Pharmacology & Toxicology Department Theses and Dissertations

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The advanced degree programs at the Indiana University of Medicine Department of Pharmacology and Toxicology prepare scientists for careers across the spectrum of biomedical research. The Master of Science (M.S.) degree is a thesis research degree that gives a student the intellectual background to understand and participate in ongoing research projects. The Doctor of Philosophy (Ph.D.) degree is offered for the student who wants to pursue an independent career in research. Students with the Ph.D. degree are prepared for an academic career combining research with teaching or for a career in industrial pharmaceutical research. A combined M.D./Ph.D. degree is open to qualified individuals who ultimately seek to direct biomedical research with a clinical emphasis.

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Analysis of the cryptic promoter in the 5'-UTR of P27
(2012-03-19) Francis, Zachary T.; Zhang, Jian-Ting; Safa, Ahmad R.; Hocevar, Barbara A.
Cyclin Dependent Kinase regulation is often manipulated by cancer cells to promote unlimited proliferation. P27 is an important regulator of Cyclin E/CDK 2, which has been found in low amounts in many types of malignant cancers. Lovastatin has been shown to cause cell cycle arrest in the G1 phase of the cell cycle by increasing the P27 protein. There has been some question, however, if lovastatin regulates P27 at the transcriptional or translational level. Although it has been claimed that P27 expression regulation is due to an IRES located in its 5’UTR, other studies suggested that P27 expression is regulated at the level of transcription. To further investigate the regulation mechanism of P27 expression, the 5’-UTR of P27 and its deletion mutants were examined using a luciferase reporter gene in HeLa cells following exposure to lovastatin. It was found that lovastatin stimulated a significant 1.4 fold increase in its promoter activity of the full length 5’UTR (575). Deletion of 35 nucleotides from the 5’ end of the UTR eliminated the lovastatin-induced increase in promoter activity. Further mapping analyses of the first 35 bases showed that two regions, M1 (575-559) and M3 (543-527), were less sensitive to lovastatin than the other mutated constructs. Since M1 and M3 still showed some activity, a construct was created with deletions in both the M1 and M3 regions. This showed no increase in luciferase activity when exposed to lovastatin. Looking at RNA levels, there was a 1.5 fold increase in RNA when the full length 5’UTR was inserted into HeLa cells and exposed to 81 µM of lovastatin. In contrast, there was no increase in RNA when M1/M3 (575-559; 543-527) was inserted into HeLa cells and exposed to 81 µM of lovastatin. In addition, there was a 1.6 fold increase in endogenous P27 RNA levels after HeLa cells were exposed to 81 µM of lovastatin. In all of these experiments, there seems to be two promoters that work cooperatively: M1 (575-559) and M3 (543-527).
AP2IX-4, a cell cycle regulated nuclear factor, modulates gene expression during bradyzoite development in toxoplasma gondii
(2017-01-10) Huang, Sherri Y.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Lu, Tao; Takagi, Yuichiro; Zhang, Jian-Ting
Toxoplasma gondii is a ubiquitous, protozoan parasite contributing significantly to global human and animal health. In the host, this obligate intracellular parasite converts into a latent tissue cyst form known as the bradyzoite, which is impervious to the immune response. The tissue cysts facilitate wide-spread transmission through the food chain and give rise to chronic toxoplasmosis in immune compromised patients. In addition, they may reactivate into replicating tachyzoites which cause tissue damage and disseminated disease. Current available drugs do not appear to have appreciable activity against latent bradyzoites. Therefore, a better understanding of the molecular mechanisms that drive interconversion between tachyzoite and bradyzoite forms is required to manage transmission and pathogenesis of Toxoplasma. Conversion to the bradyzoite is accompanied by an altered transcriptome, but the molecular players directing this process are largely uncharacterized. Studies of stage-specific promoters revealed that conventional cis-acting mechanisms operate to regulate developmental gene expression during tissue cyst formation. The major class of transcription factor likely to work through these cis-regulatory elements appears to be related to the Apetala-2 (AP2) family in plants. The Toxoplasma genome contains nearly 70 proteins harboring at least one predicted AP2 domain, but to date only three of these T. gondii AP2 proteins have been linked to bradyzoite development. We show that the putative T. gondii transcription factor, AP2IX-4, is localized to the parasite nucleus and exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had negligible effect on tachyzoite replication, but resulted in a reduced frequency of bradyzoite cysts in response to alkaline stress induction – a defect that is reversible by complementation. Microarray analyses revealed an enhanced activation of bradyzoite-associated genes in the AP2IX-4 knockout during alkaline conditions. In mice, the loss of AP2IX-4 resulted in a modest virulence defect and reduced brain cyst burden. Complementation of the AP2IX-4 knockout restored cyst counts to wild-type levels. These findings illustrate the complex role of AP2IX-4 in bradyzoite development and that certain transcriptional mechanisms responsible for tissue cyst development operate across parasite division.
