Control of Autophagic Cell Death by Caspase-10 in Multiple Myeloma
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Louis Staudt
PROVIDER: E-GEOD-43878 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Comparing the gene expression of primary human B cell populations using human Lymphochip cDNA spotted arrays. Primary samples (Cy5) were compared to a reference pool of polyA+ RNA (Cy3). Keywords: cell type comparison design PolyA+ RNA was extracted from primary B cell populations (Cy5) and compared to a reference pool of polyA+ RNA (Cy3) on human Lymphochip cDNA spotted arrays. Each population was labelled and hybridized in duplicate.
2008-08-31 | E-GEOD-8959 | biostudies-arrayexpress
Project description:The goal of this thesis is to study the response of a human keratinocyte cell line (HaCaT cells) after UVB irradiation. In order to develop a system for UV irradiation, we firtstly studied the UV illumination system to find out a stable and reliable condition for UV irradiation experiments. Further, we found out the proper dose of UVB radiation and time points after UVB irradiation by performing MTT assay and trypan blue viability test. According to the results of MTT assay and trypan blue viability test, the cellular activity as well as survival rate of UVB-irradiated cells were in proportion to the radiation doses within a lower UVB dose range. We wanted to find out the possible reasons for this phenomenon by using cDNA microarray. We hope the thesis could be a guide for the following researches, and be useful in the clinical studies about the UV damage. Keywords: dose response and time course Loop-designed microarray experiments were performed in our study. Twenty-one samples are arranges as seven small loops and one large loop, with three and seven microarray slides for each small and large loop, respectively. This set of loop-designed microarray experiment contains one control untreated (non UVB-treated) samples and two UVB-treated samples (one low dose and one high dose UVB-treated) at each time point (total seven time points).
2010-05-16 | E-GEOD-4223 | biostudies-arrayexpress
Project description:The p53 homologue, p63, is critical for normal epidermal development. While overexpression of deltaNp63alpha has been reported in squamous cell cancers, the contribution of p63 to cancer pathogenesis remains unclear. We previously demonstrated that overexpressed deltaNp63alpha aberrantly maintains proliferation of primary epidermal keratinocytes under conditions that normally induce growth arrest and differentiation. To identify target genes impacted by dysregulated deltaNp63alpha that may contribute to squamous cancer development and progression, microarray analyses were performed. Herein we report that elevated deltaNp63alpha differentially regulates genes in primary mouse keratinocytes involved in a variety of cellular functions, including cell cycle regulation, differentiation, skin barrier formation and function, apoptosis, host defense/inflammation, adhesion, migration and invasion. Of note, multiple protease inhibitor mRNAs were coordinately downregulated under both proliferating and differentiating culture conditions. These downregulated genes include two serine protease inhibitors: maspin (serpinB5) and plasminogen activator inhibitor-2 (PAI-2; serpinB2), as well as a tissue inhibitor of metalloproteinase-3 (TIMP-3). Correspondingly, secreted levels of TIMP-3 and PAI-2 protein declined in the presence of dysregulated deltaNp63alpha, while secreted maspin protein levels remained stable. Intracellular maspin protein expression decreased in response to overexpressed deltaNp63alpha, as did intracellular PAI-2. Unlike PAI-2 and maspin, TIMP-3 protein was not detected intracellularly in control or deltaNp63alpha-overexpressing keratinocytes, supporting a solely extracellular function for TIMP-3. Electrophoretic mobility shift assays using a p53/p63 consensus DNA binding sequence from the maspin promoter revealed binding of an endogenous transcription factor(s) to the consensus sequence in normal keratinocytes that was disrupted by overexpressed deltaNp63alpha. This binding was also interrupted by the addition of a p73 antibody, but not antibodies to p63 or p53, and was absent in samples derived from p73(-/-) keratinocytes, confirming p73 as a constituent of the endogenous transcription factor complex. These data suggest protease inhibitors as novel targets of dysregulated deltaNp63alpha in cancer pathogenesis. Keratinocytes were transduced with adenoviruses encoding deltaNp63alpha. Keratinocytes were either maintained in low calcium conditions (0.05 mM for 24 hours) (n=6) or high calcium conditions (0.12 mM for 24 hours) (n=6). Control samples consisted of keratinocytes transduced with adenoviruses encoding beta-galactosidase also treated with low calcium (n=6) and high calcium (n=6) conditions. Twelve technical repeat microarray experiments were conducted, pairing high-calcium deltaNp63alpha samples with high-calcium beta-galactosidase samples (n=6) and low-calcium deltaNp63alpha samples with low-calcium beta-galactosidase samples (n=6). Six of the technical repeats were conducted as reverse-fluoro experiments.
