Browsing by Subject "hypoxia"
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Item Classic and targeted anti-leukaemic agents interfere with the cholesterol biogenesis metagene in acute myeloid leukaemia: Therapeutic implications(Wiley, 2020-05-25) Chen, Fangli; Wu, Xue; Niculite, Cristina; Gilca, Marilena; Petrusca, Daniela; Rogozea, Adriana; Rice, Susan; Guo, Bin; Griffin, Shawn; Calin, George A.; Boswell, H. Scott; Konig, Heiko; Medicine, School of MedicineDespite significant advances in deciphering the molecular landscape of acute myeloid leukaemia (AML), therapeutic outcomes of this haematological malignancy have only modestly improved over the past decades. Drug resistance and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. While low O2 compartments, such as bone marrow (BM) niches, are well‐recognized hosts of drug‐resistant leukaemic cells, standard in vitro studies are routinely performed under supra‐physiologic (21% O2, ambient air) conditions, which limits clinical translatability. We hereby identify molecular pathways enriched in AML cells that survive acute challenges with classic or targeted therapeutic agents. Experiments took into account variations in O2 tension encountered by leukaemic cells in clinical settings. Integrated RNA and protein profiles revealed that lipid biosynthesis, and particularly the cholesterol biogenesis branch, is a particularly therapy‐induced vulnerability in AML cells under low O2 states. We also demonstrate that the impact of the cytotoxic agent cytarabine is selectively enhanced by a high‐potency statin. The cholesterol biosynthesis programme is amenable to additional translational opportunities within the expanding AML therapeutic landscape. Our findings support the further investigation of higher‐potency statin (eg rosuvastatin)–based combination therapies to enhance targeting residual AML cells that reside in low O2 environments.Item Effects of choline kinase activity on phospholipid metabolism and malignant phenotype of prostate cancer cells(2010-10) Bansal, Aditya; DeGrado, Timothy R.; Harris, Robert A. (Robert Allison), 1939-; Bosron, William F.; Klaunig, James E.High choline uptake and increased choline kinase activity have been reported in many cancers. This has motivated the use of choline as a biomarker for tumor imaging. Tumors in general are heterogeneous in nature with respect to oxygen tension. There are regions of hypoxia and normoxia that are expected to have different metabolism but regulation of choline metabolism under hypoxia is poorly understood. It is important to clarify the status of choline metabolism in hypoxic microenvironment as it will have an impact on potential of choline as a cancer biomarker. The primary goal was to determine the status of choline phosphorylation in hypoxic cancer cells and its effect on uptake of choline. This was examined by tracer studies in cancer cells exposed to hypoxia. It was observed that hypoxia universally inhibits choline uptake /phosphorylation in cancer cells. Decreased choline phosphorylation resulted in transient uptake of choline radiotracers in cultured cancer cells and 9L tumors suggesting potential problem in using choline as a biomarker for cancers in hypoxic microenvironment. To investigate the mechanism behind decrease in choline phosphorylation, steady state levels of choline metabolites were measured and choline kinase catalyzed choline phosphorylation step was found to be rate-limiting in PC-3 cells. This suggested that modulation in choline kinase levels can alter choline metabolism in hypoxic cancer cells. Expression and activity assays for choline kinase revealed that choline kinase expression is down-regulated in hypoxia. This regulation involved transcriptional level mediation by HIF1 at the conserved HRE7 site in choline kinase promoter. To further understand the importance of down-regulation of choline kinase in hypoxia, stable prostate cancer cell lines over-expressing choline kinase were generated. Effect of over-expression of choline kinase in hypoxia was evaluated in terms of malignant phenotypes like proliferation rate, anchorage independent growth and