Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene expression in signaling subsets of AML blasts induced by G-CSF

ABSTRACT: While the response to G-CSF is a significant predictor of AML outcome, the nature of cells responding differentially to this and other cytokines remains unclear. Based on gene expression signatures observed in G-CSF Responsive Cells (RC) and non-G-CSF Responsive Cells (NRC) a cohort of AML samples were segregated into two types. In Type1 both cell subsets displayed an undifferentiated phenotype. In the Type2 AML the RC showed a very similar undifferentiated progenitor-like phenotype as in Type1 RC, whereas the NRC exhibited a more differentiated hematopoietic phenotype. The frequency of RC per sample was a determinant of whether a patient belonged to Type1 or Type2. Type2 RC had higher expression of CD321, higher colony formation capacity and greater responsiveness to cytokines secreted by NRC. Further analysis of clinical mRNA databases revealed that CD321 was a prognostic indicator for AML and a potential marker of clonogenic activity associated with this AML cancer lineage. Cryopreserved PBMC from 11 AML patinets were stimulated with G-CSF. Following stimulation cells were fixed, permeabilized and stained for selected surface markers (CD3, CD33, CD45) and and phospho-Stat3/5. After staining subsets positive and negative for phospo-Stat3/5 were sorted out for further microarray analysis. T-cells were sorted from each sample to control for the RNA quality by comparisons to RNA prepared from live T-cells sorted form healthy donors.

ORGANISM(S): Homo sapiens

SUBMITTER: Yury Goltsev

PROVIDER: E-GEOD-42140 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Primary mitochondrial respiratory chain (RC) diseases are heterogeneous in etiology and manifestations but collectively impair cellular energy metabolism. To identify a common cellular response to RC disease, systems biology level transcriptome investigations were performed in human RC disease skeletal muscle and fibroblasts. Global transcriptional and post-transcriptional dysregulation in a tissue-specific fashion was identified across diverse RC complex and genetic etiologies. RC disease muscle was characterized by decreased transcription of cytosolic ribosomal proteins to reduce energy-intensive anabolic processes, increased transcription of mitochondrial ribosomal proteins, shortened 5'-UTRs to improve translational efficiency, and stabilization of 3'-UTRs containing AU-rich elements. These same modifications in a reversed direction typified RC disease fibroblasts. RC disease also dysregulated transcriptional networks related to basic nutrient-sensing signaling pathways, which collectively mediate many aspects of tissue-specific cellular responses to primary RC disease. These findings support the utility of a systems biology approach to improve mechanistic understanding of mitochondrial RC disease. To identify a common cellular response to primary RC that might improve mechanistic understanding and lead to targeted therapies for human RC disease, we performed collective transcriptome profiling in skeletal muscle biopsy specimens and fibroblast cell lines (FCLs) of a diverse cohort of human mitochondrial disease subjects relative to controls. Systems biology investigations of common cellular responses to primary RC disease revealed a collective pattern of transcriptional, post-transcriptional and translational dysregulation occurring in a highly tissue-specific fashion. Affymetrix Human Exon 1.0ST microarray analysis was performed on 29 skeletal muscle samples and Fibroblast cell lines from mitochondrial disease patients and age- and gender-matched controls.

Project description:Identifying the targets of immune response after allogeneic hematopoietic cell transplantation (HCT) promises to provide relevant immune therapy candidate proteins. We used protein microarrays to serologically identify Nucleolar and Spindle Associated Protein 1 (NuSAP1) and Chromatin Assembly Factor 1, subunit B (p60) [CHAF1b] as targets of new antibody responses that developed after allogeneic HCT. Western blots and ELISA validated their post-HCT recognition and enabled ELISA testing of 120 other allo-HCT patients. CHAF1b specific antibodies were predominantly detected in AML patients whereas NuSAP1 specific antibodies were exclusively detected in AML patients one year post-transplant (p<0.0001). Complete genomic exon sequencing failed to identify a nonsynonymous SNP for NuSAP1 and CHAF1b between the donor and recipient cells. Expression profiles and RT-PCR showed NuSAP1 was predominately expressed in the bone marrow CD34+CD90+ hematopoietic stem cells (HSC), leukemic cell lines and B lymphoblasts as compared to other tissues or cells. Thus, NuSAP1 is recognized as an immunogenic antigen in 65% AML patients and suggests a tumor antigen role. In conclusion, clinically important tumor antigens can be identified as new antibody targets after allogeneic HCT using high density protein microarrays Plasma Profiling using ProtoArrays: Protein microarrays were obtained (ProtoArraysâ¢ V3, Invitrogen Corporation, Carlsbad, CA) displaying 5,056 full-length human proteins, derived from the Ultimate ORF collection (Invitrogen Corp., Carlsbad, CA) printed in duplicate with N-terminal GST epitopes expressed in Baculovirus and affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. The arrays also contained control spots consisting of buffer, empty spots, and human IgG printed in four different concentrations (IgG[1] to IgG[4]). The arrays were incubated with blocking buffer for one hr followed by application of the plasma sample diluted 1:150 for 90 min. After washing the array four times for 10 min each with PBST buffer, secondary antibody was added (goat anti-human Alexa 647, Molecular Probes, Eugene, OR) for 90 min. After washing the slides with PBST buffer, the arrays were dried and fluorescent intensity was detected using a microarray scanner (GenePix 4000B, Molecular Devices, CA). All incubations were carried out on a rotating platform at 4 ÂºC. ProtoArray data acquisition and measurement: The slides were scanned at a PMT gain of 60% with a laser power of 90% and a focus point of 0 Î¼m. Fluorescence intensity data were acquired using GenePix Pro 6.0 software (Molecular devices, Sunnyvale, CA) with the appropriate â.galâ file downloaded from the ProtoArray central portal on the Invitrogen website (http://www.invitrogen.com/protoarray) by submitting the barcode of each ProtoArray slide

Dataset Information

Gene expression in signaling subsets of AML blasts induced by G-CSF

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure