Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: RT-PCR analysis of PRL-3 mRNA expression levels in 19 bone marrow samples from AML patients either negative (ITD NEG; n = 12) or positive (ITD POS; n = 7) for FLT3-ITD mutation. MOLM-14 and MV4-11 AML cell lines were used as FLT3-ITD positive controls. β-actin, loading control. (a–d) Microarray data analysis of PRL-3 mRNA levels in FLT-ITD-positive (POS) or FLT3-ITD-negative (NEG) patients in four independent patient cohorts (total n = 1158). (a) Cohort 1 AML patient with normal karyotype ( n = 101, p = 0.001). (b) GSE1159 AML patient cohort ( n = 285, p < 0.001). (c) GSE6891 AML patient cohort ( n = 521, p < 0.001). (d) GSE15434 AML patient cohort ( n = 251, p < 0.001). Statistical differences between ITD-POS and ITD-NEG patients were determined using Chi-square test. PRL-3 expression level is divided into four groups: very high, high, intermediate, low. Western blot analysis of PRL-3 protein levels in four AML cell lines. Western blot analysis of PRL-3 in MOLM-14 and MV4-11 cells upon siRNA-mediated knock-down of FLT3 expression. NS, control non-silencing siRNA. GAPDH, loading control.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Mutagenesis, Microarray, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: A–C. Kaplan–Meier analysis of overall survival (OS) in normal karyotype AML patients for PRL-3 mRNA expression in (A) Cohort 1 AML patients, n = 101, (B) GSE6891, n = 227 and (C) GSE12417, n = 163. Statistically significant p values (using the log-rank test) are indicated in the figures.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: TF1-ITD and MOLM-14 cells were incubated with various concentrations of FLT3 inhibitors (PKC412, CEP-701) or Src inhibitors (SU6656, PP2) for 24 h. Whole cell lysates were subjected to Western blot analysis with indicated antibodies. GAPDH, loading control. (a–d) Western blot analysis of AML cells upon FLT3 inhibition. PKC412 and CEP-701 inhibited the phosphorylation of both FLT3 and STAT5 as well as PRL-3 protein levels in a dose-dependent manner in both TF1-ITD (a, b) and MOLM-14 (c, d) cells (a–d) Western blot analysis of AML cells upon FLT3 inhibition. PKC412 and CEP-701 inhibited the phosphorylation of Src, but not JAK, in a dose-dependent manner in both TF1-ITD (a, b) and MOLM-14 (c, d) cells. (a, b) Western blot analysis of AML cells upon Src inhibition. SU6656 (a) and PP2 (b) inhibited the phosphorylation of Src and STAT5 as well as PRL-3 protein levels in a dose-dependent manner in TF1-ITD cells.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Incubation, Western Blot, Inhibition

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: Two putative STAT5 binding sites (S1 and S2; DNA sequences illustrated) in a distal 5′-flanking region of PRL-3, as predicted by TRANSFAC. EMSA analysis using S1 and S2 biotinylated DNA probes (S1 and S2) incubated with nuclear extracts from either TF-1 or TF1-ITD cells. Arrow, shifted protein/probe complex. EMSA analysis as in (B) in the presence of 10-fold molar excess of unlabelled STAT5 competitor. Western blot analysis of streptavidin–agarose pull-down fractions (unbound or bound) using probe S1. Left panel: schematic diagram of a −5.4 kb upstream sequence of PRL-3 and its 5′-sequential deletion sequence with luciferase reporter vector (pGL3-S1a, S1b, S1c and S1d), respectively. Right panel: STAT5A or STAT5B expression vectors were co-transfected with PRL-3 luciferase reporter vector to TF-1 cells and luciferase activity measured. Error bars represent the mean ± SD from three independent experiments. PRL-3 expression is down-regulated upon siRNA-mediated STAT5 depletion in AML cells. NS, control non-silencing siRNA. (a) Quantitative real time PCR analysis of PRL-3 mRNA level after knock-down of STAT5 gene, normalized to GAPDH mRNA. Statistical differences between two groups were determined using Student's t -test (mean ± SD, n = 3). *** p < 0.001 (for TF1-ITD); ** p = 0.011 (for MOLM-14); * p = 0.038 (for MV4-11). (b) Western blot analysis of PRL-3 protein level after knock-down of STAT5 gene. GAPDH, loading control.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Binding Assay, Incubation, Western Blot, Sequencing, Luciferase, Plasmid Preparation, Expressing, Transfection, Activity Assay, Real-time Polymerase Chain Reaction

