Browsing by Subject "Cell Proliferation"
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Item Defining the Developmental Signals of the Cardiac Fibroblast(2012-08-31) Baek, Seung Tae; Tallquist, MichelleCardiac fibroblasts play a central role as a mediator of inflammatory and fibrotic response and also secrete extracellular matrix components that provide structural support for regeneration and remodeling of the wound. Despite the importance of the cardiac fibroblast in heart disease, very little is known about factors that are essential for differentiation along the cardiac fibroblast lineage. Using a combination of gene knockout and cardiac fibroblast-detecting methods, we have identified genes that are involved in the formation of cardiac fibroblasts. Our results demonstrate that in the absence of Tcf21, a basic helix-loop-helix transcription factor, cardiac fibroblast progenitors fail to migrate into the myocardium resulting in a specific loss of the cardiac fibroblast population. Loss of the receptor tyrosine kinase Pdgfr? also results in loss of the cardiac fibroblast population. Interestingly, Tcf21 and Pdgfra are involved in the epithelial to mesenchymal transition (EMT) of epicardial cells. The epicardium (outer surface of the heart) functions as a pool of progenitor cells for the coronary vasculature and interstitial connective tissue during embryonic development. Although several signaling pathways have been identified that disrupt EMT, no component has been reported that negatively regulates EMT, which may also involved in the cardiac fibroblast development. Using a conditional knockout of neurofibromin 1 (Nf1) in the epicardium, we identified Nf1 as a key mediator of epicardial EMT. We found that the process of EMT occurred earlier in Nf1 mutant hearts, with an increase in epicardial cells entering the compact myocardium. Moreover, loss of Nf1 caused increased epicardial-derived cell proliferation and resulted in the expansion of cardiac fibroblasts and coronary vascular smooth muscle cells. In addition to revealing the function of Nf1, Tcf21 and Pdgfra in epicardial EMT and cardiac fibroblast development, we generated and established mouse models to study the role of cardiac fibroblasts and the function of these genes during heart pathogenesis. Because developmental processes are often recapitulated in normal and pathological conditions, a better understanding of the epicardium and cardiac fibroblast development may help identify targets for therapeutics to treat heart disease.Item The Effects of Imatinib Mesylate on Antigen-Specific Cd8+ T Cell Responses(2006-12-20) Sinai, Parisa; Forman, JamesImatinib mesylate (IM) is a chemical compound designed to inhibit the constitutive tyrosine kinase activity of the Bcr-Abl oncogene in Chronic Myelogenous Leukemia (CML). While IM is very potent in treating CML, little is understood concerning the effects of IM on the immune response. In this study, I have examined the influence of IM on antigen specific CD8 T cells. Mature OT-1 TCR transgenic T cells were transferred into B6.Thy1.1 recipients. This transfer was followed by an infection with Listeria monocytogenes (LM) expressing the cognate epitope OVA 257-264 (LM-OVA) to assess whether IM affects the specific CD8 T cell response generated to this intracellular pathogen. In vitro studies revealed that IM had no effect on proliferation, apoptosis, or IFN-gamma secretion of na?or memory OT-1 T cells at doses of =5muM, although at higher doses inhibition was observed. Adoptive transfer and in vivo infection studies demonstrated that in the presence of IM the primary response of OT-1 T cells in vivo to LM-OVA infection was unaltered as measured by OT-1 T cell percentages in spleen and blood, their expression of IFN-gamma and their proliferation. However, IM influenced the primary OT-1 response by decreasing the expression of the IL-7Ralpha memory marker on OT-1 cells. IM treatment for >28 days resulted in a decreased percentage of OT-1 memory T cells before recall in blood. Furthermore, the response of memory OT-1 cells after LM-OVA rechallenge was diminished as measured by OT-1 T cell percentages in blood. In addition, IM treatment reduced the expression of IL-7Ralpha , a receptor required for memory cell survival, on effector and memory OT-1 cells. The function of memory OT-1 cells as measured by IFN-gamma expression was unaltered as was their proliferation as measured by incorporation of Brdu. Infection assays revealed that clearance of LM-OVA by memory mice is not altered by IM. In addition, the in vivo proliferation of memory OT-1 cells was not altered by IM. While IM did not alter the percentage of CD4 T cells in spleen and