• A Molecular Basis of Chemoresistance in Bladder Cancer

      Lahorewala, Sarrah; Biochemistry and Cancer Biology (Augusta University, 2020-12)
      Background: In advanced bladder cancer (BC), development of resistance to the frontline chemotherapeutic drugs Gemcitabine and Cisplatin contributes to the poor prognosis of patients. Newly discovered chondroitinase, HYAL-4 V1 (V1), drives malignant transformation in BC. We evaluated V1’s role and the downstream molecules involved in the mechanistic regulation of chemoresistance in BC. Experimental Design: HYAL-4 expression was evaluated by RT-qPCR and IHC in metastatic muscle-invasive BC patients who received Gemcitabine plus Cisplatin chemotherapy. HYAL-4 wild-type and V1 were stably expressed or silenced in three BC and one normal urothelial cell line. Transfectants were analyzed for Gemcitabine and Cisplatin sensitivity, and for Gemcitabine influx and efflux to determine the mechanism of Gemcitabine resistance. The effect of cytidine deaminase (CDA) inhibition on Gemcitabine sensitivity was evaluated in vitro and in xenograft models. Results: HYAL-4 expression was an independent predictor of disease-specific mortality and treatment failure in our clinical cohort, and stratified patients into higher risk for both those outcomes. V1-expressing BC and normal urothelial cells were resistant to Gemcitabine due to the upregulation of cytidine deaminase (CDA) expression and activity, resulting in increased Gemcitabine metabolism and efflux; treating cells with tetrahydrouridine (THU), a CDA inhibitor, abrogated the chemotherapeutic resistance. Gemcitabine-resistant V1 cells demonstrated increased expression of V1’s substrate CD44 and phosphorylated STAT3. Si-RNA mediated CD44 knockdown and STAT3 inhibition both sensitized cells to Gemcitabine in vitro. In xenograft models, treatment with a combination of Gemcitabine and THU completely inhibited tumor growth. Conclusions: This project discovered V1 as a novel determinant of Gemcitabine resistance and potential predictor of treatment response in BC. V1 drives resistance to Gemcitabine through CD44-STAT3 mediated upregulation of CDA, and inhibiting this pathway sensitizes tumor cells to the therapy in preclinical models of BC.
    • Activation of Arginase and the Endothelin System in Models of Ischemic Retinopathy

      Patel, Chintan; Department of Biochemistry and Molecular Biology; Department of Biochemistry and Molecular Biology (2014-07)
      Ischemic retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP) are characterized by microvascular degeneration, followed by an abnormal hypoxia-induced neovascularization (NV). Although the triggering insult varies among the diseases, they share a common end result of capillary loss due to increased oxidative stress, cellular inflammation and vascular injury and dysfunction. We have linked activation of the urea hydrolase enzyme arginase to the latter complications in models of DR. Both arginase and nitric-oxide synthase (NOS) enzymes utilize L-arginine as substrate. NOS dysfunction due to limitations in L-arginine availability has been implicated in the pathogenesis of diabetic complications. Our studies in streptozotocin-induced diabetic mice and high glucose treated retinal ECs have demonstrated signs of retinal vascular activation and injury. These were associated with increased arginase activity and expression, decreased bioavailable nitric oxide (NO), increased superoxide formation and increased leukostasis. Blockade of the arginase pathway prevented these alterations, suggesting a primary role of arginase in retinal vascular dysfunction and injury. Our studies have also shown that endothelium-dependent retinal vasorelaxation was impaired in diabetic mice, however, deletion of arginase improved retinal vessel function and improved blood flow. During ischemic retinopathies, disturbances in retinal blood flow can result in vasoconstriction, ischemia, tissue hypoxia and formation of neovascularization (NV). Such alterations have been linked to development of ROP, a blinding disease that adversely affects premature infants due to oxygen-induced damage of the immature retinal vasculature resulting in pathological NV. Our studies using a mouse model of ROP, the oxygen-induced retinopathy (OIR) model indicate that a potent vasoactive and angiogenic factor endothelin (EDN) is responsible for pathological NV. Our analysis revealed significant increases in EDN1, EDN2 and endothelin A receptor (EDNRA) mRNA and EDN2 protein expression during ischemia. EDN2 was localized to endothelial cells and retinal glia in OIR retinas. Treatment of OIR mice with EDNRA blocker, BQ-123, significantly increased vessel sprouting resulting in enhancement of physiological angiogenesis and decreased pathological NV. OIR triggered a significant increase in STAT3 activation and VEGFA production and increased mRNA expression of angiogenic and inflammatory mediators, which were all reduced by BQ-123 treatment. These studies suggest that EDNRA activation during OIR promotes vessel degeneration and pathological NV. Collectively, both arginase and endothelins are increased in models of ischemic retinopathies. These two pathways could be interrelated through an unknown cross-talk mechanism that needs to be elucidated.
    • AKAP350 Targets to the Golgi Apparatus Where it Interacts with CLIC5B and CIP4/S

