Nucleophosmin as a Biomarker of the Metastatic Phenotype

Nucleophosmin (NPM; Nucleolar phosphoprotein B23) is a multifunctional protein found primarily in the nucleolus. NPM may be a clinically useful tumor biomarker. NPM is over-expressed in many types of solid tumors, and altered forms of NPM are found in certain lymphomas and leukemias. In one type of leukemia, a fragment of NPM accumulates in the cytoplasm. Objectives: (1) Use a monoclonal antibody (mAb) against NPM to localize NPM in cultured cells from rat adenocarcinoma clones that have differing metastatic potentials. (2) Use the mAb and ELISA to measure NPM levels in extracts from the cells. Methods: (1) Cells were cultured on CultureSlides, washed, fixed and permeabilized, incubated with mAb, washed, and incubated with rabbit anti-mouse IgG Ab linked to horseradish peroxidase (HRP). (2) Cells were extracted with urea/thiourea/CHAPS detergent, and the proteins were concentrated by acid precipitation. A sandwich ELISA was developed using purified rabbit polyclonal anti-human recombinant (hr) NPM as the capture Ab, mouse mAb to rat NPM as the detection Ab, goat anti-mouse IgG linked to HRP to measure the bound mAb, and a hr-NPM-GST (glutathione transferase) fusion protein as the standard. Results: NPM was localized in the nuclei of both non-metastatic and highly-metastatic cells. NPM was 1.8 ± 0.2 microg/mg protein in the non-metastatic MTC cells, 2.5 ± 0.4 microg/mg protein in the low metastatic-potential MTLn2 cells, and 2.9 ± 0.3 microg/mg protein in the highly metastatic MTLn3 cells. Conclusions: NPM levels were higher in cells of the two metastatic clones, suggesting that NPM may be useful not only as a tumor biomarker but as a biomarker of the metastatic phenotype.

Supported by the UTHSC Dental Alumni Endowment Fund for Research and the Sam D. Mount, Jr. Endowment