blood, IFN-gamma expression by CD4 T cells after recall was decreased. Together, these data demonstrate that IM reduces the number of OT-1 cells following a secondary challenge to an LM-OVA infection, and this may be a result of decreased IL-7Ralpha expression on effector and memory OT-1 cells.Item The Endoplasmic Reticulum Udpase ENTPD5 Promotes Cancer Cell Growth and Survival in the PI3K/PTEN(2011-02-01T19:32:25Z) Shen, Zhirong; Wang, XiaodongPI3 Kinase and PTEN lipid phosphatase control the level of cellular phosphatidylinositol (3,4,5)-trisphosphate, an activator of AKT kinase that promotes cell growth and survival. Mutations activating AKT are commonly observed in human cancers. Activation of AKT and downstream PI3 Kinase signaling promotes protein translation, resulting in increased protein flux into ER; this will lead to decreased efficiency of protein folding and accumulation of unfolded proteins in the ER and finally lead to ER stress. How does cancer cell solve this problem of increased folding during rapid growth to avoid ER stress? We discovered that ENTPD5, an endoplasmic reticulum (ER) enzyme, is up-regulated in cell lines and primary human tumor samples with active AKT. AKT upregulates ENTPD5 by relieving transcriptional inhibition by FoxO transcription factors. ENTPD5 hydrolyzes UDP to UMP to promote protein N-glycosylation and folding in ER. Knockdown of ENTPD5 in PTEN-null cells causes ER stress and loss of receptor tyrosine kinases through ER-associated degradation pathway under stress conditions. Consequently, the growth of PTEN-null cells is inhibited both in vitro and in mouse xenograft tumor models. ENTPD5 is therefore an essential component for PI3K/AKT active cancer cells and a potential drug target for anti-cancer therapy.Item Examining the Role of Regulatory Lymphocytes in a Mouse Model of BCL1 Tumor Dormancy(2012-07-09) BitMansour, Andrew; Vitetta, Ellen S.Cancer dormancy is a clinical state where residual tumor cells persist for long periods but do not cause detectable disease. However, tumor regrowth can occur and is accompanied by resistance to treatment and high mortality rates. The mechanisms that mediate tumor dormancy have been studied using the mouse B cell lymphoma (BCL1) model of tumor dormancy. However, the events that lead to cancer relapse are not known since the tumor microenvironment consists of many cell types that either facilitate or prevent tumor progression or have a dual role depending on the disease stage. Regulatory T (Treg) cells play a key role in maintaining systemic immune tolerance and have been described to promote cancer progression. The objective of this study was to determine the role of Treg cells in preventing BCL1 tumor dormancy by suppressing the anti-tumor immune responses. Surprisingly, we found that the total numbers of Treg cell were highest in mice bearing dormant tumor cells, whereas mice with the highest BCL1 tumor burden had the lowest number of Treg cells in their tumor microenvironment. Moreover, we compared the functional differences between Treg cells isolated from mice bearing dormant tumors and those bearing non-dormant tumors. We found that they equally suppressed T cell subsets from their respective tumor microenvironments. Treg cells have also been shown to suppress B cells. Since the BCL1 tumor cells are malignant B cells, we examined the effects of Treg cells on the tumor cells themselves. We found that Treg cells did not suppress the proliferation nor inhibit IgM secretion by the tumor cells. Interestingly, we then found that the BCL1 tumor cells shared features with regulatory B (Breg) cells. Like Treg cells, Breg cells also induce immune tolerance by suppressing effector T cells. The BCL1 tumor cells homogeneously expressed the characteristic phenotype (CD1dhiCD5+) and cytokine profile (secretion of high levels of IL-10) of the B10 subset of Breg cells. Moreover, the tumor cells directly induced T cell apoptosis through a cell-contact dependent, caspase-3-mediated pathway. Therefore, the adoption of Breg cell characteristics may be another approach that BCL1 tumor cells employ to evade immune responses. [Keywords: B cell lymphoma, tumor dormancy, regulatory T cells, regulatory B cells, suppression assays, immunization, flow cytometry, cytokine profile]Item Novel Roles of Gastrin and Cholecystokinin in Islet Beta Cell Proliferation(2007-06-12) Presley, Brent Kevin; Newgard, Christopher B.Type 1 diabetes and the latter stages of Type 2 diabetes share a common theme: an insufficient beta cell mass to maintain glucose homeostasis. In Type 1 diabetes this deficiency arises from autoimmune destruction of the beta cells. In the latter stages of Type 2 diabetes, there