      Shanks, Ryan; Department of Biochemistry and Molecular Biology (2002-03)
      A-kinase anchoring proteins (AKAPs) are defined by their ability to scaffold PKA, but their function depends upon their targeting of PKA and other scaffolded signaling proteins to specific subcellular compartments. We have investigated one AKAP, AKAP350, which can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. The AKAP350 gene is multiply spliced to create three carboxyl terminal splice variants which we have designated AKAP350A, AKAP350B and AKAP350C. Immunocytochemistry in HCA-7 cells demonstrated that AKAP350A was localized specifically to the Golgi apparatus. GFP-fusion proteins representing the carboxyl terminus of AKAP350A identified a carboxyl terminal region responsible for the Golgi apparatus targeting of AKAP350A. Yeast two-hybrid analysis was utilized to screen a rabbit gastric parietal cell library with a 3.2kb segment of AKAP350 (nucleotides 3611-6813) which is weakly homologous to pericentrin. This screen yielded two positive clones representing rabbit chloride intracellular channel 1 (CLIC1) and rabbit Cdc42 interacting protein 5 (CIP5). Further yeast-two hybrid binary analysis determined that CLIC1 and CIP5 bound to AKAP350 through adjacent domains located with in the PHR. CLIC1 belongs to a family of proteins which all contain a high degree of homology in their carboxyl termini, and this conserved domain is responsible for several CLIC family member’s ability to bind AKAP350. We isolated the human homologue of bovine p64, CLIC5B, from an HCA-7 colonic adenocarcinoma cell cDNA. A splice variant of CLIC5, the predicted molecular weight of CLIC5B corresponds to the molecular weight of a major CLIC immunoreactive protein in HCA-7 cells. Immunocytochemistry determined that CLIC5B colocalized with AKAP350 at the Golgi apparatus. Yeast-two hybrid binary analysis determined that the final 120 amino acids of CLIC5B interacted with AKAP350. Furthermore, expression of a GFP-fusion protein containing the final 120 amino acids targeted to the Golgi apparatus in HCA-7 cells. CIP5 contains high homology to the human protein Cdc42 interacting protein 4 (CIP4). Yeast-two hybrid binary analysis determined that the first 117 amino acids of both human CIP4 and CIP5 interacted with AKAP350. Immunocytochemistry in HCA-7 cells determined with an antibody recognizing CIP4 and CIP5 localized to the Golgi apparatus. These results suggest that AKAP350 associates with CLIC proteins and CIP4/5, and these proteins interact with the AKAP35A splice variant at the Golgi apparatus.
    • AKAP350: A Centrosome Associated Scaffold Protein

      Schmidt, Hank; Department of Biochemistry and Molecular Biology (2000-06)
      A-kinase anchoring proteins (AKAPs) are recognized as key components of compartmentalization and transduction in intracellular cAMP signaling. They allow localization of the Type II c AMP-dependent protein kinase to specific subcellular domains, effectively positioning the enzyme near its substrate to await activation by cAMP. The role of AKAPs as protein scaffolds allows binding of multiple enzymes, regulatory molecules, and structural elements, functioning as a virtual platform for modulation of specific cellular events (i.e. membrane channel activity, receptor clustering). We have cloned a novel 350 kDa AKAP (AKAP350) from human gastric cDNA, and identified partial clones in human lung and rabbit parietal cells. The genomic region containing AKAP350, found on chromosome 7q21, is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, an NMDA receptorassociated protein. We identified three unique AKAP350 C-termini (AKAP350A, -B, and -C) resulting from alternative splicing of the 3' end of the gene. AKAP350 is associated with centrosomes, as well as with the cleavage furrow during anaphase and telophase by immunocytochemistry. Polyclonal antibodies to individual AKAP350 Cterminal splice variants demonstrate tissue dependent combinations of centrosomal and non-centrosomal distribution. In the polarized HCA-7 colon cell line AKAP350A is purely non-centrosomal while AKAP350B and -C are centrosomal. Anti-AKAP350C is limited to mitotic cells, suggesting that this isoform may be expressed only at entry into M phase. A yeast two-hybrid screen of a rabbit parietal cell library identified a novel TACC (Transforming Acidic Coiled coil Containing) protein family member as a ligand of the final pair of arginine residues in the AKAP350A splice variant. A GFP fusion with the novel AKAP interacting protein verified co-localization with AKAP350 at the centrosome exclusively during mitosis. Microinjection of dividing sea urchin embryos with GST fused to the AKAP interacting protein arrested cell division. Therefore, the AKAP350 protein scaffold may function as a large docking station, providing kinase / phosphatase signals for coordination of cytoskeletal dynamics as well as cell division.
    • Artificial Chromosome Transgenesis Reveals Long-Distance Negative Regulation of ragl in Zebrafish

      Jessen, Jason R.; Department of Biochemistry and Molecular Biology (1999-11)
      Despite the essential roles played by the recombination activating genes (ragl and rag2) during V(D)J recombination, the mechanisms that restrict their expression to lymphoid cells are undefined. Using a novel approach to achieve artificial chromosome transgenesis in zebrafish, we demonstrate that distal regulatory elements are critical to suppress ragl expression in inappropriate tissues. In contrast to smaller reporter gene constructs, 125 and 75 kb artificial chromosomes containing the zebrafish rag genomic locus directed GFP expression in a pattern reflective of endogenous rag 1. Mapping experiments identified a positive element 5' of ragl that enhances GFP expression in both lymphoid and non-lymphoid tissues and a negative element 5' of ra g l that specifically suppresses GFP expression in the skeletal muscle. Our transgenic zebrafish also express GFP in olfactory neurons which we show represent an authentic ra g l expression site in zebrafish.
    • Biosynthesis and Modification of Helicobacter pylori Lipid A