invasion potential. Both over-expression of choline kinase and hypoxia had a pronounced effect on malignant phenotypes of prostate cancer cells. Further study showed that increased choline kinase activity and hypoxic tumor microenvironment are important for progression of early-stage, androgen-dependent LNCaP prostate cancer cells but confer little survival advantage in undifferentiated, androgen-independent PC-3 prostate cancer cells.Item Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture(Elsevier, 2017-04) Dawes, Camron S.; Konig, Heiko; Lin, Chien-Chi; Department of Biomedical Engineering, School of Engineering and TechnologyHypoxia is a critical condition governing many aspects of cellular fate processes. The most common practice in hypoxic cell culture is to maintain cells in an incubator with controlled gas inlet (i.e., hypoxic chamber). Here, we describe the design and characterization of enzyme-immobilized hydrogels to create solution hypoxia under ambient conditions for in vitro cancer cell culture. Specifically, glucose oxidase (GOX) was acrylated and co-polymerized with poly(ethylene glycol)-diacrylate (PEGDA) through photopolymerization to form GOX-immobilized PEG-based hydrogels. We first evaluated the effect of soluble GOX on inducing solution hypoxia (O2 < 5%) and found that both unmodified and acrylated GOX could sustain hypoxia for at least 24 h even under ambient air condition with constant oxygen diffusion from the air-liquid interface. However, soluble GOX gradually lost its ability to sustain hypoxia after 24 h due to the loss of enzyme activity over time. On the other hand, GOX-immobilized hydrogels were able to create hypoxia within the hydrogel for at least 120 h, potentially due to enhanced protein stabilization by enzyme ‘PEGylation’ and immobilization. As a proof-of-concept, this GOX-immobilized hydrogel system was used to create hypoxia for in vitro culture of Molm14 (acute myeloid leukemia (AML) cell line) and Huh7 (hepatocellular carcinoma (HCC) cell line). Cells cultured in the presence of GOX-immobilized hydrogels remained viable for at least 24 h. The expression of hypoxia associated genes, including carbonic anhydrase 9 (CA9) and lysyl oxidase (LOX), were significantly upregulated in cells cultured with GOX-immobilized hydrogels. These results have demonstrated the potential of using enzyme-immobilized hydrogels to create hypoxic environment for in vitro cancer cell culture.Item Generation and Exploration of a Novel Low Oxygen Landscape for Hematopoietic Stem and Progenitor Cells(2022-10) Dausinas, Paige Burke; Elmendorf, Jeffrey; O'Leary, Heather; Bidwell, Joseph; Wan, Jun; Zhang, JiHematopoietic stem (HSC) and progenitor (HSPC) cells reside in low oxygen (~1- 4%, low O2) bone marrow niches which provide critical signals for maintenance, selfrenewal, and differentiation. Exposure of HSC/HSPCs to air (~21%) for less than 10 minutes irreversibly diminishes numbers of phenotypic and functional stem cells, a phenomenon termed extra physiologic oxygen stress/shock. Yet, most studies harvest and analyze HSC/HSPCs in air and often in fixed cells, leaving endogenous signaling mechanisms unidentified. To better understand the endogenous mechanisms regulating HSCs and HSPCs, we generated the first low O2 landscape of phenotypic/functional/signaling alterations in live, low O2 harvested/sorted HSC/HSPCs utilizing novel technology. HSC (LSKCD150+) and HSC/HSPC (LSK) expression, frequency, and stem cell maintenance retention were enhanced in low O2 relative to historic data and our air data. Transcriptomics uncovered low O2 differential pathway regulation of HSC/HSPCs and HSCs with analysis identifying low O2 enrichment of genes/pathways including Ca2+ ion binding, altered sodium hydrogen (Na+/H+) activity, viral entry, and transmembrane receptor activity in both HSCs and HSPCs. In exploring the low O2 landscape, we investigated differential low O2 regulation of Ca2+ and SARS-CoV-2 related pathways/mechanisms in HSCs and HSPCs. Differential Ca2+ regulation was observed in our transcriptional/proteomic analysis corroborated by phenotypic/functional data demonstrating