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Theses and Dissertations

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Browsing Theses and Dissertations by Author "Anthony, Russ, committee member"

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    Oncofetal proteins regulate proliferation and differentiation in placental cells
    (Colorado State University. Libraries, 2018) West, Rachel Claire, author; Winger, Quinton, advisor; Bouma, Gerrit, advisor; Anthony, Russ, committee member; Hamilton, Karyn, committee member
    The chromatin associated transcription factor HMGA2 is a downstream target of let-7 miRNAs and binds to chromatin to regulate gene expression, inducing rapid cell proliferation during embryogenesis. Inhibition of let-7 miRNAs by RNA binding proteins LIN28A and LIN28B is necessary during early embryogenesis to ensure stable expression of HMGA2 and proper cell proliferation. In addition to LIN28, HMGA2 is regulated by a BRCA1/ZNF350/CtIP repressor complex. In normal tissues, the BRCA1/ZNF350/CtIP complex binds to the HMGA2 promoter to prevent transcription. However, in many cancers the oncomiR miR-182 targets BRCA1, preventing BRCA1 translation and allowing for increased HMGA2. Little is known about the regulation of HMGA2 during early placental development therefore we hypothesized that both LIN28 and BRCA1 can regulate HMGA2 in placental cells. Using siRNA and CRISPR gene editing techniques, we found that knockdowns of both LIN28A and LIN28B increase HMGA2 levels in ACH-3P cells. These cells also demonstrated deficiencies in cell differentiation towards the syncytiotrophoblast, secreting higher amounts of hCG and displaying upregulated ERVW-1. Additionally, we found that a knockout of both LIN28A and LIN28B caused a significant increase of miR-182 and a decrease in BRCA1 which allows HMGA2 mRNA levels to increase and protein levels to remain the same. Using chromatin immunoprecipitation, we saw binding of the BRCA1 repressor complex to HMGA2. We also saw a decrease in binding to HMGA2's promoter in the LIN28A/B knockout cells. These findings suggest a novel role for BRCA1 during early human placental development. To test this hypothesis, we used CRISPR-Cas9 gene editing to knockout BRCA1 in the Swan71 cell line as the Swan71 cells had significantly higher BRCA1 levels compared to ACH-3P cells. HMGA2 mRNA and protein was significantly increased in the BRCA1 KO cells compared to control cells. Chromatin immunoprecipitation was used with an antibody for ZNF350 and PCR was run using primers for the promoter region for HMGA2. We saw a loss of BRCA1 repressor complex binding to HMGA2 in the knockout cells compared to our control cells, leading us to conclude that increased HMGA2 was due to decreased binding of the BRCA1 repressor complex. Additionally, we tested levels of apoptosis in our cells. After serum starving cells for 16 hours, we found that Caspase 3 and 7 levels were significantly higher in our BRCA1 KO cells compared to controls. This data suggests that BRCA1 is an important factor in the regulation of the oncofetal protein HMGA2 and promotes cell survival in human placental cells.
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    Translocation of insulin receptors into plasma membrane microdomains in response to insulin and to insulin-enhancing vanadium and chromium compounds
    (Colorado State University. Libraries, 2010) Al-Qatati, Abeer S. A., author; Roess, Deborah, advisor; Crans, Debbie, committee member; Graham, James, committee member; Anthony, Russ, committee member
    We have examined the translocation of insulin receptors into specialized, cholesterol-enriched membrane microdomains called lipid rafts following treatment of RBL-2H3 cells with insulin, bis-maltolatooxovanadium (BMOV) and tris(pyridinecarbxylato) chromium(III) (Cr(pic)3). Isopycnic sucrose gradient ultracentrifugation was used to subfractionate membrane fragments and insulin receptors were identified within low or high buoyant density membrane fractions using insulin receptor-specific antibodies and western blotting. Single particle tracking methods were used to confirm the confinement of individual insulin receptors within small membrane compartments on intact, viable RBL-2H3 cells. We demonstrated that insulin receptors translocate into lipid rafts upon binding insulin or following exposure to BMOV or Cr(pic)3 Phosphorylated insulin receptors also appeared in membrane raft fragments in response to insulin and/or insulin-mimicking compounds. Extraction of cholesterol from lipid rafts disrupted these microdomains and caused a decrease in the number of unphosphorylated and phosphorylated insulin receptors within these compartments. In addition to their ability to induce translocation of insulin receptors into lipid rafts, BMOV and Cr(pic)3 caused an increase in the number of phosphorylated IRS-1 molecules within these membrane fragments. To determine why Cr(pic)3 and BMOV might affect the distribution of insulin receptors in non-raft and raft compartments, membrane fluidity was evaluated in Cr(pic)3 and BMOV treated cells. Fluidity, as suggested by a decrease in lipid packing, was increased following treating 2H3 cells with either BMOV or Cr(pic)3 These results suggest that changes in lipid packing resulting from exposure of cells to either Cr(pic)3 and BMOV may affect the distribution of receptors in non-raft and raft compartments. Increased receptor localization in rafts or small membrane compartments evaluated by single particle tracking studies, would result in increased likelihood of insulin receptor phosphorylation within these signaling platforms. Thus rafts may be an important membrane structures involved in cell signaling events mediated by insulin receptors.