      Stead, Christopher Michael; Department of Biochemistry and Molecular Biology (2010-05)
      The secondary acylation steps of Helicobacter pylori lipid A biosynthesis are poorly understood because H. pylori only has one homolog (Jhp0265) to the Escherichia coli secondary acyl transferases LpxL and LpxM. Jhp0265 was shown to be responsible for the transfer of a secondary C18 acyl chain to the 2′-linked acyl chain of lipid A, making Jhp0265 homologous to LpxL. An activity was also demonstrated for the addition of a secondary acyl chain to the 3′-linked acyl chain of H. pylori lipid A, although the enzyme responsible for the transfer remains unknown. After synthesis, H. pylori lipid A is modified by the action of five enzymes. Mutation of the candidate modification enzyme Jhp0634 demonstrated that the enzyme catalyzes the removal of the 3′-linked acyl chains of H. pylori lipid A, producing a tetra-acylated lipid A species. Continuing with the characterization of H. pylori lipid A modification enzymes, we were also able to demonstrate an activity for a Kdo trimming enzyme in vitro. Requirement for a Kdo hydrolase in vivo was confirmed after the Kdo transferase of H. pylori was shown to be bifunctional despite the presence of only one Kdo sugar in H. pylori lipopolysaccharide. Attempted identification of the Kdo hydrolase revealed that both Hp0579 and Hp0580 were required for the removal of the Kdo sugar, which occurred in the periplasm. A Kdo hydrolase mutant revealed two unexpected phenotypes related to interaction with the innate immune system. The first was an increased sensitivity to cationic antimicrobial peptides, which was explained by a downstream effect on modification to the 4′- phosphate group of lipid A. The second phenotype related to the expression of Oantigen on the bacterial cell surface. The Kdo hydrolase mutants produced a reduced amount of fully extended lipopolysaccharide and conversely, an increased amount of core-lipid A. The type of O-antigen epitope displayed was also affected by a Kdo hydrolase mutation, in a strain specific manner.
    • Biosynthesis of the Vibrio cholerae Kdo-lipid A Domain and its Role in Pathogenesis

      Hankins, Jessica V.; Department of Biochemistry and Molecular Biology (2011-05)
      Bacteria assemble remarkable surface structures that interface with their surrounding environment. One such structure is the glycolipid lipopolysaccharide (LPS) that covers the surface of Gram-negative bacteria. LPS is anchored to the bacterial cell by its lipid anchor known as lipid A. Since lipid A is the bioactive component of LPS, modulation of its structure can have a profound impact on disease by altering the host immune response. Additionally, LPS structure directly impacts the outer membrane permeability barrier and bacterial resistance to host antimicrobial peptides. Although the lipid A domain of Escherichia coli has been well characterized, the Vibrio cholerae lipid A biosynthetic pathway has received little attention. The late stages of lipid A biosynthesis include the transfer of the 3-deoxy-Dmanno- octulosonic acid (Kdo) sugars and the secondary acyl chains to the lipid A backbone. Here, the V. cholerae Kdo transferase (Vc0233) was shown to be monofunctional, transferring one Kdo residue to the lipid A precursor, lipid IVA. V. cholerae encode a Kdo kinase (Vc0227) responsible for the phosphorylation of the Kdo residue. The functionality of Vc0227 was shown to be required for the activity of the V. cholerae lipid A LpxL homologue, Vc0213. Interestingly, the addition of the phosphate group on the Kdo sugar was shown to be essential for lipid A secondary acylation in Haemophilus influenzae and Bordetella pertussis. Vc0213 was shown to catalyze the transfer of a myristate (C14:0) to the 2′-position of the V. cholerae phosphorylated Kdolipid A domain. A second protein, Vc0212, acts as an LpxM homologue and transfers 3- hydroxylaurate (3-OH C12:0) to the 3′-position creating hexa-acylated V. cholerae lipid A domain. Although lipid A secondary acyltransferases have been characterized among various Gram-negative bacteria, this is the first report of a lipid A secondary hydroxyacyltransferase. Further, the transfer of 3-hydroxylaurate (3-OH C12:0) was demonstrated to be essential for antimicrobial peptide resistance in V. cholerae and required for activation of the innate immune receptor TLR4.
    • The c-MYC oncogene deregulates global DNA methylation and hydroxymethylation to control genome-wide gene expression for tumor maintenance in leukemia/lymphoma

      Poole, Candace Jean; Biomedical Sciences (Augusta University, 2019-05)
      Aberrant DNA methylation is a characteristic feature of tumor cells. However, our knowledge of how DNA methylation patterns are established and maintained to contribute to tumorigenesis is limited. Inactivation of the c-MYC oncogene triggers tumor regression in T-cell acute lymphoblastic leukemia (T-ALL) resulting in dramatic changes to the chromatin landscape including DNA methylation. In this study, I investigated how MYC regulates DNA methylation and hydroxymethylation patterns to contribute to gene expression programs important for tumor maintenance in T-ALL and Burkitt lymphoma. I report that MYC maintains 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) patterns by regulating the DNA methylation machinery, which is important for gene expression in T-ALL. DNA methyltransferases (DNMTs) initiate 5mC marks, while Ten-eleven translocation methylcytosine dioxygenases (TETs) oxidize 5mC to produce 5hmC as an intermediate modification, ultimately leading to active DNA de-methylation. I demonstrated that DNMT1 and DNMT3B are MYC target genes and that their expression is dependent on high MYC levels. Knockdown of DNMT3B in T-ALL reduced cell proliferation through cell cycle arrest and caused the reactivation of gene transcription through reversing promoter/CpG island methylation. Furthermore, I demonstrated that TET1 and TET2 expression is MYC-dependent, as high TET1 and low TET2 levels depend on oncogenic MYC. Knockdown of TET1 in T-ALL reduced cell proliferation through cell cycle arrest and caused genome-wide changes in 5mC and 5hmC corresponding to changes in gene programs important for ribosomal biosynthesis and protein synthesis. In contrast, ectopic expression of TET2 reduced tumor cell proliferation through apoptosis/necrosis and caused genome-wide changes in 5mC and 5hmC corresponding to changes in transcriptional regulatory gene programs. My finding that a coordinated interplay between components of the DNA methylation machinery is necessary for MYC-driven tumor maintenance highlights the potential of targeting specific DNMT or TET proteins for therapeutic strategies.
    • Canonical Wnt Signaling in Antigen Presenting Cells Regulates Microbiota-Induced Inflammation and Immune Cell Homeostasis in the Colon

