QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Millot, G. A. Articles by Svinarchuk, F. Search for Related Content PubMed PubMed Citation Articles by Millot, G. A. Articles by Svinarchuk, F.

Distinct effects of thrombopoietin depending on a threshold level of activated Mpl in BaF-3 cells

INSERM U362, Laboratoire Hématopoïèse et Cellules Souches, Institut Gustave Roussy, 39 rue Camille Desmoulins, Villejuif, France

View larger version (33K): [in a new window]   Fig. 1. A threshold number of cell-surface Mpl receptors is necessary for TPO-induced proliferation of BaF-3 cells. (A) Flow cytometric analysis of Mpl-expressing clones. A Flag-Mpl receptor construct was introduced into BaF-3 cells and clones were derived. Cell surface expression of Mpl was examined for each clone by Flag-PE immunostaining: broken line, unlabeled cells; solid line, labeled cells. (B) Proliferation assay. Cells were incubated with the indicated concentration of TPO for 48 hours and cell proliferation was quantitated by [3H]thymidine incorporation. The results shown are the means±s.d. of triplicate experiments. (C) Western blots analysis of signaling pathways activated in clones stimulated by TPO. Cells were washed three times, deprived of cytokines for 3 hours, and then stimulated with 50 ng/ml of TPO for 15 minutes. Lysates (60 µg of protein per lane) were fractionated by SDS-PAGE. Phosphorylated (upper panel) and total (lower panel) STAT-5, ERK and AKT were analyzed by immunoblotting. ns, no stimulation. The positions of ERK1 (p44) and ERK2 (p42) are indicated.

View larger version (25K): [in a new window]   Fig. 2. Proliferation and high activation of signaling pathways are restored in previously underexpressing clones that were retransduced to increase expression of Mpl. (A) Proliferation assay. Cells were incubated with the indicated concentration of TPO for 48 hours and cell proliferation was quantitated by [3H]thymidine incorporation. The results shown are the means±s.d. of triplicate experiments. (B) Western blots analysis of signaling pathways activated in clones stimulated by TPO. Cells were washed three times, deprived of cytokines for 3 hours, and then stimulated with 50 ng/ml of TPO for 15 minutes. Activation of STAT-5, ERK and AKT were determined as described in Fig. 1. ns, no stimulation.

View larger version (21K): [in a new window]   Fig. 3. Effect of TPO on cell survival in clones with low levels of Mpl expression on their cell surface. Cells were washed three times and left deprived of cytokines or stimulated with 10 ng/ml of TPO. At the indicated time, cells were collected and labeled with propidium iodide. Percentages of apoptotic cells in the sub-G1 peak were determined by flow cytometry. Results are representative of two independent experiments.

View larger version (17K): [in a new window]   Fig. 4. TPO-dependent survival and proliferation of clones expressing Mpl. (A) Cells were washed three times and stimulated with various concentrations of TPO. At the indicated times, cells were either labeled with PI and percentages of apoptotic cells determined by flow cytometry (right axix) or cell proliferation was quantitated by [3H]thymidine incorporation (left axis). The results shown for [3H]thymidine incorporation are means±s.d. of triplicate experiments. Results are representative of two independent experiments.

View larger version (32K): [in a new window]   Fig. 5. Western blot analysis of signaling pathway intermediates activated in Mpl-expressing clones after long-term TPO stimulation. Cells were washed three times and stimulated with 10 ng/ml of TPO for 3 or 9 hours. Cells were lysed and 60 µg of protein per lane was fractioned by SDS-PAGE. Lysate of clone A deprived of cytokines for 3 hours was used as a control. (A) Activation of STAT-5, ERK and AKT after 9 hours of TPO stimulation was analyzed as described in Fig. 1. (B) Expression level of Bcl-XL and BAD phosphorylation analyzed by immunoblotting with anti-Bcl-x and anti-BAD antibodies. As a control, blots labeled with the anti-Bcl-x antibody were stripped and relabeled with a mouse anti-actin antibody. ns, no stimulation. Positions of unphosphorylated (BAD), singly (BADP1) and doubly phosphorylated BAD (BADP2) are indicated.

View larger version (25K): [in a new window]   Fig. 6. The P13K/AKT pathway is involved in the TPO-mediated survival effect observed in clones with low levels of Mpl expression. (A) Proliferation assays. Cells of clone A were washed three times and left cytokine-deprived or incubated for 24 hours with 10 ng/ml of TPO, solvent alone (0.14% DMSO), or the indicated concentration of inhibitors. Cell proliferation was quantitated by [3H]thymidine incorporation. The results shown are means±s.d. of triplicate experiments. (B) Western blot analysis of STAT-5, ERK and AKT activation. Cells from clone A were washed three times, deprived of cytokines for 3 hours and stimulated with 50 ng/ml of TPO for 15 minutes, DMSO (inhibitor solvent) alone, 50 µM of the MEK inhibitor PD98059 or 10 µM of the P13K inhibitor LY294002. Activation of STAT5, ERK and AKT was analyzed as described in Fig. 1. ns, no stimulation. (C) Apoptosis analysis. Cells were washed three times and left cytokine-deprived or stimulated for 22 hours with 10 ng/ml of TPO, solvent alone (0.14% DMSO), or the indicated concentration of inhibitors. Cells were then labeled with PI and percentages of apoptotic cells were determined by flow cytometry. Results are representative of two independent experiments.