Patel H, Marley S B, Greener L, Gordon M Y
Department of Haematology, Faculty of Medicine, Imperial College, Hammersmith Campus, London, UK.
Leukemia. 2008 Mar;22(3):559-71. doi: 10.1038/sj.leu.2405057. Epub 2007 Dec 6.
We analysed the subcellular distribution of p210(BCR-ABL) protein using a junction-specific anti-BCR-ABL monoclonal antibody and confocal laser scanning microscopy (CLSM). Our studies have shown that p210(BCR-ABL) is arranged in discrete foci in the cytoplasm of cell lines and primary CD34(+) cells but not mononuclear cells suggesting the foci may be a feature of immature chronic myeloid leukaemia cells. We have devised a strategy to score the foci and found the mean number of foci varies between the cell types. The number of foci per cell is directly related to the level of p210(BCR-ABL) expression. CLSM was also used to analyse the distribution and colocalization of CT10 regulator-like (CRKL) p210(BCR-ABL). CRKL-p210(BCR-ABL) foci were completely or partially associated, touching or separate in different regions of the same cell. We also analysed the distribution of phosphorylated CRKL (pCRKL) with p210(BCR-ABL) and unexpectedly found only a small proportion of pCRKL in complex with p210(BCR-ABL). The foci distribution and high levels of uncomplexed p210(BCR-ABL), pCRKL and CRKL protein suggested the possibility of a dynamic equilibrium. Imatinib promoted nuclear transport of p210(BCR-ABL)-positive foci. It also disrupted complex formation between p210(BCR-ABL) and casitas B-cell lymphoma and CRKL but not between p210(BCR-ABL) and GRB2. Our observations of the CRKL and p210(BCR-ABL) complex may be important for understanding the function of CRKL.
我们使用一种连接特异性抗BCR-ABL单克隆抗体和共聚焦激光扫描显微镜（CLSM）分析了p210(BCR-ABL)蛋白的亚细胞分布。我们的研究表明，p210(BCR-ABL)在细胞系和原代CD34(+)细胞的细胞质中呈离散的灶状排列，但在单核细胞中则不然，这表明这些灶状结构可能是未成熟慢性髓性白血病细胞的一个特征。我们设计了一种对这些灶状结构进行评分的策略，发现不同细胞类型之间灶状结构的平均数量有所不同。每个细胞的灶状结构数量与p210(BCR-ABL)的表达水平直接相关。CLSM还用于分析CT10调节因子样（CRKL）与p210(BCR-ABL)的分布和共定位。CRKL-p210(BCR-ABL)灶状结构在同一细胞的不同区域完全或部分相关，相互接触或分离。我们还分析了磷酸化CRKL（pCRKL）与p210(BCR-ABL)的分布，意外地发现只有一小部分pCRKL与p210(BCR-ABL)形成复合物。灶状结构的分布以及大量未形成复合物的p210(BCR-ABL)、pCRKL和CRKL蛋白提示可能存在动态平衡。伊马替尼促进了p210(BCR-ABL)阳性灶状结构的核转运。它还破坏了p210(BCR-ABL)与卡斯他斯B细胞淋巴瘤和CRKL之间的复合物形成，但不影响p210(BCR-ABL)与GRB2之间的复合物形成。我们对CRKL和p210(BCR-ABL)复合物的观察结果可能对理解CRKL的功能很重要。