      Swafford, Daniel Joseph; Department of Biochemistry and Molecular Biology / Cancer Center (8/3/2018)
      Aberrant Wnt/β-catenin-signaling occurs in several inflammatory diseases including inflammatory bowel disease (IBD) and IBD-associated colon carcinogenesis. However, its role in shaping mucosal immune responses to commensals in the gut remains unknown. Here, we investigated the importance of canonical Wnt signaling in CD11c+ antigen presenting cells (APCs) in controlling intestinal inflammation. Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt-signaling in intestinal CD11c+ antigen presenting cells (APCs) controls intestinal inflammation by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt co-receptors, low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6) in CD11c+ APCs in mice (LRP5/6ΔCD11c mice) resulted in enhanced intestinal inflammation with increased histopathological severity of colonic tissue. This was due to microbiota-dependent increased production of pro-inflammatory cytokines and decreased expression of immune regulatory factors such as IL-10, retinoic acid (RA), and IDO. In addition, loss of LRP5/6-mediated signaling in CD11c+ APCs resulted in altered microflora and T cell homeostasis, which led to a loss of systemic tolerance to oral antigen. Furthermore, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice resulted in reduced acute intestinal inflammation with decreased histopathological severity of colonic tissue. Loss of canonical Wnt signaling in CD11c+ APCs also results in an increase in colonic polyp formation and exacerbation of chronic inflammation/injury. This was also heavily dependent on the presence and composition of the gut microbiota, as fecal transfers from LRP5/6ΔCD11c mice to floxed control (LRP5/6FL/FL) mice that were administered an antibiotic cocktail produces a polyp load and weight loss similar to that of LRP5/6ΔCD11c mice without treatment. Additionally, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice reduces severity of inflammation-associated colon carcinogenesis in these mice. Furthermore, we show that treatment of LRP5/6ΔCD11c mice with either RA or IL-10 reduces severity of inflammation-associated colon carcinogenesis. Mechanistically, RA and IL-10 may independently reduce key inflammatory factors at the acute phase of colitis. These results ultimately reveal a mechanism by which intestinal APCs control intestinal inflammation and immune homeostasis via the canonical Wnt signaling pathway, which may serve as a promising target for chronic inflammatory disorders.
    • Characterization o f Zebrafish Mutant m erlot as a Non-Mammalian Vertebrate Model for Congenital Anemia Due to Protein 4.1 Deficiency

      Shafizadeh, Ebrahim; Department of Biochemistry and Molecular Biology (2002-08)
      The zebrafish mutant merlot (mot) is characterized by onset o f a severe anemia at 96 hours post fertilization. We performed whole mount RNA in situ hybridization and showed that the process o f primitive erythropoiesis is not interrupted in the mot embryos. Blood analysis demonstrated that mot suffers from a severe congenital hemolytic anemia. Using the TU N E L assay, we detected apoptotic erythroid progenitors in the kidneys. We performed electron microscopic analysis and detected membrane abnormalities and a loss o f the cortical membrane organization in the mot cells. We used positional cloning techniques w ith a candidate gene approach to demonstrate that mot encodes the erythroid specific isoform o f protein 4.1R, a critical component o f the red blood cell membrane skeleton. Sequence analysis o f 4.IR cD N A detected nonsense point mutations in both alleles o f mot resulting in premature stop codons. We performed linkage analysis and transgenic rescue experiments to provide further confirmation that the molecular defect in the protein 4 .1R is the underlying cause o f anemic phenotype in mot fish. This study presents the zebrafish mutant merlot as the first characterized non-mammalian vertebrate model o f congenital anemia due to a defect in protein 4.1R integrity.
    • Characterization of Neurotransmitter Transporter Gene Family in C. Elegans

