CXCL12 Stimulation of CXCR4-WHIM Transduced Waldenstrom's Macroglobulinemia cell line BCWM.1
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ABSTRACT: CXCR4 cDNA transcripts were subcloned into plenti-IRES-GFP vectors, and stably transduced using a lentiviral system In addition to wild-type CXCR4, vectors with the mutations c.1013C>G (p.Ser338*), c.932_933insT (p.Thr311fs), and c.1030_1041delinsGT (p.Ser344fs) were generated. Cell lines were stimulated with 50nM of the CXCR4 ligand CXCL12 (SDF1A) (R&D Systems, Minneapolis MN) for two hours. RNA from each sample was extracted at baseline and at 2 hours. At baseline and following stimulation, RNA was extracted and processed for analysis using Affymetrix Human Gene ST 2.0 arrays (Affymetrix, Santa Clara, CA). Data was analyzed using Oligo and Limma bioconductor packages in R using TMM normalization.
Project description:CXCR4 cDNA transcripts were subcloned into plenti-IRES-GFP vectors, and stably transduced using a lentiviral system In addition to wild-type CXCR4, vectors with the mutations c.1013C>G (p.Ser338*), c.932_933insT (p.Thr311fs), and c.1030_1041delinsGT (p.Ser344fs) were generated. Cell lines were stimulated with 50nM of the CXCR4 ligand CXCL12 (SDF1A) (R&D Systems, Minneapolis MN) for two hours. RNA from each sample was extracted at baseline and at 2 hours.
Project description:Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and non-peptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key-factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine-triphosphate (ATP) and uridine-triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation of migrating CD34+ cells and increased cell adhesion to fibronectin. In vivo, pre-incubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Gαi) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5â-triphosphatases (GTPase) Rac2 and downstream effectors Rho GTPaseâactivated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the migration of HSCs and their homing to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases. Experiment Overall Design: Highly purified CD34+ cells from 6 healthy donors were seeded at 1000000 cells/ml in serum free medium (EX vivo 15) w/o cytokines and treated with 10 mM UTP, 150ng/ml CXCL12, or 10 mM UTP plus 150ng/ml CXCL12 respectively for 24 hours. As a control, CD34+ untreated cells were maintained in the same culture conditions at the same time.
Project description:Breast tumors are produced by an uncontrollable cell proliferation mechanism and can be classified as benign (TMB) or malignant (TMM). TMM or breast cancer is the neoplasia with the highest incidence and mortality in Mexican women. Over time, some types of TMB can transform into a TMM. However, the mechanisms involved in such processes remain elusive and limited studies have examined the molecular differences between TMB and TMM. Hence, the aim of this study was to evaluate and compare the proteomic profile of TMB (n = 10) and TMM (n = 6) of Mexican women.
Project description:The chemokine CXCL12 and its receptor CXCR4 play important roles in signaling and migration of T-cells, but little is known about the transcriptional events involved in CXCL12-mediated T-cell migration. In this study we performed microarray analysis on CXCL12- treated T-cells, and found that the Wnt family of proteins was significantly upregulated during CXCL12 treatment. Confirmation of these results by real-time PCR and Western analysis indicated that the non-canonical Wnt pathway was specifically upregulated during CXCL12 treatment. In vitro and in vivo knockdown studies confirm that b-catenin (the key mediator of canonical Wnt signaling) is not involved in the CXCL12-mediated migration of T-cells. However, Wnt5A, a non-canonical Wnt protein, increases signaling through the CXCL12/ CXCR4 axis via Protein Kinase C (PKC). Our results demonstrated that CXCL12 required Wnt5A to mediate T-cell migration, and the treatment of T-cells with recombinant Wnt5A sensitized T-cells to CXCL12 induced migration. Additionally, Wnt5A expression was required for the sustained expression of CXCR4, both transcriptionally and translationally. These results could be translated in vivo, using EL4 thymoma metastasis as a model of T-cell migration. Taken together our data indicate, for the first time, that Wnt5A is a critical mediator of the CXCL12/ CXCR4 signaling axis. Experiment Overall Design: Primary T cells were treated with human CXCL12 (Peprotech, Rocky Hill, NJ) at 100 ng/ml per 10 million cells overnight in a humidified incubator at 37ºC with 5% CO2. Control cells were incubated in media only. Cells were harvested and washed with ice cold PBS for 2 times followed by the addition of ice cold TRIzol (Invitrogen, Carlsbad, CA) and frozen at -80ºC overnight. Total RNA was isolated using the RNA isolation kit manufactured by Qiagen (Valencia, CA). The cDNA was prepared from equal amount of RNA using a cDNA preparation kit (Bio-Rad, Hercules, CA) followed by preparation of cRNA according to manufacturerâs instructions (Agilent, Santa Clara, CA). The cRNA was amplified and labeled with either Cy-3 or Cy-5, using the Agilent low-input linear amplification kit, according to manufacturerâs protocols. Labeled cRNA were applied to the Human 44K whole genome oligo array slides (Agilent, Santa Clara, CA). Slides were hybridized in a rotating chamber overnight at 60ºC in 6X SSC. Next day, slides were washed with 0.005% Triton X-102 for 10 minutes, and then in 0.1X SSC, 0.005% Triton X-102 for 5 minutes on ice. Slides were dried using a nitrogen-filled air gun, and scanned using an Agilent scanner. Images were analyzed using the Agilent Feature Extractor Software, Version A.7.5.1 and ratios for each spot were calculated.