increases in low O2 of cytosolic and mitochondrial Ca2+ flux, ABC Transporter (ABCG2) and Na+/H+ (NHE1) expression, discovery of a novel low O2 Ca2+ high HSPC population that enhances HSC maintenance compared to Ca2+ low populations and blunting of this population and subsequent enhanced stem cell maintenance upon NHE1 inhibition (Cariporide). Multi-omics analyses also identified enhancements in COVID19-related pathways in low O2 that corresponded with enhanced expression of SARS-CoV-2 receptors/co-receptors, SARS-CoV-2 spike protein (SP) binding, and expansion of SP-bound HSC/HSPCs in low O2 compared to air, as well as enhanced stem cell maintenance of SP-bound, versus unbound, cells in low O2. Together, these data presented show low O2 harvest/retention of HSC/HSPCs enhances stem cell maintenance, which could be utilized to improve HSC expansion, and leads to differential pathway/signaling regulation of various biological pathways in HSC/HSPCs including Ca2+ and SARS-CoV-2/viral infection that results in phenotypic and functional consequences.Item Hypoxia-Inducible Factor-1α Regulates CD55 in Airway Epithelium(American Thoracic Society, 2016-12) Pandya, Pankita H.; Fisher, Amanda J.; Mickler, Elizabeth A.; Temm, Constance J.; Lipking, Kelsey P.; Gracon, Adam; Rothhaar, Katia; Sandusky, George E.; Murray, Mary; Pollok, Karen; Renbarger, Jamie; Blum, Janice S.; Lahm, Tim; Wilkes, David S.; Microbiology and Immunology, School of MedicineAirway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium.Item Progenitor cell combination normalizes retinal vascular development in the oxygen-induced retinopathy (OIR) model(American Society for Clinical Investigation, 2019-11-01) Calzi, Sergio Li; Shaw, Lynn C.; Moldovan, Leni; Shelley, William C.; Qi, Xiaoping; Racette, Lyne; Quigley, Judith L.; Fortmann, Seth D.; Boulton, Michael E.; Yoder, Mervin C.; Grant, Maria B.; Pediatrics, School of MedicineRetinopathy of prematurity (ROP) is a disorder of the developing retina of preterm infants. ROP can lead to blindness because of abnormal angiogenesis that is the result of suspended vascular development and vaso-obliteration leading to severe retinal stress and hypoxia. We tested the hypothesis that the use of the human progenitor cell combination, bone marrow–derived CD34+ cells and vascular wall–derived endothelial colony–forming cells (ECFCs), would synergistically protect the developing retinal vasculature in a mouse model of ROP, called oxygen-induced retinopathy (OIR). CD34+ cells alone, ECFCs alone, or the combination thereof were injected intravitreally at either P5 or P12 and pups were euthanized at P17. Retinas from OIR mice injected with ECFCs or the combined treatment revealed formation of the deep vascular plexus (DVP) while still in hyperoxia, with normal-appearing connections between the superficial vascular plexus (SVP) and the DVP. In addition, the combination of cells completely prevented aberrant retinal neovascularization and was more effective anatomically and functionally at rescuing the ischemia phenotype than either cell type alone. We show that the beneficial effects of the cell combination are the result of their ability to orchestrate an acceleration of vascular development and more rapid ensheathment of pericytes on the developing vessels. Lastly, our proteomic and transcriptomic data sets reveal pathways altered by the dual cell therapy, including many involved in neuroretinal maintenance, and principal component analysis (PCA) showed that cell therapy restored OIR retinas to a state that was closely associated with age-matched normal retinas. Together, these data herein support the use of dual cell therapy as a promising preventive treatment for the development of ROP in premature infants.Item QUANTITATIVE IMMUNOHISTOCHEMISTRY USING THE APERIO WHOLE SLIDE IMAGING SYSTEM EVALUATING ANGIOGENESIS AND HYPOXIA MARKERS IN PANCREATIC CARCINOMA MOUSE MODEL TREATED WITH VEHICLE CONTROL, E3330, AND A STAT 3 INHIBITOR(Office of the Vice Chancellor for Research, 2012-04-13) Spencer, Cleandrea; Sandusky, George; Fishel, Melissa; Kelley, Mark R.Investigation of the signaling pathways and molecular