Browsing by Subject "Cell Proliferation"
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Item Alterations in Neural Stem Cell Fate Following Focal Ischemia(2014-02-04) Nguyen, Derek; Vemireddy, Vamsidhara; Mashimo, Tomoyuki; Battiste, James; Maher, Elizabeth; Bachoo, RobertINTRODUCTION: The purpose of this experiment is to determine the differentiation identity of the neural stem cells (NSC) in the subventricular zone (SVZ) of adult mouse brain after a middle cerebral artery occlusion (MCAO). Injury to the brain causes a large number of changes including inflammation and apoptosis, but the reaction of NSC's has been more difficult to characterize because of the transient nature of their response. Previously, adult neural stem cells (NSC) in the SVZ have been observed to differentiate predominantly into cells with neuronal characteristics. This theory is questioned via a tamoxifen-inducible cre-recombinase (Cre-ERT2) expression mouse model system. METHOD: The Cre-ERT2 expression mouse model system is driven by the Cystatin-C promoter to label NSC's in a time specific manner and track their cell fate after MCAO. After the ischemia, these brain sections were stained with different immunohistochemicals at three separate time points. One set was co-labeled with GFAP, an astrocyte marker, and BrdU, a proliferation marker. Another set was co-labeled with DCX, a neuronal marker, and BrdU. This was used to differentiate between latent NSCs and proliferating NSCs by comparing the ipsilateral side (ischemic) with the contralateral side (control) of the brain. RESULTS: Compared to the contralateral, the ipsilateral side had a significant increase in GFAP/BrdU positive cells between day 3 and day 7 time points. The cell quantity dropped between day 7 to day 14 time points. Compared to the contralateral, the ipsilateral side had a decrease in DCX/BrdU positive cells between day 3 and day 7 time points. The cell quantity significantly increased between day 7 to day 14 time points, and the quantity at day 14 was about twice to that of the day 3 time point. DISCUSSION: This data demonstrated that after the MCAO, the stem cells are not just undergoing neurogenesis, but are for certain period of time, also differentiating into astrocytes that are migrating towards the site of injury. This phenomenon is only witnessed in the NSCs towards the day 7 time point. Afterwards and leading up to day 14, the NSCs seem to be changing their cell fate programming from the astrocyte pathway back to the intended neuronal pathway. Thus, the staining results verify that after an ischemia, NSCs within the SVZ regions of the brain undergo a constant change of programmed cell fate, alternating between immature neurons and astrocytes implicating future aims for "programmed" neurogenesis in the development of therapeutic strategies for the treatment of brain damage and disease.Item Defining the Developmental Signals of the Cardiac Fibroblast(2012-08-31) Baek, Seung Tae; Tallquist, Michelle D.Cardiac 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 Pdgfra 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(2011-02-01) 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.Item Identification of EWS-FLI1 Regions Necessary for Ewing Sarcoma Proliferation(December 2021) Bremauntz Enriquez, Alberto; Amatruda, James F.; McFadden, David G.; McKnight, Steven L.; Yu, HongtaoEwing sarcoma is pediatric bone malignancy defined by a translocation between EWS and ETS family transcription factor. EWS-FLI1 (EF) is the most common translocation and codes for a novel transcription factor that combines the N-terminus of EWS, which contains LC domain comprised of tyrosine rich peptide repeats, and C-terminal portion of FLI1, which contains the ETS DNA binding domain. EF is a key transcriptional regulator known to both activate and repress genes. While understanding of the molecular mechanism by which EF controls transcription have become clearer, targeted therapeutic interventions against EF or its transcriptional program have yet to make clinical impact. This is due in part to a poor understanding of how the N-terminus of EWS is contributing to the oncogenic program and a disparate range of reported effects after EF depletion on Ewing sarcoma cells. This report shows the adaptation of two inducible degron systems, Small Molecule Assisted