anti-Flag antibody M2. (B) DNA content analyses on mock-transfected CMK and CMK/AIM-1(K/R). Mock-transfected CMK and CMK/AIM-1(K/R) were culture with IPTG for 120 h. Also, mock-transfected CMK was treated with 1 nM TPA for 120 h. DNA content analyses were performed before and after the cultures. (C) Light micrographs of cells. Cytocentrifugation preparations of each clone, before and after the 120-h IPTG treatment, were stained with May-Grunwald-Giemsa. Bar, 10 μm.
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Figure 11: Effects of the induced expression of Flag-AIM-1(K/R) on CMK cells. (A) Western blot analysis on the induction of Flag-AIM-1(K/R) before and after IPTG treatment. CMK/AIM-1(K/R) cells were cultured with 1 mM of IPTG for the times indicated. Total cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the anti-Flag antibody M2. (B) DNA content analyses on mock-transfected CMK and CMK/AIM-1(K/R). Mock-transfected CMK and CMK/AIM-1(K/R) were culture with IPTG for 120 h. Also, mock-transfected CMK was treated with 1 nM TPA for 120 h. DNA content analyses were performed before and after the cultures. (C) Light micrographs of cells. Cytocentrifugation preparations of each clone, before and after the 120-h IPTG treatment, were stained with May-Grunwald-Giemsa. Bar, 10 μm.

Mentions: In addition to K562 cells, we also prepared several stable clones from CMK in which AIM-1(K/R) was inducibly expressed; these clone were named CMK/AIM-1(K/R). As shown in Fig. 11 A, induction of AIM-1(K/R) protein (IPTG treatment) was confirmed by immunoblot analysis using an anti-Flag antibody (Fig. 11 A). In a clone of CMK/AIM-1(K/R), DNA content analysis revealed that the induced AIM-1(K/R) provoked prominent polyploidization up to 32N (2N, 20.9%; 4N, 24.4%; 8N, 27.9%; 16N, 22.1%; and 32N, 4.7%) after the 5-d IPTG treatment (Fig. 11 B). Similar levels of polyploid formation were also observed in other clones of CMK/AIM-1(K/R) after IPTG treatment (data not shown). However, the AIM-1(K/R)–induced polyploidization lacked discrete twofold multiples of DNA content in the FACS® profile compared with the TPA-induced polyploidization (Fig. 11 B). Thus, in morphologic analyses, we found that the AIM-1(K/R)–induced mature megakaryocytes with multiple nuclei (typical cells are shown in Fig. 11 C) were more liable to result in apoptosis than the TPA-induced megakaryocytes. Because TPA has been reported to activate anti-apototic Ras/mitogen-activated protein kinase pathways, it was speculated that the AIM-1(K/R)–induced polyploid megakaryocytes were more sensitive to apotosis than the TPA-induced polyploid megakaryocytes due to the lack of anti-apoptotic signals. In addition to K562 and CMK cells, the induced expression of AIM-1(K/R) was able to provoke polyploidization of F-36P-mpl cells, as in the case of TPO (data not shown). Taken together, these results implied that the suppression of AIM-1 activities by the induced AIM-1(K/R) was, at least in part, sufficient for inducing polyploidization of K562, CMK, and F-36P-mpl cells.


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

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

Effects of the induced expression of Flag-AIM-1(K/R) on CMK cells. (A) Western blot analysis on the induction of Flag-AIM-1(K/R) before and after IPTG treatment. CMK/AIM-1(K/R) cells were cultured with 1 mM of IPTG for the times indicated. Total cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the anti-Flag antibody M2. (B) DNA content analyses on mock-transfected CMK and CMK/AIM-1(K/R). Mock-transfected CMK and CMK/AIM-1(K/R) were culture with IPTG for 120 h. Also, mock-transfected CMK was treated with 1 nM TPA for 120 h. DNA content analyses were performed before and after the cultures. (C) Light micrographs of cells. Cytocentrifugation preparations of each clone, before and after the 120-h IPTG treatment, were stained with May-Grunwald-Giemsa. Bar, 10 μm.
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Figure 11: Effects of the induced expression of Flag-AIM-1(K/R) on CMK cells. (A) Western blot analysis on the induction of Flag-AIM-1(K/R) before and after IPTG treatment. CMK/AIM-1(K/R) cells were cultured with 1 mM of IPTG for the times indicated. Total cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the anti-Flag antibody M2. (B) DNA content analyses on mock-transfected CMK and CMK/AIM-1(K/R). Mock-transfected CMK and CMK/AIM-1(K/R) were culture with IPTG for 120 h. Also, mock-transfected CMK was treated with 1 nM TPA for 120 h. DNA content analyses were performed before and after the cultures. (C) Light micrographs of cells. Cytocentrifugation preparations of each clone, before and after the 120-h IPTG treatment, were stained with May-Grunwald-Giemsa. Bar, 10 μm.
Mentions: In addition to K562 cells, we also prepared several stable clones from CMK in which AIM-1(K/R) was inducibly expressed; these clone were named CMK/AIM-1(K/R). As shown in Fig. 11 A, induction of AIM-1(K/R) protein (IPTG treatment) was confirmed by immunoblot analysis using an anti-Flag antibody (Fig. 11 A). In a clone of CMK/AIM-1(K/R), DNA content analysis revealed that the induced AIM-1(K/R) provoked prominent polyploidization up to 32N (2N, 20.9%; 4N, 24.4%; 8N, 27.9%; 16N, 22.1%; and 32N, 4.7%) after the 5-d IPTG treatment (Fig. 11 B). Similar levels of polyploid formation were also observed in other clones of CMK/AIM-1(K/R) after IPTG treatment (data not shown). However, the AIM-1(K/R)–induced polyploidization lacked discrete twofold multiples of DNA content in the FACS® profile compared with the TPA-induced polyploidization (Fig. 11 B). Thus, in morphologic analyses, we found that the AIM-1(K/R)–induced mature megakaryocytes with multiple nuclei (typical cells are shown in Fig. 11 C) were more liable to result in apoptosis than the TPA-induced megakaryocytes. Because TPA has been reported to activate anti-apototic Ras/mitogen-activated protein kinase pathways, it was speculated that the AIM-1(K/R)–induced polyploid megakaryocytes were more sensitive to apotosis than the TPA-induced polyploid megakaryocytes due to the lack of anti-apoptotic signals. In addition to K562 and CMK cells, the induced expression of AIM-1(K/R) was able to provoke polyploidization of F-36P-mpl cells, as in the case of TPO (data not shown). Taken together, these results implied that the suppression of AIM-1 activities by the induced AIM-1(K/R) was, at least in part, sufficient for inducing polyploidization of K562, CMK, and F-36P-mpl 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