is a precipitous drop in beta cell mass, resulting from the combination of several factors. In the last decade, islet transplantation has re-emerged as a viable option for the treatment of Type 1 diabetes, thanks to greatly improved islet isolation protocols and immunosuppressive regimens. Despite these advancements, the supply of available islet beta cells for transplantation is greatly eclipsed by the demand. Consequently, the identification of genes or external factors that promote beta cell proliferation and survival is a key step toward developing a replenishable population of beta cells that can be used for transplantation for Type 1 diabetes. Additionally, any method discovered to promote beta cell growth or enhance beta cell function is directly applicable in the treatment of Type 2 diabetes. Through a broad-based microarray screen, the preprogastrin gene was found to be differentially expressed in our model of beta cell cytokine resistance, the INS-1res cell lines compared to unselected INS-1 cells. As a result of this finding, preprogastrin was initially evaluated for its involvement in cytokine resistance and beta cell survival. During the course of this analysis, preprogastrin was determined to exhibit significant mitogenic properties when overexpressed in INS-1 cell lines and isolated rat islets. These results led to the discovery that the related protein, preprocholecystokinin, also promotes impressive beta cell growth, in addition to enhanced beta cell function, as measured by improved glucose-stimulated insulin secretion. Several additional experiments suggest that traditional plasma membrane receptors and signaling pathways for gastrin and cholecystokinin do not explain the effect of overexpression of these prohormones on islet replication, including a lack of effect of exogenously added gastrin and cholecystokinin peptides. These results could be attributable to an intracrine mode of signaling that will require further investigation as a possible therapeutic target for the treatment of Type 1 and Type 2 diabetes.Item The Role of Protein Phosphatases in Regulation of Drosophila S6 Kinase by Nutrient Signaling Pathways(2006-05-15) Bielinski, Vincent Anthony; Mumby, MarcThe regulation of cellular growth is a highly complex, but fundamental, process and the inability of cells to initiate or repress cell growth in the presence of appropriate signals underlies many disease states including cancer and diabetes. Many studies have investigated the mechanisms by which cells activate growth; however, few have focused upon the means by which cells turn off growth in response to environmental signals. As many of the protein components of nutrient signaling pathways are proto-oncogenes, of equal importance is identifying and characterizing the components utilized to down-regulate growth, as mutations in these genes may also lead to genesis of disease. Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase implicated in the regulation of numerous processes, including cellular growth. Elucidating the role of PP2A in nutrient signaling pathways will aid in the understanding of the biochemical processes that eukaryotes employ to properly respond to their environment. I have cloned the Drosophila homolog of the Tap42/a4 gene (dTap), which retains 28% amino acid identity to the human a4 gene product. Utilizing a combination of RNA interference (RNAi) and nutrient starvation conditions, I have tested the sequestration model of dTap as a repressor of PP2A activity and shown that in S2 cells, dTap does not antagonize nutrient signaling pathways by repressing PP2A activity. Although dTap does not act as an inhibitor of PP2A activity towards dS6K, loss of dTap conferred rapamycin resistance to S2 cells and prolonged loss of dTap resulted in cell death. I have also utilized RNAi to screen the gene products of the PP2A-like family of phosphatases and Drosophila PP2A regulatory subunits for effects upon nutrient signaling pathways. I have shown that the PP2A catalytic subunit is required for the dephosphorylation of Drosophila S6 kinase (dS6K) during periods of inhibition of growth. The ablation of two other gene products, the PP2A-B56-2 regulatory subunit and the dPP4 catalytic subunits, results in hypophosphorylation of dS6K during amino acid starvation, suggesting that the activities of these two gene products are required for maintenance of nutrient signaling pathways and proper growth in Drosophila.Item Small Molecule Regulator of ENTPD5, and ER Enzyme in the PTEN/AKT Pathway(2011-02-01T19:34:58Z) Huang, Song; Wang, XiaodongPI3K signaling plays a crucial role in effecting alterations in a broad range of cellular functions in response to diverse