Project description:This study reports the improvement in function and antitumor efficacy of CXCR4 overexpression in therapeutic CD8+ T cells. Polyclonal CD8+ T cells from OT-I C57BL/6 (B6) mice were transduced with a modified pMP71 retroviral vector containing murine Cxcr4 and Gfp reporter sequences (T_CXCR4) or with a control vector containing Gfp alone (T_Control), and co-transferred into to B6 mice that were then vaccinated with peptide-pulsed DC. 90 days following DC vaccination, lymphocytes were isolated from the spleen and resting memory OT-I T_CXCR4 and OT-I T_Control cells were FACS sorted to perform gene expression profiling.
Project description:The objective of this experiment is to identify genes that are regulated by the LRR-receptor-like protein TOO MANY MOUTHS (TMM) to control stomatal precursor (meristemoid) cell fate and behavior. An interesting aspect of the tmm phenotype is that mutant plants overproduce stomata on leaves, but lack stomata entirely on inflorescence stems and in other locations on the plant. Despite the failure to produce mature stomata, mutant stems produce presumptive stomatal precursors through asymmetric divisions. This suggests that normal TMM signaling is required to maintain meristemoid cell fate but not to execute formative asymmetric divisions. Because stem stomata develop in a morphogenetic wave from tip to base, it is possible to harvest tissues that contain meristemoids but not guard mother cells or mature stomata. This provides an opportunity to compare the gene expression profiles of tissues with meristemoids that maintain their cellular identity and ultimately produce stomata (Col gl1), to tissues in which meristemoids form but rapidly lose their cellular identity due to a failure in TMM signaling (tmm-1). Because guard mother cells and stomata are not present in the tissue harvested, genes that change in abundance during these developmental stages will be filtered out. Ultimately we hope to reveal genes that operate downstream of TMM to confer meristemoid cell fate or to provide 'niche factors' suitable for their development into stomata. For analysis, approximately 90 developing inflorescence stem tips were collected and flower buds and meristems removed prior to flash freezing for each replicate sample. The region harvested had been previously determined to contain developing meristemoids but not stomata or guard mother cells by microscopic observation. An identical region was harvested from tmm-1 mutant plants grown at the same time, in the same flat. 4 samples were used in this experiment.
Project description:Canine keratinocyte cell line (CPEK, CELLnTEC Advanced Cell Systems, Bern, Switzerland) : Unstimulated control (UC) vs. Samples stimulated by 4µg/mL recombinant human periostin (PO) (R&D Systems, Minneapolis, MN) for 6 or 24 hrs.
Project description:To assess the impact of AdV-VP55 mediated degredation of host miRNAs on the murine whole lung transcriptome profile Non-replicateive Adeno vectors were delivered intranasally to 6-8 week old C57BL/6 mice for 48hrs. Whole lung tissue was isolated and used for transcriptome profiling.
Project description:We carried out a single-cell (sc) multiomic analysis on a series of MYD88 mutated Waldenström macroglobulinemia (WM) cases and identified two distinct subtypes of disease, memory B-cell-like (MBC-like) and plasma cell-like (PC-like), based on their expression of key lineage defining genes. Biologically, the subtypes are characterized by their variable capacity to differentiate fully towards a plasma cell (PC) and exhibit unique transcriptomic, chromatin accessibility, and genomic profiles. The MBC-like subtype is unable to differentiate beyond the memory B-cell (MBC) stage, upregulates key MBC genes, and is characterized by upregulated BCR and AKT/mTOR signaling. In contrast, the PC-like subtype can partially differentiate towards a PC, upregulates key PC genes, has enhanced NF-kB signaling, and has an upregulated unfolded protein response. Pseudotime trajectory analysis of combined scRNA-sequencing and scATAC-sequencing supports the variable differentiation capacity of each subtype and implicate key transcription factors SPI1, SPIB, BCL11A, and XBP1 in these features. The existence and generalizability of the two disease subtypes were validated further using hierarchical clustering of bulk RNA-seq data from a secondary set of cases. The biological significance of the subtypes was further established using whole genome sequencing, where it was shown that CXCR4, NIK, and ARID1A mutations occur predominantly in the MBC-like subtype and 6q deletions in the PC-like subtype. We conclude that the variable differentiation blockade seen in WM manifests itself clinically as two disease subtypes with distinct epigenetic, mutational, transcriptional, and clinical features with potential implications for WM treatment strategies.
Project description:Telomere maintenance mechanisms (TMM) are a hallmark of high-risk neuroblastoma, and are conferred by activation of telomerase or alternative lengthening of telomeres (ALT). However, detection of TMM is not yet part of the clinical routine, and consensus on TMM detection, especially on ALT assessment, remains to be achieved. Based on our results we here propose a workflow to reliably detect TMM in neuroblastoma. We show that unambiguous classification is feasible following a stepwise approach that determines both, activation of telomerase and ALT. The workflow proposed in this study can be used in clinical routine and provides a framework to systematically evaluate telomere maintenance mechanisms in risk stratification and treatment allocation of neuroblastoma patients.