PRL-2 increases Epo and IL-3 responses in hematopoietic cells

Abstract

Dual specificity protein tyrosine phosphatase PRL-2 is overexpressed in pediatric acute myeloid leukemia (AML) and is located at human chromosome 1p35, a region often rearranged or amplified in malignant lymphoma and B-cell chronic lymphocytic leukemia (B-CLL). Little is known of the significance of PRL-2 expression in hematopoietic malignancies. Herein we demonstrated that ectopic expression of PRL-2 in murine pre-B-cell line Baf3ER and mouse bone marrow cells induced key features associated with malignant progression and metastasis. PRL-2-transfected Baf3ER cells had augmented growth responses to hematopoietic growth factors Epo or IL-3 with shortened cell cycle, reduced requirement (5x) for Epo in cell survival, increased cell migration (3x), reduced cell adhesion (5x), and conversion to an immature cell morphology in association with increased expression (3x) of stem cell marker Bmi-1. When transduced into mouse bone marrow cells, PRL-2 increased Epo-induced colony formation (4x) and gave rise to larger colonies. These observations provide evidences implicating PRL-2 as a pathogenic molecule in hematopoietic malignancies and suggest its potential as a novel therapeutic target.

Fig 1

PRL-2 transfectants (PRL-2a and PRL-2b clones) of Baf3ER cells have enhanced proliferation in proportion to the PRL-2 protein levels. (A) The schema of the exogenous PRL-2 protein tagged at the N-terminal region with the FLAG epitope. (B) Single cell clones (PRL-2a and PRL-2b) of Baf3ER stable transfectants of the FLAG-PRL-2 expression construct or control vector (V)-transfected Baf3ER cells (pooled transfectants) were generated. Total cell lysates (TCL) of the cells were analyzed by SDS-PAGE/Western blotting using antibodies as indicated. (C) Growth of V, PRL-2a and PRL-2b cells in the presence of Epo at (0.5 U/ml) for 3 days was quantified by MTT assays. Data represent the mean values ± SD of triplicate samples.

Fig 2

PRL-2b cells are hyper-proliferative in responses to either Epo or IL-3 with accelerated cell cycle progression. PRL-2b and vector control cells (V) were cultured in the absence or presence of Epo (A) or IL-3 (B) for 6 days prior to quantification of viable cells by MTT assay. Data represents mean ± SD of triplicate samples. PRL-2b and V cells cultured in the presence of Epo during active growth were subjected to DNA content analysis by flow cytometry (C).

Fig 3

PRL-2b cells exhibit improved cell viability, enhanced cell migration and reduced cell adhesion. (A) PRL-2b and vector control cells (V) were cultured in diminishing amounts of Epo for 48 hours. Viable cells were then quantified by trypan blue exclusion assays. (B) Percentages of PRL-2b and vector control cells invaded through Matri-gel during 24 hr incubation as quantified by cell migration assays. (C) Percentages of PRL-2b and vector-control cells adherent to fibronectin-coated plates after 30 min incubation as quantified by MTT assays. Data represents mean ± SD of triplicate samples.

Fig 4

PRL-2b cells have a less mature morphology and higher Bmi-1 expression. Representative images (1 × 1,000) of vector control (A) or PRL-2b cells (B) during active growth in culture as captured by microscopy after fixation and staining. The transfectants were also analyzed by SDS-PAGE/Western blotting with antibodies as indicated (C).

Fig 5

Hyper-phosphorylation of Stat5 in PRL-2b cells was induced by Epo or IL-3 stimulation. Growth factor-deprived BaF3ER transfectants were starved and stimulated with Epo (0.05 U/ml) (A) or IL-3 (1% WCM) (B) for the indicated period and subjected to SDS-PAGE/Western blotting with indicated antibodies.

Fig 6

PRL-2 transfection of mouse bone marrow cells promotes colony formation by hematopoietic progenitor cells. Mouse bone marrow cells retrovirally transduced with vector (MSCV-IRES-GFP) or PRL-2 expression construct (MSCV-IRES-GFP-hPRL-2) were evaluated in methylcellulose cultures for colony formation responses to Epo. (A) Total number of GFP-positive colonies that contain at least 50 cells as scored on day 7. Data represent mean ± SD of replicates. (B) Representative images (50 ×) of the colonies of bone marrow progenitor cells transduced with vector (left) or PRL-2 (right).