      Jiang, Gouliang; Department of Biochemistry and Molecular Biology (2005-10)
      GABA functions as an inhibitory neurotransmitter in body muscles and as an excitatory neurotransmitter in enteric muscles in C. elegans. No transporter specific for this neurotransmitter has been identified to date in this organism. Here we report on the cloning and functional characterization of a GABA transporter from C. elegans (ceGAT- 1) and on the functional relevance of the transporter to the biology of body muscles and enteric muscles. ceGAT-1 is coded by snf-11 gene, a member of the sodium-dependent neurotransmitter symporter gene family in C. elegans. The cloned ceGAT-1 functions as a Na+/C f -coupled high-affinity transporter selective for GABA with aK t of~15 uM. The Na+:C1':GABA stoichiometry for ceGAT-1-mediated transport process is 2:1:1. The process is electrogenic as evidenced from GABA-induced inward currents in X laevis oocytes that express ceGAT-1 heterologously. The transporter is expressed exclusively in GABAergic neurons and in two other additional neurons. We also investigated the functional relevance of ceGAT-1 to the biology of body muscles and enteric muscles by ceGAT-1-specific RNAi in rrf-3 mutant, a strain of C. elegans in which neurons are not refractory to RNAi as in wild type strain. Downregulation of ceGAT-1 by RNAi leads to an interesting phenotype associated with altered function of body muscles and enteric muscles and also with altered sensitivity to aldicarb-induced paralysis. These findings provide unequivocal evidence for a modulatory role of GABA and ceGAT-1 in the biology of cholinergic neurons and in the function of body muscles and enteric muscles in this organism. We also cloned and functionally characterized for the first time a sodium-coupled transporter for amino acids in C. elegans. This transporter, designated ceNAT-1 (sodiumcoupled amino acid transporter-1), is identified in Worm database as snf-5, also a member of the sn f gene family (sodium/neurotransmitter symporter gene family). When expressed heterologously in mammalian cells, ceNAT-1 mediates the uptake of a broad spectrum of neutral amino acids in a Na+ dependent manner. The transport process exhibits a Na+: amino acid stoichiometry of 1:1. There is no involvement of C f in the transport process. When expressed heterologously in A! laevis oocytes, ceNAT-1 induces inward currents in response to neutral amino acids under voltage-clamp conditions, indicating that the transport process is electrogenic. Based on functional features, NAT-1 seems to be the C. elegans counterpart of the amino acid transporter B°AT in mammals. Mutations in the gene coding for B°AT cause Hartnup disease in humans. The clinical phenotype of Hartnup disease varies markedly depending on the environmental conditions. The present study shows that RNAi-mediated knockdown of NAT-1 or genetic deletion of NAT-1 in C. elegans is not associated with any detectable phenotype. This may be similar to the situation in humans where environmental conditions influence the clinical outcome of Hartnup disease. Further studies with altered experimental conditions are needed to determine if C. elegans with deletion of NAT-1 is a useful model system for investigations of Hartnup disease. Recently, a second isoform of B°AT has been identified in mammals. This transporter is expressed predominantly in the brain. Therefore, it is not clear at present whether the ceNAT-1 represents the worm counterpart of the Hartnup gene or the recently identified second isoform. We also report here on the cloning and functional characterization of a C. elegas betaine transporter which is encoded by snf-3, another member of the C. elegans sn f gene family. We named this transporter ceBGT-1. ceBGT-1 exhibits high specificity for betaine when expressed heterologouly in mammalian expression system and the uptake process mediated by ceBGT-1 is dependent on both sodium and chloride. The Na+: Cl': betaine stoichiometry for ceBGT-1-mediated transport process is 2:1:las confirmed y two-microelectrode voltage-clamp study. The Kt of ceBGT-1 for betaine is about 0.32 mM. Consistent with its role in osmoregulation, in vivo expression study using transgenic GFP fusion technique shows ceBGT-1 is expressed in the canal cells of C. elegans which represent the excretory represent the excretory organ in this organism. Investigation of the effects of hypertonicity on the expression of ceBGT-1 shows that hypertonicity increases its expression in C. elegans cultured with medium containing 350 mM NaCl compared to C. elegans cultured under normal conditions (50 mM NaCl).
    • CHOLINESTERASE INHIBITOR TOXICITY: MECHANISTIC STUDIES AND THERAPEUTIC STRATEGIES FOCUSED ON AXONAL TRANSPORT

      Naughton, Sean X; Biomedical Sciences (Augusta University, 2019-12)
      Organophosphates (OPs) are a broad class of chemicals with a variety of uses that include pesticides, chemical warfare agents, fuel additives, and plasticizers. Due to their sheer number of applications and known toxicological profile, OPs represent a persistent concern to human health worldwide. Furthermore, the effects of OPs that occur independently of their well-known mechanism of acute toxicity (AChE inhibition) have not been well studied. The presented research seeks to expand upon our understanding of AChE-independent mechanisms of OP toxicity as well as to identify potential therapies for treating these negative effects. In Manuscript 1 we demonstrate that the OP diisopropylfluorophosphate (DFP) induced axonal transport deficits occur in vivo at exposure levels that were not associated with cholinergic toxicity. Additionally, we observed deficits in white matter integrity following sub-acute DFP exposure. In Manuscript 2 we present a series of experiments, which were conducted to identify potential therapeutic compounds for the treatment of OP induced deficits in axonal transport. Here, we utilized a phenotypic drug-screening assay in order to identify compounds that could be protective against DFP. In Manuscript 3 we present data which demonstrates that the carbamate physostigmine does not impair axonal transport, as has been previously demonstrated with OPs. These experiments were critical to demonstrating the AChE independence of OP-induced axonal transport deficits and further elucidate the unique nature of OP toxicity in comparison to other AChE inhibitors. Collectively, these studies contribute to a better understanding of the full spectrum of toxicological effects of OPs and provide insightful findings into potential therapeutics for the treatment of OP related toxicity.
    • Cloning, Expression and Characterization of Bovine aB-crystallin

