Abstract
Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.
Highlights
Megakaryocytes (MKs) are one of the few cell types that undergo a modified form of the cell cycle called endomitosis, in which cells do not undergo the late stages of mitosis and instead become polyploid [1]
SP600125 was used to treat four different cell lines: Dami and CMK cells, which are acute megakaryocytic leukemia (AMKL) cell lines, Meg-01 cells, which is a leukemic cell line derived from a patient with chronic myelogenous leukemia with a megakaryocytic phenotype [13, 14, 15], and HEL cells, which is a cell line with significant similarity to MKs that was derived from an erythroleukemia patient (Fig. S1) [16]
We further found that the SP600125induced polyploidization of Dami and CMK cells was accompanied by the phosphorylation of S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389 and that H-89 blocked the SP600125-induced polyploidization of Dami and CMK cells by concomitantly increasing the phosphorylation of S6K1 at Thr389 and decreasing the phosphorylation at Thr421/Ser424
Summary
Megakaryocytes (MKs) are one of the few cell types that undergo a modified form of the cell cycle called endomitosis, in which cells do not undergo the late stages of mitosis and instead become polyploid [1]. In nocodazole-induced polyploidization, the cells are multinucleated (karyokinesis is not affected), while in MKs, the nuclei are polylobulated. Synchronized polyploid cell models with polylobulated nuclei were established from Dami and CMK cells, and these cells were more similar to primary MKs that have undergone physiological polyploidization than polyploid. We found that SP600125 induced polyploidization of more differentiated cell lines through the phosphorylation of S6K1 at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 synergistically with other signaling pathways
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