Gene expression profile of normoxic versus hypoxic HeLa cells
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ABSTRACT: To identify new primary HIF target genes we cultured HeLa cells under short period (6 hours) of mild hypoxia (1% oxygen) Hypoxia-induced gene expression in HeLa cells was measured after 6h exposure to 1% oxygen and compare to normoxic
Project description:Sialic acids on vertebrate cell surfaces mediate many biological roles. Altered expression of certain sialic acid types or their linkages can have prognostic significance in human cancer. A classic but unexplained example is enhanced α2-6-sialylation on N-glycans, resulting from over-expression of the Golgi enzyme β-galactoside:α2-6-sialyltransferase (ST6Gal-I). Previous data supporting a role for the resulting Siaα2-3Galβ1-4GlcNAc (Sia6LacNAc) structure in tumor biology were based on in vitro studies in transfected carcinoma cells, in which increased Sia6LacNAc on β1-integrins enhanced their binding to ligands, and stimulated cell motility. Here we examine for the first time the in vivo role of the ST6Gal-I enzyme in the growth and differentiation of spontaneous mammary cancers in mice transgenic for an MMTV-promoter-driven polyoma-middle-T antigen, a tumor in which beta1-integrin function is important for tumorigenesis, and in maintaining the proliferative state of tumor cells. Tumors induced in St6gal1 null animals were more differentiated in comparison to those in the wild-type background, both by histological analysis and by protein expression profiles. Furthermore, we show the St6gal1 null tumors have selectively altered expression of genes associated with focal adhesion signaling, and have decreased phosphorylation of FAK, a downstream target of β1-integrins. This first in vivo evidence for a role of ST6Gal-I in tumor progression was confirmed using a novel approach, which conditionally restored St6gal1 in cell lines derived from the null tumors. These findings indicate a role for ST6Gal-I as a mediator of tumor progression, with its expression causing a less differentiated phenotype, via enhanced β1-integrin function. Experiment Overall Design: Messenger RNA from tumors arising in wild-type or St6gal1 null littermates was used for the gene expression analysis on the Mouse Genome 430 2.0 Array (Affymetrix). Six PyV-mT St6gal1 null and wild-type tumor pairs were used, in four independent pools of three tumors each. Each sample is a pool of three different tumors from three different animals. For the first set of microarray analysis samples GSM239969 and GSM239970 were being compared, and in the second set, samples GSM239971 and GSM239972 were compared.
Project description:The purpose of experiment is to compare the mRNA expression differences between HeLa cells conditioned with normoxia 21% oxygen and hypoxia 1% oxygen at 6h and 20h.
Project description:We used HeLa cells that overexpress GFP-tagged SRSF6 (4-fold) were grown in normal conditions (Normoxia, 21% oxygen) or hypoxic conditions (Hypoxia, 0.2% oxygen). compared the binding pattern of SRSF6 between normoxia, 4h hypoxia and 24h hypoxia.
Project description:The purpose of experiment is to compare the mRNA expression differences between HeLa cells conditioned with normoxia 21% oxygen and hypoxia 1% oxygen at 6h and 20h. Total RNA was obtained from HeLa cells for 4 different conditions, 3 biological replicate experiments were performed, resulting in a total of 12 samples.
Project description:We used HeLa cells that express GFP-tagged SRSF6 at physiological levels and subjected them to 4 h or 24 h hypoxia (0.2% oxygen). Cells grown in normal conditions were used as control (Normoxia, 21% oxygen). We performed iCLIP using anti-GFP antibodies and compared the binding pattern of SRSF6 between normoxia, 4h hypoxia and 24h hypoxia.
Project description:Histone deacetylases (HDACs) have been identified as therapeutic targets due to regulatory function in DNA structure and organization. We have analyzed the role of the LBH589, a novel pan inhibitor of class I and II HDACs, in Acute Lymphoblastic Leukemia. In vitro, LBH589 was shown to induce a dose dependent antiproliferative and apoptotic effect which was associated with an increase in the acetylation of H3 and H4 histone acetylation which was uniformly in every genetic subgroup of ALL. In vivo administration of LBH589 in BALB/c-RAG2-/-γc-/- mice in which T and B-cell leukemic cell lines were injected induced a significant reduction in tumor growth (TOM-1, p<0.01 and MOLT-4 p<0.05). Leukemic cells from patients were employed to establish a xenograft model of human leukemia in BALB/c-RAG2-/-γc-/- mice and further transplanted in consecutive generations of mice. Treatment of these xenografts with LBH589 induced an increase in the acetylation of H3 and H4 and prolonged the survival of mice in comparison with the animals treated with Vincristine and Dexametasone (p<0.05) and this effect was significantly higher when LBH589 was combined with Vincristine and Dexametasone (p<0.001). Our results that the use of LBH589 in combination with standard chemotherapy represents an attractive option for treatment of patients with ALL. Two primary samples of ALL (one ALL-B and one ALL-T) and two samples of each leukemia after passages in immunodeficient mice.