2013-02-26 | E-GEOD-34862 | biostudies-arrayexpress
Project description:Abstract: Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable, however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2-treatment using human and non-human primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34+ hCB following xenotransplantation. In non-human primate autologous transplantation, dmPGE2-treated CD34+ MPBSCs showed stable multilineage engraftment over one year post-infusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and non-human primates, and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial. In order to identify the potential mechanism of action of dmPGE2 on gene expression and HSC function, and to possibly explain the muted response of rhesus CD34+ MPBSCs to dmPGE2 exposure, microarray gene expression analysis was conducted. Human and rhesus CD34+ MPBSCs were exposed to DMSO control or dmPGE2 at a dose of either 10 and 50 uM as described above, and processed for microarray analysis at 0, 2, 6, 12 and 24 hours post treatment. Four human donors contributed 8 to 10 samples each at varying time points and dosage for a total of 37 samples (2 samples were technical repeats). Six rhesus macaques contributed 3 to 9 samples each at varying time points and dosage for a total of 28 samples (4 samples were technical repeats). Pooled PBMC's from 6 donors aphereses were used as a reference sample.
2012-06-24 | E-GEOD-27126 | biostudies-arrayexpress
Project description:Using RNA interference to identify IRF4 target genes. Keywords: time series design Gene expression was analyzed using Lymphochip cDNA spotted arrays. Myeloma cell lines were infected with control (shluc, Cy3) or shIRF4 (Cy5) constructs, and changes in gene expression were monitored over time after induction of the shRNA with doxycyclin.
2008-07-07 | E-GEOD-8958 | biostudies-arrayexpress
Project description:The activated B cell-like (ABC) subgroup of diffuse large B cell lymphoma (DLBCL) is characterized by constitutive activation of the NF-êB pathway. Here we show that the NF- êB pathway induces the expression of the cytokines IL-6 and IL-10 in ABC DLBCL cell lines, which also have high levels of total and phosphorylated STAT3 protein, suggesting autocrine signaling. Using RNA interference for STAT3, we defined a gene expression signature of IL-6 and IL-10 signaling through STAT3. Based on this signature, we constructed a molecular predictor of STAT3 signaling that defined a subset of ABC DLBCL tumors with high expression of STAT3, IL-6 and/or IL-10, and their downstream targets. Although the STAT3-high and STAT3-low subsets had equivalent expression of genes that distinguish ABC DLBCL from GCB DLBCL, STAT3-high ABC DLBCLs had higher expression of signatures that reflected NF-kB activity, proliferation, and glycolysis. A smallmolecule inhibitor of JAK signaling, which blocked STAT3 signature expression, was toxic only for ABC DLBCL lines, and synergized with an inhibitor of NF-kB signaling. These findings suggest that the biological interplay between the STAT3 and NF-kB pathways may be exploited for the treatments of a subset of ABC DLBCLs. Activated B cell-like (ABC) subgroup of diffuse large B cell lymphoma (DLBCL) cell lines were used as model systems to study the cytokine pathways in these cells. We expressed inducible IkB super-repressor for 1 to 24 hours to identify NF-kB target genes in OCI-Ly3 and OCI-Ly10 cells for a total of 13 arrays with replicates of each time point. We treated OCI-Ly3 cells with IL-10 for 1 to 96 hours for a total of 10 arrays with replicates of each time point. We treated OCI-Ly10 cells with IL-6 for 30 minutes to 24 hours for a total of 8 arrays with replicates of each time point. OCI-Ly10 cells transfected with no siRNA versus STAT3-siRNA for 8, 24, or 48 hours for a total of 5 arrays with replicates for 24 and 48 hours. We treated OCI-Ly10 cells with JAK inhibitor I for 3 and 6 hours for a total of 4 arrays with replicates of each time point.
2010-05-27 | E-GEOD-10009 | biostudies-arrayexpress
Project description:A subtype of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like (ABC) DLBCL, depends on constitutive NF-kB pathway signaling for survival. Small molecule inhibitors of IkB kinase b (IKKb), a key regulator of the NF-kB pathway, kill ABC DLBCL cells and hold promise for the treatment of this lymphoma type. We conducted an RNA interference genetic screen to investigate potential mechanisms of resistance of ABC DLBCL cells to IKKb inhibitors. We screened a library of small hairpin RNAs (shRNAs) targeting 500 protein kinases for shRNAs that would kill an ABC DLBCL cell line in the presence of a small molecule IKKb inhibitor more effectively than in its absence. Two independent shRNAs targeting IKKa synergized with the IKKb inhibitor to kill three different ABC DLBCL cell lines but were not toxic by themselves. Surprisingly, IKKa shRNAs blocked the classical rather than the alternative NF-kB pathway in ABC DLBCL cells, as judged by inhibition of IkBa phosphorylation. IKKa shRNA toxicity was reversed by coexpression of wild type but not kinase inactive forms of IKKa, suggesting that IKKa may directly phosphorylate IkBa under conditions of IKKb inhibition. These results suggest that therapy for ABC DLBCL may be improved by targeting both IKKa and IKKb. Keywords: compound treatment design Gene expression profiling of OCI-Ly3 cells with or without expressing IKKa shRNA in the presence or absence of 12.5 uM IKKb inhibitor for 2 and 3 days. Four samples were analyzed.
2009-06-01 | E-GEOD-11902 | biostudies-arrayexpress
Project description:Biotechnology-derived therapeutics manufacturing is highly regulated to assure product quality, safety and efficacy. Process conditions are closely monitored as they can influence product characteristics. Culture of mammalian cells at different scales is a vital part of biomanufacturing. Currently, scale up of bioreactors is largely done based on engineering parameters such as oxygen transfer and mixing characteristics. There is a lack of genomic translational studies in mammalian cell culture scale-up that can help delineate measurable cellular attributes for improved process understanding. These could be used for quantifying physiological response of cells due to changes in bioreactor environment. In this study, we identified 18 process-sensitive sentinel genes using microarray statistical analysis, which we further verified by qRT-PCR. These were differentially expressed transcripts between a typical 5 liter bench-scale bubble-aerated and impeller-agitated bioreactor and novel 35 mL high-throughput minibioreactors. Using expression changes and biochemical-pathway guidance of these sentinel genes, we were able to tune engineering parameters such that the improved scale-down resulted in both process parameter and sentinel gene profile convergence between the two systems, further solidifying the functionality evidence of these sentinel genes. This study serves as a starting point for developing qRT-PCR assays as comparability metrics that could be performed near-at-line during the cell culture process itself leading to enhanced process control. Additional broad applications of sentinel genes could be to validate different manufacturing facilities for the same product, and allow better process definition by fingerprinting the manufacturing process for more rapid biogenerics and vaccine approvals. We ran consecutive bioreactor (5L and HTCB) runs, each with an independent vial thaw, to achieve multiple biological replicates per time-point. Bioreactors were sampled approximately every 12 hours for RNA extraction. For the 5L bioreactors, microarray samples were run for day 1 (n=2), day 2 (n=2), day 3 (n=3), and day 3.5 (n=3). Here 2 or 3 of the three biological replicates run for each time-point were included in the analysis, based on >70% genes found. For the High-Throughput Controlled Minibioreactors (HTCB), microarray samples were run for day 1 (n=3), day 2 (n=3), day 3 (n=3), day 4 (n=2), and day 4.5 (n=2). Here 2 to 4 of the 4 biological replicate runs were included in the analysis, based on >70% genes found. We define early exponential as day 1, peak exponential as day 2 and day 3 and late stationary as day 3.5.
2012-10-01 | E-GEOD-33063 | biostudies-arrayexpress
Project description:Microarray was used to analyze azole resistance of Candida glabrata oropharyngeal isolates from 7 hematopoietic stem cell transplant recipients receiving fluconazole prophylaxis. Transcriptional profiling of the sequential-paired clinical isolates by microarray revealed 19 genes upregulated in the majority of resistant isolates compared to their paired-susceptible isolates. All seven resistant isolates had greater than two fold upregulation of CgPDR1, a master transcriptional regulator of PDR network, and all 7 resistant isolates showed upregulation of known CgPDR1-target genes. The altered transcriptome can be explained in part by the observation that all 7 resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 ORF. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, S391L) and one in the activation domain (G943S) while two mutations (N764I, R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive and five resistant isolates into a laboratory azole-sensitive strain (cgpdr1) via integrative transformation. The cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic background, upregulation of CgPDR1 and CgPDR1-target genes varied between the 5 transformants, independent of the domain locations in which the mutations occurred. In sum, gain-of-function mutations in CgPDR1 not only contributed to the clinical azole resistance but different mutations had varying degrees of impact on the CgPDR1-target genes. Seven pairs of oropharyngeal sequential isolates were chosen for study because each pair came from an individual hematopoietic stem cell transplant recipient receiving fluconazole. Seven groups consisted of 5 biological replicates in which a sensitive sample was paired with a resistant sample. Each group included 1 to 2 reciprocally labeled samples.
2010-07-07 | E-GEOD-21352 | biostudies-arrayexpress
Project description:Clinical applications of human interferon (IFN)-alpha have met with varying degrees of success. Nevertheless, key molecules in IFN-alpha-induced cell death have not been clearly identified. Our previous study indicated that IFN (alpha, beta and omega) receptor (IFNAR) 1/2- and IFN regulatory factor (IRF) 9-RNA interference (RNAi) completely inhibited the antiproliferative (AP) activity of IFN-alpha in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-alpha., followed by transcription of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, IFNAR1/2- and IRF9-RNAi inhibited the gene expression of TRAIL, but not of Fas ligand (FasL), following IFN-alpha treatment. In fact, TRAIL but not FasL inhibited the proliferation of OVCAR3 cells. IFN-alpha notably up-regulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. Following TRAIL signaling, Caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly abrogated both AP activities of IFN-alpha and TRAIL. Furthermore, BID-RNAi prevented both IFN-alpha and TRAIL from collapsing the mitochondrial membrane potential (Delta Psi m). Finally, we provide important new evidence that BID overexpression led to a major enhancement of both AP activities of IFN-alpha and TRAIL in human lung carcinoma A549 cells resistant to IFN-alpha. Thus, this study suggests that BID is crucial in IFN-alpha-induced cell death, indicating a notable potential to be a targeted therapy for IFN-alpha resistant tumors. Biological replicate samples were created by treating OVCAR3 cells with IFN-alpha2c (n=8); IFNAR1-RNAi and IFN-alpha2c (n=4); IFNAR2-RNAi and IFN-alpha2c (n=5); ISGF3gamma-RNAi and IFN-alpha2c (n=3); and Negative RNAi and IFN-alpha2c (n=3). For analysis, the eight IFN-alpha2c treated OVCAR3 samples were paired with an untreated OVCAR3 control sample. The 15 RNAi treated OVCAR3 samples were paired with a Negative RNAi control sample.
2012-05-21 | E-GEOD-29790 | biostudies-arrayexpress