Shut-Off (SMASh) and Auxin-inducible degron (AID), into the endogenous locus of EF in a series of Ewing sarcoma cell lines to define the phenotype of EF depletion. Across multiple cell line and degradation mechanisms, EF depletion in Ewing sarcoma cells leads to decreased cell proliferation through G1/S arrest that can be rescued through re-expression of EF. Having established a baseline for EF depleted cells, I developed a proliferation-based assay to test the functionality of mutant EF constructs on their ability to drive proliferation in the setting of endogenous EF depletion. I tested a series of EF truncations, with single or multiple exon deletions in the EWS portion of the translocation. Expression of EF constructs with loss of any single exon was tolerated and allowed for continued proliferation, but loss of at least exons 1-4 on the N-terminus and loss of exons 5-7 from the C-terminus resulted in non-functional EF constructs. Given that there appear to be redundant elements within the N-terminus of EF, I tested truncated and mutant version of a minimal rescue construct containing only exon 1-5 of the N-terminus of EF. Mutation of only 3 tyrosines to serine within the minimal construct was sufficient to prevent proliferation in Ewing cell lines.Item In Cell Stress Conditions, VEGFR2 Exerts Pronounced Effects on Cell Growth in Dysplastic Barrett's Epithelial Cells(2015-01-26) Belli, Olivia; Zhang, Qiuyang; Souza, Rhonda F.BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF), a potent inducer of angiogenesis, recently has been shown to exert direct pro-proliferative and pro-survival effects on cancer cells through binding to its receptors, VEGFR1 and VEGFR2. In earlier studies, we showed that VEGF/VEGFR2 signaling exerts direct pro-proliferative effects on transformed Barrett's and adenocarcinoma cells in an autocrine fashion, with no significant effects on apoptosis. To explore the potential contribution of VEGFR signaling to cell growth of dysplastic Barrett's cells, we knocked down the VEGFR1 or VEGFR2 and studied the effects on cell morphology, cell number, proliferation, and apoptosis. METHODS: We used 3 high-grade dysplastic Barrett's epithelial cell lines (CP-B, CP-C, and CP-D). VEGFR1 and VEGFR2 were knocked down through stable infection with retroviral shRNA vectors. Knockdown was assessed through qRT-PCR for VEGFR1 and Western blot for VEGFR2. Cell morphology was assessed by optic microscopy. Cell numbers were assessed by cell counts at 48 hours in full growth media (FM) and under cell stress conditions (1% FM); proliferation was assessed by BrdU incorporation and apoptosis was assessed by a cell death Elisa in 1%FM. RESULTS: Knockdown of VEGFR1 was seen in 34% of CP-C, 22% of CP-D, and 0% of CP-B. Thus, we used CP-C only to assess effects of VEGFR1 knockdown. By Western blotting, we observed knockdown of VEGFR2 in CP-B and in CP-D, but not in CP-C. Thus, we used CP-B and CP-D to assess effects of VEGFR2 knockdown. We did not observed any morphology changes in the VEGFR knockdown cell lines compared to controls. In FM, dysplastic cells containing either VEGFR1 or VEGFR2 knockdown had higher cell numbers compared to control cells. In 1%FM, there was no significant difference in cell number between CP-C cells containing VEGFR1 knockdown and controls. In contrast, at 48 hours in 1%FM, CP-B and CP-D containing VEGFR2 knockdown had lower cell numbers (19.9 ± 1.3 X 104 and 10.5± 1.4 cells, respectively) compared to controls (41.4 ± 4.4 and 16.9 ± 1.6 cells). Compared to controls, VEGFR2 knockdown significantly increased BrdU incorporation in CP-B cells, even though overall cell number was decreased, whereas in CP-D, BrdU incorporation was decreased (p=0.059) along with overall cell number. Compared to controls, VEGFR2 knockdown increased apoptosis in CP-B and CP-D cells. CONCLUSIONS: VEGFR2, but not VEGFR1, contributes to cell growth of dysplastic Barrett's cells, but only under conditions of cell stress, with both pro-survival and pro-proliferative effects. These findings support a potential role for anti-VEGFR2 therapies in the treatment of high grade dysplasia in Barrett's esophagus.