      Kelley, Patrick; Department of Biochemistry and Molecular Biology (2000-01)
      The lens protein, aB-crystallin, plays central role in slowing the formation of pre-cataractous protein aggregates. By binding to newly exposed, hydrophobic regions of damaged proteins, aB-crystallin creates stable, soluble complexes that are resistant to further unfolding and aggregation. Several regions o f aB-crystallin have been identified as possible sites for this chaperone-like binding, three of which are explored further in the present work using assays for both the physical properties and in vitro function of sitedirected mutants of aB-crystallin. A priority in this study was the generation of a bovine aB-crystallin cDNA clone. One of the regions of putative chaperone binding in aB - crystallin, residues 24-32, which was implicated by deuterium exchange experiments, contains several hydrophobic residues. Since hydrophobic residues are thought to play a central role in chaperone binding, the present work investigates a mutant of one of these residues, F28, which was changed to serine. Two charged residues within this same domain, E30, and E34 were each separately mutated to glutamine in order to assess the role of negatively charged residues in chaperone activity. Another two regions implicated as possible binding sites in aB-crystallin, residues 59-68 and residues 92-108, were previously identified using SAED binding studies. In the present study, one hydrophobic residue from each of these domains was mutated to a less hydrophobic residue: 16IS and L94Q. The results show that at 25°C, a serine as position 28 (F28S) causes moderate alterations in secondary structure, tertiary structure and oligomeric assembly. At 58°C, however, this mutant suffers from a disintegration o f oligomeric structure as well as a loss of chaperone function. The data implicate F28 as a critical residue for maintaining the structural integrity of aB-crystallin. The other mutants, with the exception o f L94Q, behaved similar to wild type aB-crystallin whether assayed at 37 ° or 58 °. The L94Q mutant displayed slightly better chaperone activity than wild type in the high temperature (58°) assay. The present work supports the idea that the chaperone-like behavior of aBcrystallin requires intact oligomeric structure and that the activity may not be associated with a discreet binding site but instead a diverse array of residues spread out over the surface of aB-crystallin.
    • Combinational immunotherapy of anti-OX40 antibody and IDO inhibitor synergistically enhances anti-tumor immune T cell-mediated response

      Berrong, Zuzana Jirina; Department of Biochemistry and Molecular Biology (2016-05)
      One of the major goals of cancer immunotherapy is to disrupt the immunosuppressive environment that allows tumors to thrive in and to generate potent and enduring antitumor specific-immune responses. Cancer vaccines may elicit antigen-specific immune responses; however, this is, in many tumor models and in human cancer, insufficient for positive outcomes due to existence of multiple immune-inhibitory mechanisms in tumors. A relatively recently introduced strategy to increase the therapeutic efficacy of tumor vaccination is to combine different immunological approaches that target different immunosuppressive pathways and to enhance the efficacy of vaccines by T cell agonists. OX40 is a co-stimulatory receptor expressed on T cells that can lead to proliferation and enhancement ofT cell effector function when bound by its ligand or targeted with agonist antibody. Here, we show that different doses of anti-OX40 antibody (Ab) elicit differential impacts on the T cell immune response resulting in either efficacious or detrimental therapeutic effect in immunized tumor-bearing mice. We demonstrate that treating tumor-bearing mice with an optimal dose of 1 mg/kg anti-OX40 Ab leads to a potent therapeutic and immune anti-tumor effect when combined with vaccine, whereas higher dose at 2.5 mg/kg of anti-OX40 Ab with vaccine increases the accumulation of regulatory T cells in the tumor and diminishes the therapeutic effect. Furthermore, we proposed that OX40 downstream molecular signaling through AKT activation in T cells may elucidate the differential T cell response when stimulated with anti-OX40 Ab. After optimizing the dose of agonist anti-OX40 Ab to stimulate the immune system toward maximal anti-tumor response when combined with vaccine, we strategized to improve the combinational therapy by targeting the so far untouched immunosuppressive environment. One of the immune suppressive molecules correlating with cancer progression is indoleamine-(2,3)-dioxygenase (IDO) enzyme. The catalytic activity of IDO hinders effector T cells from properly eliciting an anti-tumor effect. Here, we further evaluated the therapeutic outcome and immune mechanisms of the vaccineinduced immune response enhancement by agonist anti-OX40 antibody, while inhibiting the immunosuppressive IDO enzyme. We demonstrate that therapeutic efficacy of this combinational treatment leads to a profound inhibition of tumor growth and complete regression of established tumors in 60% of treated mice. We show that the mechanisms responsible for this therapeutic potency are: i) an increase in vaccine-induced tumorinfiltrating effector T cells that is facilitated by anti-OX40 antibody, and ii) a decrease of IDO enzyme activity within the tumor and the enhancement in the functionality of effector T cells that are facilitated by 1-methyl tryptophan (1-MT, IDO inhibitor). Our findings provide a promising and translatable strategy that can enhance the overall efficacy of cancer immunotherapy.
    • CXCR2 EXPRESSING TUMOR CELLS DRIVE VASCULAR MIMICRY IN ANTI-ANGIOGENIC THERAPY RESISTANT GLIOBLASTOMA