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: SEAP reporter assay results measuring AP-1 activity in TF-1 cells overexpressing GFP (TF1-GFP) or GFP-PRL-3 (TF1-PRL-3). Error bars represent the mean ± SD from three independent experiments. (a, b) PRL-3 specifically upregulates c-Jun but not c-Fos. (a) Western blot analysis of TF1-GFP, TF1-PRL-3 and TF1-ITD cells. (b) Western blot analysis after knock-down of endogenous PRL-3 in TF1-ITD cells. GAPDH, loading control. NS, control non-silencing siRNA. (a–d) PRL-3-mediated upregulation of c-Jun is dependent on ERK and JNK pathways. (a) Western blot analysis after knock-down of endogenous PRL-3 in TF1-ITD and MOLM-14 cells. (b) Western blot analysis of TF1-GFP and TF1-PRL-3 cells. (c, d) Western blot analysis after knock-down of ERK1/2 or JNK in TF1-PRL-3 cells. GAPDH, loading control. NS, control non-silencing siRNA. (a–c) MTS assay results reflecting numbers of viable TF1-PRL-3 cells after treatment with ERK-specific inhibitor (U0126), JNK-specific inhibitor (SP600125) or general AP-1 inhibitor (curcumin) for the various time points. Error bars represent the mean ± SD from three independent experiments.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Reporter Assay, Activity Assay, Western Blot, MTS Assay

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: Right panel: MTS assay results reflecting numbers of viable TF1-GFP and TF1-PRL-3 cells after culture in the absence of cytokines for various durations. Error bars represent the mean ± SD from three independent experiments. Left panel: Western blot analysis of TF1-GFP and TF1-PRL-3 cells. GAPDH, loading control. Flow cytometry analysis of propidium iodide-stained TF1-GFP and TF1-PRL-3 after 48 h culture in the absence of cytokines. Note the difference in sub-G1 peak/population, reflective of apoptotic cells. Representative data from three independent experiments are shown. Left panel: flow cytometry analysis of annexin-V- and 7-AAD-stained TF1-GFP and TF1-PRL-3 after 48 h culture in the absence of cytokines. The percentage in the upper left quadrant indicates the fraction of annexin-V-positive apoptotic cells in the entire cell population analysed. Right panel: quantitation of annexin-V-positive apoptotic population in three independent experiments. Statistical differences between two groups were determined using Student's t -test (mean ± SD, n = 3, *** p = 0.0012).

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: MTS Assay, Western Blot, Flow Cytometry, Staining, Quantitation Assay

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: The growth of PRL-3-depleted MOLM-14 and MV4-11 FLT3-ITD-positive AML cells was analysed by MTS assay and flow cytometry. (a) Knock-down of PRL-3 decreased cell number in FLT3-ITD positive MOLM-14 cells (mean ± SD, n = 3). (b) Depletion of PRL-3 accumulated cells in G1 phase in MOLM-14 cells. (a) Knock-down of PRL-3 decreased cell number in FLT3-ITD positive MV4-11 cells (mean ± SD, n = 3). (b) Depletion of PRL-3 accumulated cells in G1 phase in MV4-11 cells. Representative data (right panel) from three independent experiments are shown.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: MTS Assay, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: (a, b) Results of immunotherapy on liver and spleen sizes in a mouse model of AML. (a) Representative images of livers and spleens harvested from normal nude mice (upper left panel) or nude mice 12–14 days after i.v. injection of TF1-ITD cells together with bi-weekly i.v. administration of control IgG (upper right panel), PRL-3 mAb (lower left panel) or FLT3 mAb (lower right panel). (b) Quantitation of liver and spleen weights of mice as described in (a). Statistical differences between data groups were determined using Student's t -test from three independent experiments. * p < 0.001; ** p = 0.00121. (a, b) Results of immunotherapy and PRL-3 knock-down on leukaemic infiltration in mouse bone marrow (BM) cells in a mouse model of AML. (a) BM cells from nude mice 12–14 days after i.v. injection of (I) TF1, TF1-ITD cells together with bi-weekly i.v. administration of (II) control IgG or (III) PRL-3 mAb (lower left panel) or (IV) TF1-ITD cells depleted of endogenous PRL-3 were analysed using flow cytometry analysis using the human-specific marker CD45+ to distinguish TF1 human-derived AML cells. Percentages indicate proportion of CD45+ cells in the BM population analysed. (b) Quantitation of CD45+ engrafted cells as described in (a). Statistical differences between two groups were determined using Student's t -test (mean ± SD, n = 5, * p < 0.001). Kaplan–Meier survival analysis of PRL-3 mAb-treated ( n = 7) or control IgG-treated ( n = 7) mice in the TF1-ITD leukaemia mouse model ( p < 0.001).

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Injection, Quantitation Assay, Flow Cytometry, Marker, Derivative Assay