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 Nur77 Prevents Excessive Osteoclastogenesis by Inducing Ubiquitin Ligase Cbl-b to Mediate NFATc1 Self-Limitation(2020-07-21) Li, Xiaoxiao; Mendelson, Carole R.; Zinn, Andrew R.; Kliewer, Steven A.; Wan, YihongOsteoclasts are bone-resorbing cells essential for skeletal remodeling. However, over-active osteoclasts can cause bone degenerative disorders. Therefore, the level of NFATc1, the master transcription factor of osteoclast, must be tightly controlled. Although the activation and amplification of NFATc1 have been extensively studied, how NFATc1 signaling is eventually resolved is unclear. Here, we uncover a novel and critical role of the orphan nuclear receptor Nur77 in mediating an NFATc1 self-limiting regulatory loop to prevent excessive osteoclastogenesis. Nur77 deletion leads to low bone mass owing to augmented osteoclast differentiation and bone resorption. Mechanistically, NFATc1 induces Nur77 expression at late stage of osteoclast differentiation; in turn, Nur77 transcriptionally up-regulates E3 ubiquitin ligase Cbl-b, which triggers NFATc1 protein degradation. These findings not only identify Nur77 as a key player in osteoprotection and a new therapeutic target for bone diseases, but also elucidate a previously unrecognized NFATc1→Nur77→Cblb→NFATc1 feedback mechanism that confers NFATc1 signaling autoresolution.Item The Role of Protein Phosphatases in Regulation of Drosophila S6 Kinase by Nutrient Signaling Pathways(2006-05-15) Bielinski, Vincent Anthony; Mumby, Marc C.The 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 The Role of Pyruvate Dehydrogenase in Cell Growth(2014-07-28) Rajagopalan, Kartik N.; Amatruda, James F.; DeBerardinis, Ralph J.; Brown, Michael S.; Lum, LawrenceOtto Warburg's observation that tumor cells have increased rates of glucose uptake and lactate secretion in comparison to normal cells spawned his notion that tumors have dysfunctional mitochondria. However, in addition to metabolizing glucose to lactate, tumors in vivo exhibit mitochondrial glucose oxidation, indicating activity of pyruvate dehydrognase (PDH), which gates entry of glucose derived carbon into the tricarboxylic acid (TCA) cycle. To test whether cells require glucose oxidation for proliferation, the work in this thesis establishes a model wherein PDH activity is suppressed using RNA interference. Small hairpin RNAs against the transcript encoding the PDHE1α protein were cloned into a retroviral vector which allowed doxycycline-inducible control of expression. Metabolism of cancer cells was studied in vitro using a combination of metabolomics and metabolic flux analysis. Growth in monolayer culture was performed in medium containing lipid-replete serum as well as serum in which lipids had been depleted. As expected, suppression of PDH activity reduced flow of carbon from glucose to the TCA cycle and to de novo fatty acid synthesis. Surprisingly, H460 lung cancer cells could tolerate a 60% reduction of PDH flux without any significant effect on proliferation rate, as long as lipids were present in the medium. Further examination of the effects of PDH silencing on the overall network of central carbon metabolism revealed enhanced channeling of carbon from glutamine to fatty acids and an increase in scavenging free fatty acids. Lipid depletion caused a reduction in growth rate of PDH deficient cells, and this defect was completely rescued by supplying free fatty acids to the medium. Together the data indicate that proliferating cells exhibit PDH activity that allows transfer of glucose carbon to citrate and the TCA cycle as well as ultimately into fatty acids. Importantly, suppression of PDH activity limits growth in conditions in which cancer cells do not have access to extracellular lipids. This work illustrates that compensatory pathways that sustain cell proliferation are activated during suppression of mitochondrial oxidation of glucose.Item Single-Cell Image Analysis Enables High-Throughput Phenotypic Drug Screen and Elucidates Cell-Fate Decision Principles(2019-05-20) Hsu, Chien-Hsiang; Martinez, Elisabeth; Wu, Lani; Altschuler, Steven J.; Amatruda, James F.; Xie, YangCellular phenotypes encode information that can be used to infer the external signals cells experience. Here we applied quantitative image analysis to enable a one-pass multi-class phenotypic drug screen. Our combined experimental and computational approach can functionally annotate large compound libraries across diverse drug classes in a single-pass