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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 effects of the induced expression of WT AIM-1 and STK15 on TPA-induced polyploidization of K562 cells. (A) Northern blot analysis on the induction of Flag-AIM-1(WT) and Flag-STK15(WT) before and after IPTG treatment. Total cellular RNA was isolated before and after the 4-h IPTG treatment and subjected to Northern blot analysis. The filters were hybridized with 32P-labeled probes for AIM-1 or STK15. Exo, exogenous mRNA; Endo, endogenous mRNA. (B) Western blot analysis on the induction of Flag-AIM-1(WT) and Flag-STK15(WT) before and after IPTG treatment. The cells were cultured with 1 mM IPTG for the times indicated. Total cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the anti-Flag antibody M2. (C) Flow cytometric analysis of DNA content for each clone before and after TPA treatment in the culture, with or without IPTG. Cells were cultured with or without 1 mM of IPTG for 24 h, and then TPA was added to the culture medium (1 nM, final concentration). 96 h after adding TPA, the cultured cells were subjected to DNA content analysis.
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Figure 6: The effects of the induced expression of WT AIM-1 and STK15 on TPA-induced polyploidization of K562 cells. (A) Northern blot analysis on the induction of Flag-AIM-1(WT) and Flag-STK15(WT) before and after IPTG treatment. Total cellular RNA was isolated before and after the 4-h IPTG treatment and subjected to Northern blot analysis. The filters were hybridized with 32P-labeled probes for AIM-1 or STK15. Exo, exogenous mRNA; Endo, endogenous mRNA. (B) Western blot analysis on the induction of Flag-AIM-1(WT) and Flag-STK15(WT) before and after IPTG treatment. The cells were cultured with 1 mM IPTG for the times indicated. Total cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the anti-Flag antibody M2. (C) Flow cytometric analysis of DNA content for each clone before and after TPA treatment in the culture, with or without IPTG. Cells were cultured with or without 1 mM of IPTG for 24 h, and then TPA was added to the culture medium (1 nM, final concentration). 96 h after adding TPA, the cultured cells were subjected to DNA content analysis.

Mentions: Because the sustained reduction of AIM-1 and STK15 expression was coupled with polyploidization of megakaryocytes, it was speculated that downregulation of AIM-1 and/or STK15 might be a prerequisite for abortive mitosis and consequent polyploidization of megakaryocytes. To examine this possibility, we inducibly expressed Flag-tagged AIM-1(WT) and STK15(WT) in K562 cells with a Lac-inducible system in which expression of the target protein was induced by IPTG treatment, and investigated their effects on TPA-induced polyploidization of K562 cells; the clones were designated K562/AIM-1(WT) and K562/STK15(WT), respectively. As shown in Fig. 6 A, the expression of AIM-1 and STK15 mRNA was induced after the 4-h IPTG treatment at levels almost similar to or more than that of respective endogenous mRNA in unsynchronized cells. Also, immunoblotting with anti-Flag antibody demonstrated that AIM-1 and STK15 proteins were effectively induced by IPTG treatment after 12–120 h (Fig. 6 B). With or without the 24-h IPTG pretreatment, K562/AIM-1(WT), K562/STK15(WT), and a control clone K562/mock transfected with an empty vector were cultured with TPA in the presence or absence of IPTG for 4 d. Without IPTG treatment, DNA content analysis showed that TPA treatment induced similar levels of polyploidization in K562/mock, K562/AIM-1(WT), and K562/STK15(WT) cells (Fig. 6 C). In contrast, the induced expression of AIM-1(WT) reduced the proportion of 8N fraction from 16.3 ± 1.5% to 5.7 ± 0.6%, with a significant difference (P < 0.01, two-sample rank test) (Fig. 6 C and Table ). In addition, IPTG-induced AIM-1(WT) was required to inhibit TPA-induced polyploidization as observed in several K562/AIM-1(WT) clones (data not shown). In contrast, STK15(WT) did not show an apparent effect on TPA-induced polyploidization in K562/STK15(WT) cells (the proportion of 8N fraction: IPTG[−] 11.9 ± 1.1% versus IPTG[+] 11.7 ± 1.3%) (Fig. 6 C and Table ). Consistent with these data, in morphologic analysis, IPTG-induced AIM-1(WT) significantly suppressed TPA-induced polyploidization, whereas the induced STK15 showed little or no effect (several typical cells are shown in Fig. 7). These results suggested that downregulation of AIM-1, but not STK15, might be required for TPA-induced polyploidization of K562 cells.