Project description:Insulin and IGF-1 promote adipocyte differentiation via complex and overlapping signalling networks. Here we used microarray analysis of brown preadipocytes derived from wild-type and insulin receptor substrate (IRS) knockout (KO) animals, which exhibited progressively impaired differentiation, to define the set of genes that predict adipogenic potential in these cells. 374 genes/ESTs were identified whose expression in preadipocytes correlated with their ultimate ability to differentiate. Many of these genes were related to early adipogenic events, including genes involved in extracellular matrix, cytoskeletal organization, growth arrest, post-mitotic clonal expansion, and inhibitors of adipogenesis, including preadipocyte factor-1 and multiple members of the Wnt-signalling pathway. Reconstitution of IRS-1 KO cells with IRS-1 reversed these changes and restored the ability to differentiate. Several of these genes showed concordant changes in brown adipose tissue in vivo. Necdin was markedly increased in IRS-1 KO cells that could not differentiate, and knockdown of necdin restored brown adipogenesis with down-regulation of Pref-1 and Wnt10a expression. We demonstrated a necdin-E2F4 interaction repressing PPARg transcription. IRS proteins regulated necdin via a CREB dependent pathway, defining a signalling network involved in brown preadipocyte determination.
Project description:Oxygen is vital for the development and survival of mammals. In response to hypoxia, the brain initiates numerous adaptive responses at the organ level as well as at the molecular and cellular levels, including the alteration of gene expression. Astrocytes play critical roles in the proper functioning of the brain; thus the manner in which astrocytes respond to hypoxia is likely important in determining the outcome of brain hypoxia. Here, we used microarray gene expression profiling and data-analysis algorithms to identify and analyze hypoxia-responsive genes in primary human astrocytes. We also compared gene expression patterns in astrocytes with those in human HeLa cells and pulmonary artery endothelial cells (ECs). Remarkably, in astrocytes, five times as many genes were induced as suppressed, whereas in HeLa and pulmonary ECs, as many as or more genes were suppressed than induced. More genes encoding hypoxia-inducible functions, such as glycolytic enzymes and angiogenic growth factors, were strongly induced in astrocytes compared with HeLa cells. Furthermore, gene ontology and computational algorithms revealed that many target genes of the EGF and insulin signaling pathways and the transcriptional regulators Myc, Jun, and p53 were selectively altered by hypoxia in astrocytes. Indeed, Western blot analysis confirmed that two major signal transducers mediating insulin and EGF action, Akt and MEK1/2, were activated by hypoxia in astrocytes. These results provide a global view of the signaling and regulatory network mediating oxygen regulation in human astrocytes.. This SuperSeries is composed of the following subset Series:; GSE3045: Astrocytic response to hypoxia; GSE3051: HeLa response to hypoxia Experiment
Project description:Histone deacetylases (HDACs) have been identified as therapeutic targets due to regulatory function in DNA structure and organization. We have analyzed the role of the LBH589, a novel pan inhibitor of class I and II HDACs, in Acute Lymphoblastic Leukemia. In vitro, LBH589 was shown to induce a dose dependent antiproliferative and apoptotic effect which was associated with an increase in the acetylation of H3 and H4 histone acetylation which was uniformly in every genetic subgroup of ALL. In vivo administration of LBH589 in BALB/c-RAG2-/-gammac-/- mice in which T and B-cell leukemic cell lines were injected induced a significant reduction in tumor growth (TOM-1, p<0.01 and MOLT-4 p<0.05). Leukemic cells from patients were employed to establish a xenograft model of human leukemia in BALB/c-RAG2-/-gammac-/- mice and further transplanted in consecutive generations of mice. Treatment of these xenografts with LBH589 induced an increase in the acetylation of H3 and H4 and prolonged the survival of mice in comparison with the animals treated with Vincristine and Dexametasone (p<0.05) and this effect was significantly higher when LBH589 was combined with Vincristine and Dexametasone (p<0.001). Our results that the use of LBH589 in combination with standard chemotherapy represents an attractive option for treatment of patients with ALL. Three different biological replicates of ALL derive cell lines TOM-1 and MOLT-4 after and before treatment with LBH589