screen with high prediction accuracy confirmed via orthogonal, secondary validation assays. We further investigated how heterogeneity arose from an isogenic population. We monitored the dynamics of p21 to understand the proliferation-senescence cell-fate decision in single cells under non-lethal dose of chemotherapy via time-lapse microscopy before, during and days after treatment. Surprisingly, while high p21 is associated with senescence at late times, we find the opposite at early times during drug treatment: most senescence-fated cells have low p21 levels, while proliferation-fated cells have much higher p21 expression. Further, we identify a p21 "Goldilocks zone" for proliferation, in which increasing p21 levels has the undesirable effect of increasing proliferative outcomes. Our study identifies a counter-intuitive role for early p21 dynamics in cell-fate decision and pinpoints the source of proliferative cancer cells that emerge after exposure to non-lethal doses of chemotherapy.Item Small Molecule Regulator of ENTPD5, and ER Enzyme in the PTEN/AKT Pathway(2011-02-01) 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 Ubiquitin Conjugase UBE2K Is Essential for Normal Rat Development and Spermatogenesis(2016-04-14) Chaudhary, Jaideep; Repa, Joyce J.; Mendelson, Carole R.; Wan, Yihong; Hamra, F. KentAnimal models allow us to investigate questions about human physiology using in vivo systems. Following the advent of gene targeting by homologous recombination in mouse pluripotent embryonic stem cells during the early 1980's [Reviewd by (Hamra, 2010)], the laboratory mouse has emerged as the most widely published animal model in science (NCBI, PubMed). Interestingly, prior to the year 2000, annual publications in the laboratory rat outnumbered annual publications in the mouse by greater than 2-3 fold in almost every field of science spanning each decade in the 20th century (NCBI, PubMed). Popularity of the laboratory rat as a model organism in science derived from its many attributes for modeling human physiology and disease in a laboratory scale mammal. However, genetic tools to effectively manipulate the rat genome lagged behind that of the mouse for almost 4 decades up until ~2009 due to inabilities to maintain pluripotent rat cells in culture, and inefficient methods for micro-manipulating rat early embryos. Now, several laboratories have made large strides to advance technologies to genetically engineer rats (Geurts et al., 2009; Tong et al., 2011), including our laboratory, which succeeded in applying stem cell-based technologies to the rat's "genomic toolbox" by using donor stem spermatogonia as vectors to directly genetically modify the rat's germline (Chapman et al., 2015; Hamra et al., 2002; Izsvak et al., 2010). For this dissertation, I focused on phenotyping the growth and reproduction defects in the UBE2K knockout rat strain, which happens to represent the first knockout rat strain reported using genetically selected germline stem cells form culture (Izsvak et al., 2010). I reasoned that phenotyping UBE2K rats in more detail would allow me to formulate testable hypotheses on the cellular and molecular mechanisms by which UBE2K functioned to regulate rat body growth and reproduction. UBE2K is an ubiquitin ligase, and knocking its gene (Ube2k) out in rats resulted in stunted growth, compromised motor capacity of hind legs and infertility. I found infertility in male UBE2K-deficient rats to be caused by an arrest during meiosis-I that prevented the zygotene to pachytene spermatocyte transition. Based on known interactions of UBE2K, and the biochemical function of UBE2K as ubiquitin ligase, I hypothesized that UBE2K is a component of the PRC1 complex that ubiquitinates H2A to prevent transcription as a mechanism necessary for germ cells to undergo meiosis. I showed using immunostaining, that there is an inverse correlation between H2A ubiquitin staining and UBE2K expression that is disrupted in UBE2K-deficient rats, supporting my hypothesis. However, future work will need to address whether UBE2K directly or indirectly associates with the PRC1 complex. Based on my research presented in this Dissertation, I propose a novel function for the UBE2K ubiquitin conjugase in meiotic transcriptional control during spermatogenesis.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.