Subcellular distribution of p210(BCR-ABL) in CML cell lines and primary CD34+ CML cells.
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.
Department of Haematology, Faculty of Medicine, Imperial College, Hammersmith Campus, London, UK.
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Patel
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Marley
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Greener
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Gordon
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis* / Q000378:metabolism; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000494:pharmacology; D001549:Benzamides; D045744:Cell Line, Tumor / Q000737:chemistry / Q000648:ultrastructure; D016922:Cellular Senescence; D016044:Fusion Proteins, bcr-abl / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D000068877:Imatinib Mesylate; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism / Q000473:pathology*; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000648:ultrastructure; D018613:Microscopy, Confocal; D014411:Neoplastic Stem Cells / Q000737:chemistry / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010879:Piperazines / Q000494:pharmacology; D025941:Protein Interaction Mapping; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011743:Pyrimidines / Q000494:pharmacology; D013347:Subcellular Fractions / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D001549:Benzamides; C082869:CRKL protein; D009687:Nuclear Proteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl