Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human myeloma cell lines treated with 500 nM pristimerin or DMSO vehicle alone for 4 hours during high-throughput chemical library screens for compounds that induce suppression of cyclin D2

ABSTRACT: As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2. The top-ranked compound was a natural triterpenoid, pristimerin. We used gene expression microarray studies to identify co-regulated pristimerin-response genes and to deduce the compoundâ??s direct molecular target(s), utilizing pattern-matching algorithms available at the Connectivity Map (Cmap). The early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor (ATF) 3 and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (<90min) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (<100nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-kB activation via inhibition of IKKa or IKKbï?¬ while proteosome inhibitors instead suppress NF-kB function by impairing degradation of ubiquitinated-IkB. By inhibiting both IKK and the proteosome pristimerin causes overt suppression of constitutive NF-kB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-kB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC50<100nM), inhibits xenografted plasmacytoma tumors in mice and is synergistically cytotoxic with bortezomib â?? providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-kB inhibitors as therapeutics for human multiple myeloma and related malignancies. Experiment Overall Design: H929 and U266 human myeloma cell lines were treated with pristimerin 500nM (~2x IC50) or DMSO vehicle and harvested at 4h, prior to the appearance of any macroscopic evidence of perturbed viability. RNA was isolated using Trizol, column purified (Qiagen, Valencia, CA) and hybridized to Hg_U133_plus_2 microarrays (Affymetrix, Santa Clara, CA) according to the manufacturerâ??s instructions. Probe-set signal intensities were PLIER16-normalized using Affymetrix Expression Console software and flagged using Affymetrix Microarray Suite 5 (MAS5) Present/Absent/Marginal detection calls. Expression data was analyzed with GeneSpring 7 (Agilent Technologies, Santa Clara CA) software, with drug-treated samples normalized per cell line to DMSO-treated controls. Gene lists were filtered to exclude non-expressed probe-sets with MAS5 â??Absentâ?? detection calls across all samples tested (treated and untreated). Probe-sets upregulated or suppressed by pristimerin in each cell line were delineated by volcano plot filter (>1.5 fold change, FDR<0.25) and the Venn intersection of probe-sets modulated by drug in both cell lines was used in deriving a pristimerin signature.

ORGANISM(S): Homo sapiens

SUBMITTER: rodger tiedemann

PROVIDER: E-GEOD-14011 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Abstract. Deregulated c-MYC is found in a variety of cancers where it promotes proliferation as well as apoptosis. In many hematological malignancies enhanced NF-kB exerts prosurvival functions. Here we investigated the role of NF-kB in mouse and human c-MYC-transformed lymphomas. The NF-kB-pathway is extinguished in murine lymphoma cells and extrinsic stimuli typically inducing NF-kB activity fail to activate this pathway. Genetic activation of the NF-kB pathway induces apoptosis in these cells, while inhibition of NF-kB by an IkBa superrepressor provides a selective advantage in vivo. Furthermore, in human BurkittÂ´s lymphoma cells we find that NF-kB activation induces apoptosis. NF-kB upregulates Fas and predisposes to Fas-induced cell death, which is caspase 8 mediated and can be prevented by CFLAR overexpression. We conclude that c-MYC overexpression sensitizes cells to NF-kB-induced apoptosis and persistent inacvtivity of NF-kB signaling is a prerequisite for MYC-mediated tumorigenesis. We could also show that low immunogenicity and Fas insensitivity of MYC driven lymphoma cells is reversed by activation of NF-kB. Our observations provide a molecular explanation for the described absence of the NF-kB signaling in BurkittÂ´s lymphoma and question the applicability of NF-kB inhibitors as candidates for treatment of this cancer. Experiment Overall Design: Ramos cells were transfected in triplicates with pRTS-GFP (A+,C+,D+) or pRTS-CA-IKK2 (E+,F+,H+), selected with hygromycin and transgene expression was induced with doxycycline (0,5mg/ml) for 48hrs. RNA was isolated with RNeasy mini kit (Qiagen, Venlo, Netherlands) and Gene expression profiling (GEP) was performed using Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA). 2 Âµg of total RNA were labeled using the GeneChipÂ® One-Cycle Target Labeling assay kit (Affymetrix). After hybridization arrays were stained and washed in a FS 450 Fluidics station (Affymetrix) before imaging on an Affymetrix GeneChip (3000) scanner. Raw data were generated using the GCOS 1.4 software (Affymetrix). Probe level data were obtained using the Robust Multichip Average (RMA) normalization algorithm and CEL files were loaded into Genesifter (GeneSifter.Net, VizX Laboratories, Seattle, WA, USA). Genes were identified as differentially expressed among the two classes if a two-sample T-test revealed a nominal significance level of 0.05 and the ratio between the two classes was at least 2 fold. Calculation of false discovery rate was done according to the method of Benjamini and Hochberg. Biological significance was determined using Gene Ontolgy reports.

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Transcription profiling of human myeloma cell lines treated with 500 nM pristimerin or DMSO vehicle alone for 4 hours during high-throughput chemical library screens for compounds that induce suppression of cyclin D2

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