extracellular stimuli (insulin, growth factors, integrins and GPCRs etc.). A key downstream effector of PI3K is the serine-threonine kinase Akt, which in response to PI3K activation, phosphorylates and regulates the activity of a number of cellular targets, through which it modulates a variety of cellular functions, including glucose metabolism, protein synthesis, cell proliferation and survival. Tumor supressor gene PTEN encodes a lipid phosphatase that antagonizes PI3K function and consequently inhibits downstream signaling through Akt. Dysregulation of this pathway has been found in a variety of human cancer, mainly by loss of function of PTEN, or amplification and activating mutations of PI3K and/or Akt. We were interested in the anti-apoptotic mechanism of PI3K/Akt signaling. We observed a defect in apoptosome formation in PTEN-null (PTEN -/-) MEF cell lysate. This defect is due to rapid depletion of ATP by a strong ATP hydrolysis activity in PTEN-null MEF lysate, which is absent in PTEN heterozygous (PTEN +/-) lysate. Following this activity, we purified three enzymes, namely ENTPD5, CMPK1 and AK1, that together forms a coupled enzymatic cycle, hydrolyzing ATP to AMP. In the cell, ENTPD5 is an ER localized UDPase that hydrolyzes UDP, the by-product of glycosyl-transferase, into UMP. Only in its monophosphate form can Uridine nucleotide exits ER through an antiporter by exchanging a molecule of UDP-sugar from cytosol. Up-regulation of ENTPD5 in PTEN-null MEF cells accelerates glycosylation substrate replenishment, therefore promotes N-glycosylation and increases ER protein folding capacity to accommendate the increase of protein synthesis resulted from active PI3K/Akt signaling. Knockdown of ENTPD5 in PTEN-null cells suppresses global N-glycosylation, resulting in ER stress and degradation of several growth factor receptors. As a consequence, the growth of PTEN-null cells is inhibited both in vitro and in mouse xenograft tumor models. Given the essential role of ENTPD5 in PI3K/Akt pathway, we performed biochemical high-throughput screen for ENTPD5 inhibitors. The newly identified inhibitors recapitulate the phenotype of ENTPD5 knockdown in vitro. Interestingly, PTEN-null MEF cells are more susceptible to these inhibitors than PTEN heterozygous MEF cells, in terms of the intensity of induced ER stress and cell death. Inhibition of ENTPD5 produces synthetic lethality with PTEN loss or PI3K/Akt hyperactivation, therefore provides a potential therapy for the cancers harboring these lesions.Item Using Light-Activated EFG to Control Cell Behavior with Automated Instrumentation(2008-05-13) Miller, Danielle Suzanne; Luebke, Kevin J.A key interest in cell biology is the ability to control cell behavior, particularly for creating functional assemblies of cells to restore, maintain or enhance tissue and organ function. Success in controlling cell behavior must include techniques that provide signals which influence the organization, growth and activities of cells. Growth factors are naturally occurring proteins that act as external chemical signals and which play a key role in regulation and control of a variety of cellular processes, such as differentiation, proliferation and migration. One of the challenges in controlling these processes using growth factors is the ability to spatially direct their timed release to the cellular environment. Another challenge then becomes the continued ability to influence these processes with the dynamic flexibility to meet the changing cellular demands during tissue development. We have developed a technology that uses light-activated epidermal growth factor (EGF) to influence cell behavior. We used peptide synthesis to incorporate a photolabile caging group on a critical residue. The caged-growth factor was inactive until converted with light, which enabled the management of its effects with the precision with which light could be directed. Since the factor was a soluble, diffusible species, it was not limited to a static pattern or substrate. Thus, dynamic control over its mitogenic and chemotactic effects on cell behavior was achieved. To utilize the light-activated EGF we developed a device for its delivery and activation. The system was a fully automated machine capable of maintaining the strict requirements of cell culture, integrated with components that achieved interchangeable, high resolution patterns, along with an optical system for photo-activating caged growth factors. The instrument was designed, characterized and then used to investigate the effect of light-activated EGF on cell patterning and mobility. Using this device, spatially resolved fibroblast cell patterning and migration were achieved.