Fuhrken Peter G, Apostolidis Pani A, Lindsey Stephan, Miller William M, Papoutsakis Eleftherios T
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, USA.
J Biol Chem. 2008 Jun 6;283(23):15589-600. doi: 10.1074/jbc.M801923200. Epub 2008 Apr 8.
The molecular mechanisms underlying differentiation of hematopoietic stem cells into megakaryocytes are poorly understood. Tumor suppressor protein p53 can act as a transcription factor affecting both cell cycle control and apoptosis, and we have previously shown that p53 is activated during terminal megakaryocytic (Mk) differentiation of the CHRF-288-11 (CHRF) cell line. Here, we use RNA interference to reduce p53 expression in CHRF cells and show that reduced p53 activity leads to a greater fraction of polyploid cells, higher mean and maximum ploidy, accelerated DNA synthesis, and delayed apoptosis and cell death upon phorbol 12-myristate 13-acetate-induced Mk differentiation. In contrast, reduced p53 expression did not affect the ploidy or DNA synthesis of CHRF cells in the absence of phorbol 12-myristate 13-acetate stimulation. Furthermore, primary Mk cells from cultures initiated with p53-null mouse bone marrow mononuclear cells displayed higher ploidy compared with wild-type controls. Quantitative reverse transcription-PCR analysis of p53-knockdown CHRF cells, compared with the "scrambled" control CHRF cells, revealed that six known transcriptional targets of p53 (BBC3, BAX, TP53I3, TP53INP1, MDM2, and P21) were down-regulated, whereas BCL2 expression, which is known to be negatively affected by p53, was up-regulated. These studies show that the functional role of the intrinsic activation of p53 during Mk differentiation is to control polyploidization and the transition to endomitosis by impeding cell cycling and promoting apoptosis.
造血干细胞向巨核细胞分化的分子机制目前仍知之甚少。肿瘤抑制蛋白p53可作为一种转录因子，影响细胞周期调控和细胞凋亡，我们之前已经表明，在CHRF-288-11（CHRF）细胞系的终末巨核细胞（Mk）分化过程中p53被激活。在此，我们使用RNA干扰降低CHRF细胞中p53的表达，并表明p53活性降低会导致多倍体细胞比例增加、平均和最大倍性升高、DNA合成加速，以及在佛波酯12-肉豆蔻酸酯13-乙酸酯诱导的Mk分化时凋亡和细胞死亡延迟。相比之下，在没有佛波酯12-肉豆蔻酸酯13-乙酸酯刺激的情况下，p53表达降低并不影响CHRF细胞的倍性或DNA合成。此外，与野生型对照相比，由p53基因敲除小鼠骨髓单个核细胞起始培养的原代Mk细胞显示出更高的倍性。与“乱序”对照CHRF细胞相比，对p53敲低的CHRF细胞进行定量逆转录PCR分析发现，p53的六个已知转录靶点（BBC3、BAX、TP53I3、TP53INP1、MDM2和P21）表达下调，而已知受p53负面影响的BCL2表达上调。这些研究表明，p53在Mk分化过程中内在激活的功能作用是通过阻碍细胞周期进程和促进细胞凋亡来控制多倍体化和向核内有丝分裂的转变。
