(A)Colony-formation of REH and RS4;11 cells following treatment with the Hh pathway ligand SHhNP

(A)Colony-formation of REH and RS4;11 cells following treatment with the Hh pathway ligand SHhNP. vast majority of these tumors lack mutational activation of Hh pathway components, and increased Hh signaling may be due to over-expression of activating ligands or SMO [15], [16]. In pre-clinical models, pathway inhibition may result in reduced tumor cell proliferation or survival. Evidence that the Hh signaling pathway plays a role in several B cell malignancies including MM and non-Hodgkin lymphoma (NHL) as well as normal early B-cell development suggests that it may be involved in precursor B-ALL [6], [8], [17]. Plxnc1 Moreover, in several human hematologic malignancies, the Hh signaling pathway has been found to regulate self-renewal required for long-term maintenance of the malignant clone [6], [18],[19]. We examined Hh signaling pathway activity in B-ALL and found that Hh signaling regulates the self-renewal of highly clonogenic tumor cells both and in precursor B-ALL cell lines was detected by reverse-transcriptase PCR. Human fetal brain (HFB) was used as a positive control for Hh pathway expression, and Rhein-8-O-beta-D-glucopyranoside a-actin was used as a control gene for experiments with cell lines and HFB with and without reverse transcriptase. Levels of and were measured in cell lines and primary clinical specimens by real-time quantitative PCR using the Step 1 1 Plus thermal cycler and Fast Taqman reagent (Applied Biosystems). Clinical specimens which expressed all three genes and were considered to positive for expression of Hh pathway components. Normal bone marrow CD34+ CD19+ progenitors from normal bone marrow donors were used as controls in real-time PCR experiments. Quantitative calculations were performed using the ??ct method. Primer sequences are listed in Supplemental Table S1. Hh pathway agonists and inhibitors Recombinant Sonic Hedgehog (ShhNP) was a gift of P. Beachy (Stanford University). The monoclonal antibody 5E1 was obtained from the Iowa Hybridoma Bank [20]. The naturally occurring SMO inhibitor cyclopamine and the semi-synthetic cyclopamine derivative IPI-926 were provided by Infinity Pharmaceuticals [21]. Transient transfection studies REH and Rhein-8-O-beta-D-glucopyranoside RS4;11 precursor B ALL cells were co-transfected with a Gli-responsive firefly luciferase vector containing 8 tandem copies of a consensus Gli binding site immediately upstream of the chicken lens crystallin promoter (pGL3-8-Gli-luciferase) and constitutive luciferase expression vectors (pRL-CMV; Promega) using the Amaxa Nucleofector Apparatus (Lonza) [22]. Transfected cells were then treated with Hh pathway modulators for 48 hours. Treated cells were then harvested and assayed for firefly and luciferase activities using the dual luciferase reporter assay (Promega). Clonogenic assays REH Rhein-8-O-beta-D-glucopyranoside and RS4;11 cells were seeded at 1105 cells/ml and treated with ShhNP, 5E1, cyclopamine (5 M) or IPI-926 (1 M) for 72 hours. Following 72 hours of treatment, cells were washed twice with media to remove drugs then 500 cells were plated in quadruplicate in 1 ml of 1 1.2% methylcellulose, 30% FBS, 1% bovine serum albumin (BSA), 0.1 mM 2-mercaptoethanol, and 2 mM L-glutamine. Samples were plated in quadruplicate onto 35 mm2 tissue culture dishes and incubated in a humidified atmosphere at 37C and 5% CO2. Colonies consisting of >40 cells were counted using an inverted microscope at 10C14 days, then harvested and replated in methylcellulose.20 Results represent colony formation during each round of replating relative to vehicle control cells. Examination of Rhein-8-O-beta-D-glucopyranoside ALDH activity by circulation cytometry REH and RS4;11 cells.