Yang Zhenyun, Li Yiping, Yin Fuqin, Chan Rebecca J
Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Exp Hematol. 2008 Oct;36(10):1285-96. doi: 10.1016/j.exphem.2008.04.016. Epub 2008 Jul 21.
Mutations in PTPN11, which encodes the protein tyrosine phosphatase Shp2, are commonly found in juvenile myelomonocytic leukemia (JMML). We hypothesized that PTPN11 mutations promote cell-cycle progression and confer enhanced survival to hematopoietic progenitors.
Murine bone marrow low-density mononuclear cells were transduced with pMIEG3, pMIEG3-WT Shp2, pMIEG3-Shp2D61Y, or pMIEG3-Shp2E76K followed by cell-cycle and survival functional analysis as well as biochemical analysis for key cell-cycle and programmed cell-death regulatory proteins.
A higher proportion of hematopoietic progenitors bearing the gain-of-function Shp2 mutants were residing in the S or G2 phase of the cell cycle in response to low doses of granulocyte-macrophage colony-stimulating factor compared to cells transduced with empty vector (MIEG3) or with WT Shp2. Likewise, Shp2D61Y- or Shp2E76K-expressing hematopoietic cells demonstrated reduced apoptosis based on Annexin-V staining and produced increased progenitor colonies after 48 hours in minimal media compared to cells transduced with empty vector or WT Shp2. To differentiate enhanced survival vs hyperproliferation, cells were stained with PKH26 to distinguish undivided cells from divided progeny. Shp2D61Y- or Shp2E76K-expressing PKH26+ cells similarly demonstrated reduced apoptosis. Upon biochemical analysis, expression of Akt- and Erk-responsive cell-cycle and programmed cell-death regulatory proteins were altered, including increased levels of cyclin D1, Bcl2, and BclXL and reduced levels of p27, p21, and Bim.
Collectively, these data demonstrate that gain-of-function Shp2 mutants promote hematopoietic progenitor cell-cycle progression and survival and imply that agents targeting the cell cycle or promoting apoptosis may have therapeutic potential in JMML.
编码蛋白酪氨酸磷酸酶Shp2的PTPN11基因突变常见于青少年骨髓单核细胞白血病（JMML）。我们推测PTPN11基因突变促进细胞周期进程并赋予造血祖细胞更强的生存能力。
用pMIEG3、pMIEG3-WT Shp2、pMIEG3-Shp2D61Y或pMIEG3-Shp2E76K转导小鼠骨髓低密度单核细胞，随后进行细胞周期和生存功能分析以及对关键细胞周期和程序性细胞死亡调节蛋白的生化分析。
与用空载体（MIEG3）或WT Shp2转导的细胞相比，携带功能获得性Shp2突变体的造血祖细胞中有更高比例处于细胞周期的S期或G2期，这是对低剂量粒细胞-巨噬细胞集落刺激因子的反应。同样，基于膜联蛋白-V染色，表达Shp2D61Y或Shp2E76K的造血细胞显示凋亡减少，并且与用空载体或WT Shp2转导的细胞相比，在基础培养基中培养48小时后产生的祖细胞集落增加。为了区分增强的生存能力与过度增殖能力，用PKH26对细胞进行染色以区分未分裂细胞和分裂后代。表达Shp2D61Y或Shp2E76K的PKH26+细胞同样显示凋亡减少。经过生化分析，Akt和Erk反应性细胞周期及程序性细胞死亡调节蛋白的表达发生改变，包括细胞周期蛋白D1、Bcl2和BclXL水平升高，以及p27、p21和Bim水平降低。
总体而言，这些数据表明功能获得性Shp2突变体促进造血祖细胞的细胞周期进程和生存，并暗示靶向细胞周期或促进凋亡的药物可能对JMML具有治疗潜力。