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

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

The effects of the induced expression of WT AIM-1 and STK15 on TPA-induced polyploidization of K562 cells. (A) Northern blot analysis on the induction of Flag-AIM-1(WT) and Flag-STK15(WT) before and after IPTG treatment. Total cellular RNA was isolated before and after the 4-h IPTG treatment and subjected to Northern blot analysis. The filters were hybridized with 32P-labeled probes for AIM-1 or STK15. Exo, exogenous mRNA; Endo, endogenous mRNA. (B) Western blot analysis on the induction of Flag-AIM-1(WT) and Flag-STK15(WT) before and after IPTG treatment. The cells were cultured with 1 mM IPTG for the times indicated. Total cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the anti-Flag antibody M2. (C) Flow cytometric analysis of DNA content for each clone before and after TPA treatment in the culture, with or without IPTG. Cells were cultured with or without 1 mM of IPTG for 24 h, and then TPA was added to the culture medium (1 nM, final concentration). 96 h after adding TPA, the cultured cells were subjected to DNA content analysis.
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Related In: Results  -  Collection

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Figure 6: The effects of the induced expression of WT AIM-1 and STK15 on TPA-induced polyploidization of K562 cells. (A) Northern blot analysis on the induction of Flag-AIM-1(WT) and Flag-STK15(WT) before and after IPTG treatment. Total cellular RNA was isolated before and after the 4-h IPTG treatment and subjected to Northern blot analysis. The filters were hybridized with 32P-labeled probes for AIM-1 or STK15. Exo, exogenous mRNA; Endo, endogenous mRNA. (B) Western blot analysis on the induction of Flag-AIM-1(WT) and Flag-STK15(WT) before and after IPTG treatment. The cells were cultured with 1 mM IPTG for the times indicated. Total cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the anti-Flag antibody M2. (C) Flow cytometric analysis of DNA content for each clone before and after TPA treatment in the culture, with or without IPTG. Cells were cultured with or without 1 mM of IPTG for 24 h, and then TPA was added to the culture medium (1 nM, final concentration). 96 h after adding TPA, the cultured cells were subjected to DNA content analysis.
Mentions: Because the sustained reduction of AIM-1 and STK15 expression was coupled with polyploidization of megakaryocytes, it was speculated that downregulation of AIM-1 and/or STK15 might be a prerequisite for abortive mitosis and consequent polyploidization of megakaryocytes. To examine this possibility, we inducibly expressed Flag-tagged AIM-1(WT) and STK15(WT) in K562 cells with a Lac-inducible system in which expression of the target protein was induced by IPTG treatment, and investigated their effects on TPA-induced polyploidization of K562 cells; the clones were designated K562/AIM-1(WT) and K562/STK15(WT), respectively. As shown in Fig. 6 A, the expression of AIM-1 and STK15 mRNA was induced after the 4-h IPTG treatment at levels almost similar to or more than that of respective endogenous mRNA in unsynchronized cells. Also, immunoblotting with anti-Flag antibody demonstrated that AIM-1 and STK15 proteins were effectively induced by IPTG treatment after 12–120 h (Fig. 6 B). With or without the 24-h IPTG pretreatment, K562/AIM-1(WT), K562/STK15(WT), and a control clone K562/mock transfected with an empty vector were cultured with TPA in the presence or absence of IPTG for 4 d. Without IPTG treatment, DNA content analysis showed that TPA treatment induced similar levels of polyploidization in K562/mock, K562/AIM-1(WT), and K562/STK15(WT) cells (Fig. 6 C). In contrast, the induced expression of AIM-1(WT) reduced the proportion of 8N fraction from 16.3 ± 1.5% to 5.7 ± 0.6%, with a significant difference (P < 0.01, two-sample rank test) (Fig. 6 C and Table ). In addition, IPTG-induced AIM-1(WT) was required to inhibit TPA-induced polyploidization as observed in several K562/AIM-1(WT) clones (data not shown). In contrast, STK15(WT) did not show an apparent effect on TPA-induced polyploidization in K562/STK15(WT) cells (the proportion of 8N fraction: IPTG[−] 11.9 ± 1.1% versus IPTG[+] 11.7 ± 1.3%) (Fig. 6 C and Table ). Consistent with these data, in morphologic analysis, IPTG-induced AIM-1(WT) significantly suppressed TPA-induced polyploidization, whereas the induced STK15 showed little or no effect (several typical cells are shown in Fig. 7). These results suggested that downregulation of AIM-1, but not STK15, might be required for TPA-induced polyploidization of K562 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