      Angara, Kartik Prasad; Department of Biochemistry and Molecular Biology (Augusta University, 7/20/2018)
      Glioblastoma (GBM) is a hypervascular and hypoxic neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation to normalize blood vessels, control abnormal vasculatures and prevent recurrence. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT-induced therapy resistance due to activation of alternative neovascularization mechanisms. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties, lay down vascular patterned networks reminiscent of host endothelial blood vessels and served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands in the event of the ensuing hypoxia resulting from AAT. In our studies, we have demonstrated that AAT accelerates VM. We observed that Vatalanib (a VEGFR2 tyrosine kinase inhibitor) induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer side. Interestingly, 20-HETE synthesis inhibitor HET0016 significantly decreased GBM tumors through decreasing VM structures both at the core and at the periphery of the tumors. During our extensive studies to understand the tumor-inherent mechanisms of AAT-induced resistance, we identified a crucial chemokine, CXCL8 or IL-8, to be highly upregulated in the GBM tumors treated with AAT. IL-8 has been well established as a highly prevalent cytokine in GBM with potent pro-migratory and pro-angiogenic functions. AAT-treated groups had significantly higher populations of CXCR2+ glioma stem cells and endothelial-like subpopulations and these populations were decreased following treatment with HET0016 and SB225002 (a CXCR2 antagonist). CXCR2+ GBM tumor cells were shown to form VM-like vascular channels carrying functional RBCs. Knocking down CXCR2 led to smaller tumor size in the animals and improperly developed vascular structures without CXCR2+ GBM cells lining them. This confirms our hypothesis that CXCR2+ GBM cells initiate VM and contribute to AAT resistance in GBM. Our present study suggests that HET0016 and SB225002 have potential to target therapeutic resistance and can be combined with other antitumor agents in preclinical and clinical trials.
    • Deciphering mechanisms of DNA methylation regulation by depletion of the DNA methyltransferases and SETD2

      Tiedemann, Rochelle Lee; Department of Biochemistry and Molecular Biology / Cancer Center (2015)
      DNA methylation (5mC) is a stabile epigenetic mark that confers differential function for gene expression and chromatin accessibility dependent on the context and locality of the mark. Promoter regions populated by CpG islands (CGIs) are highly unmethylated while the remaining ∼80% of CpGs are methylated and distributed across gene bodies, repetitive and transposable elements, and intergenic regions of the genome. The presence and/or absence of particular histone modifications also dictate the patterning of 5mC genome-wide. In cancer, a reversal of 5mC patterns occur in which hypermethylation of tumor suppressor gene CGIs confers gene silencing, and hypomethylation of repetitive and transposable elements contribute to genomic instability. The mechanisms by which 5mC becomes aberrantly regulated in cancer remain unknown. In this study, direct and indirect mechanisms of 5mC regulation were investigated. To understand the direct regulation of 5mC genome-wide, we depleted cell line models of the DNA methyltransferases (DNMTs) that are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) 5mC patterns. Profiling of 5mC patterns on the Illumina HumanMethylation450 BeadChip revealed a unique antithetical relationship between DNMT1 and DNMT3B for the regulation of both 5mC and DNA hydroxymethylation (5hmC) across gene bodies. DNMT3B mediated nonCpG methylation, while DNMT3L influenced the activity of DNMT3B toward nonCG versus CpG site methylation. DNMT3B depletion induced 5mC patterns that closely resemble those observed during cellular differentiation and occurred across gene bodies of highly expressed, H3K36me3-marked genes. SETD2, the histone methyltransferase responsible for H3K36me3 establishment across active gene bodies, was determined to influence the guidance of DNA methylation genome-wide through an indirect mechanism. SETD2 knockout induced widespread loss of H3K36me3 that did not coincide with changes in 5mC. However, paradoxical gains in H3K36me3 significantly induced hypermethylation and upregulation of underlying genes. Genes marked exclusively by the poised enhancer mark, H3K4me1, were commonly targeted for this epigenetic phenotype. DNA methylome profiling of loss-of-function SETD2 mutated clear cell renal cell carcinoma, papillary renal cell carcinoma, and lung adenocarcinoma tumors confirmed the predominance of the hypermethylation phenotype upon loss of SETD2. Collectively, these studies provide novel insight to understanding the regulatory mechanisms by which 5mC patterns are conferred.
    • Determining the Molecular Basis for Depressed Ventricular Contractile Function in Cardiac Neural Creat-Ablated Chick Embryos

      Hatcher, Cathy J; Department of Biochemistry and Molecular Biology (1998-01)
      Ablation of the cardiac neural crest (CNC A) in embryonic chicks at Hamburger- Hamilton stages 8-10 results in a high incidence of persistent truncus arteriosus, a congenital heart defect characterized by a single arterial trunk leaving the heart. Decreased ventricular contractility, which could be due to defects at the level of the contractile apparatus or in the excitation-contraction coupling (ECC) process, has been documented in this model. The first hypothesis that the decrease in isometric force produced per cross-bridge by Triton-skinned ventricular muscle preparations was due to inhibition of the contractile apparatus caused by excessive microtubules was not supported by the data: (1) The total microtubule content and the maximum calcium-activated forc generated by ventricular muscle strips of hearts from embryonic day (ED) 15 CNC A and sham-operated control embryos was not significantly different. (2) Destabilization of microtubules in ventricular muscle strips did not improve the force-producing capability of the contractile apparatus in CNCA embryos. Therefore, microtubules do not appear to be the cause for decreased isometric force production. The second hypothesis that the decrease in ventricular contractility was due to an improperly functioning FKBP1 2 .6 , a ryanodine receptor (RyR) modulatory protein which plays a role in ECC, was supported by the data: (1) FKBP1 2 .6 was present in comparable amounts in hearts from ED 15 CNCA and sham-operated embryos, (2) Dissociation of FKBP1 2 .6 from the RyR had significant effects on intact twitch force and calcium-induced calcium release from the sarcoplasmic reticulum in ED IS sham-operated, but not CNCA embryos. Therefore, FKBP1 2 .6 is present, but not functional in hearts from CNCA embryos, and, thereby, plays a major role in the impaired ECC in these hearts.
    • Development of an Instrument to Measure the Appraisal of Cancer-related Fatigue

