Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, USA.
Physiol Genomics. 2012 Jun 15;44(12):638-50. doi: 10.1152/physiolgenomics.00028.2012. Epub 2012 May 1.
During endomitosis, megakaryocytes undergo several rounds of DNA synthesis without division leading to polyploidization. In primary megakaryocytes and in the megakaryocytic cell line CHRF, loss or knock-down of p53 enhances cell cycling and inhibits apoptosis, leading to increased polyploidization. To support the hypothesis that p53 suppresses megakaryocytic polyploidization, we show that stable expression of wild-type p53 in K562 cells (a p53-null cell line) attenuates the cells' ability to undergo polyploidization during megakaryocytic differentiation due to diminished DNA synthesis and greater apoptosis. This suggested that p53's effects during megakaryopoiesis are mediated through cell cycle- and apoptosis-related target genes, possibly by arresting DNA synthesis and promoting apoptosis. To identify candidate genes through which p53 mediates these effects, gene expression was compared between p53 knock-down (p53-KD) and control CHRF cells induced to undergo terminal megakaryocytic differentiation using microarray analysis. Among substantially downregulated p53 targets in p53-KD megakaryocytes were cell cycle regulators CDKN1A (p21) and PLK2, proapoptotic FAS, TNFRSF10B, CASP8, NOTCH1, TP53INP1, TP53I3, DRAM1, ZMAT3 and PHLDA3, DNA-damage-related RRM2B and SESN1, and actin component ACTA2, while antiapoptotic CKS1B, BCL2, GTSE1, and p53 family member TP63 were upregulated in p53-KD cells. Additionally, a number of cell cycle-related, proapoptotic, and cytoskeleton-related genes with known functions in megakaryocytes but not known to carry p53-responsive elements were differentially expressed between p53-KD and control CHRF cells. Our data support a model whereby p53 expression during megakaryopoiesis serves to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. Furthermore, we identify a putative p53 regulon that is proposed to orchestrate these effects.
在核内有丝分裂过程中，巨核细胞经历多次 DNA 合成而不分裂，导致多倍体化。在原代巨核细胞和巨核细胞系 CHRF 中，p53 的缺失或敲低会增强细胞周期并抑制细胞凋亡，从而导致多倍体化增加。为了支持 p53 抑制巨核细胞多倍体化的假说，我们表明在 K562 细胞（p53 缺失细胞系）中稳定表达野生型 p53 会由于 DNA 合成减少和凋亡增加而减弱细胞在巨核细胞分化过程中经历多倍体化的能力。这表明 p53 在巨核细胞生成过程中的作用是通过细胞周期和凋亡相关的靶基因介导的，可能是通过阻止 DNA 合成和促进凋亡。为了确定 p53 通过哪些候选基因介导这些作用，我们通过微阵列分析比较了 p53 敲低（p53-KD）和诱导终末巨核细胞分化的对照 CHRF 细胞之间的基因表达。在 p53-KD 巨核细胞中下调的 p53 靶基因包括细胞周期调节剂 CDKN1A（p21）和 PLK2、促凋亡 FAS、TNFRSF10B、CASP8、NOTCH1、TP53INP1、TP53I3、DRAM1、ZMAT3 和 PHLDA3、DNA 损伤相关的 RRM2B 和 SESN1，以及肌动蛋白成分 ACTA2，而抗凋亡 CKS1B、BCL2、GTSE1 和 p53 家族成员 TP63 在 p53-KD 细胞中上调。此外，在 p53-KD 和对照 CHRF 细胞之间，一些与细胞周期相关、促凋亡和细胞骨架相关的基因也表现出差异表达，这些基因在巨核细胞中有已知功能，但不携带 p53 反应元件。我们的数据支持这样一种模型，即在巨核细胞生成过程中表达 p53 通过阻碍细胞周期并促进凋亡来控制多倍体化和从核内有丝分裂向凋亡的转变。此外，我们确定了一个假定的 p53 调节因子，该因子被提议协调这些作用。