mechanisms that are major contributors to pancreatic tumor progression and its resistance to traditional therapies is lacking. Human apurinic endonuclease/redox factor 1 (APE/Ref-1) mediates repair of radiation-induced DNA lesions and regulates transcription via redox-based activation. Transcriptional factors HIF-1α, NFκB, and AP-1 are regulated by Ref-1 and are implicated in pancreatic tu-mor growth and the response to hypoxia. CD31 and CA IX (carbonic anhy-drase) were biomarkers used in an in vivo study to evaluate the effective-ness of E3330, an APE 1 inhibitor, in a pancreatic mouse model. Immunostained slides were scanned using the Aperio automated whole slide scanning system (Scanscope CS) and were viewed using ImageScopeTM. Single fields of view from each WSDI measuring ∼10,000,000 μm2 and rep-resenting the whole area of the tumor were selected for analysis using the Aperio positive pixel algorithm. The preclinical xenograft model evaluated human pancreatic carcinoma cell lines grown in NOD/SCID mice treated with the E3330 compound, a STAT 3 inhibitor, and an untreated vehicle control group. Immunohisto-chemistry (IHC) was used to predict effectiveness of treatment for pancreat-ic carcinoma based on CD31 and CA IX biomarker expression. IHC slides were quantified using both a traditional pathology hand count and the Aperio Imaging Analysis System. The positive pixel algorithm data closely mirrored the hand count for two biomarkers (CD31 and CA IX). In the E3330 treated group, the data showed CD31 (angiogenesis) was significantly knocked down with increased CA IX expression compared to the vehicle control. Hypoxia of the tumor cells was up in both treated groups. In summary, the Aperio im-aging analysis system matched the hand count pathology data. The drug ef-fects with E3330 exhibited both anti-angiogenesis and tumor hypoxia activi-ty in the tumors. This project was supported by the Center for Research and Learning’s Diversity Scholars Re-search Program.Item Regulation of HIF1 under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual-Targeting in Patient-Derived 3D Pancreatic Cancer Models(Office of the Vice Chancellor for Research, 2016-04-08) Logsdon, Derek P.; Grimard, Michelle; Shahda, Safi; Zyromski, Nicholas; Schipani, Ernestina; Carta, Fabrizio; Supuran, Claudiu T.; Korc, Murray; Ivan, Mircea; Kelley, Mark R.; Fishel, Melissa L.Abstract Half of all patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) die within a year despite extensive surgery and/or a highly aggressive chemotherapy regimen. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including reactive stroma and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased metastatic potential and impaired therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular hypoxia response to regulate intracellular pH, promoting cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein with two major activities: an endonuclease function in DNA base excision repair and a redox signaling function that reduces oxidized transcription factors, enabling them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in survival mechanisms, tumor growth, and hypoxia signaling. We explored the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1), a critical factor in hypoxiainduced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition will result in enhanced PDAC cell killing under hypoxic conditions. Methods: We performed qRT-PCR and Western Blots to confirm changes in CA9 expression in PDAC cells following APE1/Ref-1 inhibition and hypoxia exposure. Proliferation assays were used to assess cell killing following inhibition of APE1/Ref-1 and CA9 under hypoxia. Ex vivo 3-Dimensional co-culture models including both tumor and CAFs were used to examine whether we could enhance the efficacy of APE1/Ref-1 and/or CA9 inhibition with a dual-targeting approach to kill tumor spheroids. Results: HIF1-mediated induction of CA9 is significantly diminished in PDAC cells following APE1/Ref-1 redox inhibition. Additionally, dual-targeting of APE1/Ref-1 and CA9 reduces PDAC tumor cell growth under hypoxic conditions and in 3D tumor co-cultures.