      Clark, Jane C.; Department of Biochemistry and Molecular Biology (2001-05)
      Based on a conceptual definition and model of fatigue, a three-phase descriptive study was conducted to develop items for a self-report instrument to measure the appraisal of fatigue. In Phase I, interviews with people experiencing cancer-related fatigue and a review of the literature were used to generate items to sample four constructs of the appraisal of fatigue: Fatigue, meaning, impact, and adaptability to fatigue. In Phase II, items were refined and reduced based on recommendations of a panel of content experts (N=7), instrument development expert, and pilot study results (N=20). In Phase III, reliability and validity estimates of the Fatigue Appraisal Scale were evaluated based on responses of a heterogeneous sample (N=196) of individuals diagnosed with cancer. Acceptable estimates of internal consistency reliability (Cronbach’s alpha) were determined for subscales of Fatigue (a =.91), Impact of Fatigue (a =.89), and Adaptability to Fatigue (a =.74). The internal consistency reliability of the Meaning of Fatigue subscale did not meet the required .60 for a new measurement instrument. To assess construct validity, hypotheses were generated and tested about the relationships of the subscales of the Fatigue Appraisal Scale and selected subscales of the Profile of Mood States-Shortened Form, Fatigue Assessment Instrument, and Functional Assessment of Cancer Therapy-Anemia. Results indicated low to moderate correlations (r = .20 to .70) among the subscales in the directions hypothesized with the exception of the correlation of the Adaptability to Fatigue subscale and the FACT-An Emotional and Functional Well-being subscales and the Impact of Fatigue subscale and the POMS Tension-anxiety subscale. Construct validity was examined further through factor analysis, using principal components analysis with varimax rotation. A four-factor solution with item loadings of .40 or greater on each factor was determined. Four items did not load on any of the four factors and were deleted. The factors were named: Global Fatigue, Impact of Fatigue, Adaptability to Fatigue, and Challenge of Fatigue. Items from the original Meaning of Fatigue subscale loaded on the Global Fatigue, Impact of Fatigue subscale or on the new factor, Challenge of Fatigue. Findings supported the factor structure for the instrument. Recommendations for future validation studies of the 34-item Revised Fatigue Appraisal Scale were offered.
    • Development of Novel Inhibitors of HSP90

      Patwardhan, Chaitanya A.; Department of Biochemistry and Molecular Biology (2014-02)
      Pharmacological inhibition of the Hsp90 machinery is an exciting option for cancer therapy. Clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90, and induced overexpression of anti-apoptotic proteins, Hsp70 and Hsp27, is thought to contribute to this undesired outcome. We therefore face an urgent need to develop much better inhibitors of the Hsp90 machinery that can effectively kill cancer cells with minimal side effects. The goal of this dissertation is to identify novel inhibitors of Hsp90 chaperoning machinery to efficiently kill cancer cells with minimal side effects on normal cell survival. First, we report that the natural product, gedunin, may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to the co-chaperone p23 and inactivates it, without inducing over-expression of Hsp27 and only a relatively modest induction of Hsp70. Using molecular docking and mutational analyses; we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits p23 chaperoning activity, blocks its cellular interaction with Hsp90 and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the Cterminus. These results provide important insight into the molecular mechanism of action of this promising lead compound. Second, we report the development of a novel semi-high-throughput drugscreening assay to identify small molecule inhibitors of Hsp90 and its cochaperones. Our assay quantitatively measures the ability of Hsp90 and its cochaperones to refold the progesterone receptor (PR), a physiological client of Hsp90, in an in vitro assay performed in a 96-well plate format. We tested the NIH clinical collection drug library of 446 compounds and identified capsaicin as a “hit”. Our data show that capsaicin targets the Hsp70-Hsp90 chaperone complex in cells and alters Hsp70 multi-chaperone complexes. It induces cellular destabilization of Hsp90-Hsp70 client proteins and causes degradation of the Hsp70 (induced form) but not the Hsc70 (constitutive form) protein through lysosome-autophagy pathway. Cell survival assays showed that capsaicin selectively kills cancer cells by inducing mitophagy. Taken together, our data suggest that capsaicin could be used in combination with Hsp90 inhibitors for cancer treatment.
    • The Differential Roles of PI3K P110 Isoforms in Regulating CD4 T Cell Subset Polarization

      Webb, Mason James; Department of Biochemistry and Molecular Biology (5/12/2017)
      Class IA phosphatidylinositol-4,5-bisphosphate 3-kinases, or PI3K’s, are one of the earliest bottlenecks for T cell receptor signaling transduction, without which phosphorylated phosphatidylinositides cannot be generated and the T cell activation cascade becomes impaired. Of the catalytic class IA PI3K subunits, there are three isoforms designated as p110α, p110β, and p110δ. The Khleif laboratory has discovered that these catalytic subunits display unique roles in T regulatory cells and non-polarized activated CD4+ T cells. This thesis aims to determine what differential control these p110 isoforms have upon distinct polarized CD4+ T cell subsets.