Nucleophosmin (NPM) Has Increased Expression and Shuttling Activity in Chronic Lymphocytic Leukaemia without Somatic Hypermutation of the Immunoglubulin Gene (UM-CLL) and Is Linked to Ribosomal Proteins: A Possible Role for Increased Protein biosynthesis in the Adverse Outcome of UM-CLL.

Chronic lymphocytic leukaemia (CLL) is a neoplastic proliferation of mature B-lymphocytes that has a marked variability of clinical outcome. Two distinct groups of CLL may be distinguished by the presence (M-CLL) or absence (UM-CLL) of somatic hypermutation of the immunoglobulin heavy-chain gene. UM-CLL has an adverse outcome, but the precise biological differences that underlie this more aggressive clinical-course are unclear. Using a proteomic approach, we have found that M-CLL and UM-CLL are consistently distinguished according to protein expression pattern. Of those proteins, the most important predictor of CLL class was the protein nucleophosmin (NPM). A significant increase in expression of NPM was found in UM-CLL compared with M-CLL (3.2 fold p<0.05 Mann Whitney U test). This increased expression did not reflect differences in apoptosis or proliferation, or gene mutation. However, co-immunoprecipitation experiments identified the ribosomal proteins 60s (L19 and L24) and 40s (S9) to be associated with NPM in CLL. Ribosomal proteins are known to shuttle between the nucleoli, nucleus, and cytoplasm, of cells. Standard- and confocal-immunofluorescence microscopy identified NPM to be present, as expected, in the nucleoli of all CLL cells. Additionally however, NPM was consistently detected in the nucleoplasm of CLL, and in a proportion of cells from all CLL cases, NPM was also shown to be present in the cytoplasm (range =7–52% of cells expressed cytoplasmic NPM). Morphometric analysis suggested no net transfer from the nucleolus to the cytoplasm; but did suggest trafficking from the nucleoplasmic compartment to the cytoplasm consistent with shuttling between these two cellular regions. In UM-CLL the fluorescent intensity of the cytoplasmic NPM was found to be higher than that of the M-CLL (t-test p=0.009), and there was an approximate two fold increased proportion of cytoplasmic NPM-expression in the UM-CLL subgroup (p value< 0.001). We propose therefore that NPM, in association with ribosomal proteins, displays high nucleocytoplasmic shuttling in CLL, and may reflect increased protein biosynthesis. In particular, the higher levels of NPM expression and shuttling in UM-CLL may play a role in the adverse outcome of this form of the disorder.