APE1/REF-1 redox signaling regulates HIF1A-mediated CA9 expression in hypoxic pancreatic cancer cells : combination treatment in patient-derived pancreatic tumor model
(2017-12-14) Logsdon, Derek Paul; Kelly, Mark; Fishel, Melissa; Jerde, Travis; Vasko, Michael; Fehrenbacher, Jill
Pancreatic ductal adenocarcinoma (PDAC) is an extremely deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in pancreatic tumors contributes to tumor signaling, fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and decreased therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia by regulating intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein with two major activities: endonuclease activity in DNA base excision repair and a redox signaling activity that reduces oxidized transcription factors, enabling them to bind target sequences in DNA. APE1/Ref-1 is a central node in redox signaling, contributing to the activation of transcription factors involved in tumor survival, growth, and hypoxia signaling. This work evaluates the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1a), a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition results in enhanced PDAC cell killing under hypoxic conditions. We found that HIF1a-mediated induction of CA9 is significantly attenuated following APE1/Ref-1 knock-down or redox signaling inhibition in patient-derived PDAC cells and pancreatic cancer-associated fibroblast cells. Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in enhanced acidification and cytotoxicity of PDAC cells under hypoxic conditions as well as decreased tumor growth in an ex-vivo 3-dimensional tumor co-culture model. Further experiments characterized novel analogs of clinically relevant drugs targeting the key enzymes in this pathway, resulting in improved potency. These results underscore the notion that combination therapy is essential and demonstrate the potential clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment.
Aromatase inhibitors produce hypersensitivity in experimental models of pain : studies in vivo and in isolated sensory neurons
(2014) Robarge, Jason Dennis; Flockhart, David A.; Fehrenbacher, Jill C.; Khanna, Rajesh; Skaar, Todd C.; Vasko, Michael R.
Aromatase inhibitors (AIs) are the current standard of care for the treatment of hormone receptor positive breast cancer in postmenopausal women. Nearly one-half of patients receiving AI therapy develop musculoskeletal toxicity that is characterized by joint and/or muscle pain and approximately one-fourth of patients discontinue their therapy as a result of musculoskeletal pain. Since there are no effective strategies for prevention or treatment, insight into the mechanisms of AI-induced pain is critical to improve treatment. However, there are few studies of AI effects in animal models of nociception. To determine whether AIs produce hypersensitivity in animal models of pain, I examined the effects of AI administration on mechanical, thermal, and chemical sensitivity in rats. The results demonstrate that (1) repeated injection of 5 mg/kg letrozole in male rats produces mechanical, but not thermal, hypersensitivity that extinguishes when drug dosing is stopped; (2) administering a single dose of 1 or 5 mg/kg letrozole in ovariectomized (OVX) rats also induces mechanical hypersensitivity, without altering thermal sensitivity and (3) a single dose of 5 mg/kg letrozole or daily dosing of letrozole or exemestane in male rats augments flinching behavior induced by intraplantar ATP injection. To determine whether the effects of AIs on nociceptive behaviors are mediated by activation or sensitization of peptidergic sensory neurons, I determined whether letrozole exposure alters release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons and from sensory nerve endings in rat spinal cord slices. No changes in basal, capsaicin-evoked or high extracellular potassium-evoked CGRP release were observed in sensory neuronal cultures acutely or chronically exposed to letrozole. Furthermore, letrozole exposure did not alter the ability of ATP to augment CGRP release from sensory neurons in culture. Finally, chronic letrozole treatment did not augment neuropeptide release from spinal cord slices. Taken together, these results do not support altered release of this neuropeptide into the spinal cord as mediator of letrozole-induced mechanical hypersensitivity and suggest the involvement of other mechanisms. Results from this dissertation provide a new experimental model for AI-induced hypersensitivity that could be beneficial in delineating mechanisms mediating pain during AI therapy.
BASE EXCISION REPAIR APURINIC/APYRIMIDINIC ENDONUCLEASES IN APICOMPLEXAN PARASITE TOXOPLASMA GONDII
(2012-03-19) Onyango, David O.; Sullivan, William J., Jr.; Chou, Kai-Ming; Georgiadis, Millie M.; Queener, Sherry F.; Vasko, Michael R.
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Toxoplasma infection is a serious threat to immunocompromised individuals such as AIDS patients and organ transplant recipients. Side effects associated with current drug treatment calls for identification of new drug targets. DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen species produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host immune response. Most of the oxidative DNA damage is repaired through the base excision repair (BER) pathway, of which, the apurinic /apyrimidinic (AP) endonucleases are the rate limiting enzymes. Toxoplasma possesses two different AP endonucleases. The first, TgAPE, is a magnesium-dependent homologue of the human APE1 (hAPE1), but considerably divergent from hAPE1. The second, TgAPN, is a magnesium-independent homologue of yeast (Saccharomyces cerevisiae) APN1 and is not present in mammals. We have expressed and purified recombinant versions of TgAPE and TgAPN in E. coli and shown AP endonuclease activity. Our data shows that TgAPN is the more abundant AP endonuclease and confers protection against a DNA damaging agent when over-expressed in Toxoplasma tachyzoites. We also generated TgAPN knockdown Toxoplasma tachyzoites to establish that TgAPN is important for parasite protection against DNA damage. We have also identified pharmacological inhibitors of TgAPN in a high-throughput screen. The lead compound inhibits Toxoplasma replication at concentrations that do not have overt toxicity to the host cells. The importance of TgAPN in parasite physiology and the fact that humans lack APN1 makes TgAPN a promising candidate for drug development to treat toxoplasmosis.
Biochemical and pharmacological characterization of the Atg8 conjugation system in toxoplasma gondii
(2017-06-28) Varberg, Joseph M.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Mosley, Amber; Safa, Ahmad; Vasko, Michael R.
Toxoplasma gondii is an important human pathogen that infects millions of people worldwide and causing severe and potentially lethal disease in immunocompromised individuals. Recently, a homologue for the autophagy protein Atg8 (TgAtg8) was identified in Toxoplasma that is required for both canonical and noncanonical processes essential for parasite viability. Importantly, TgAtg8 functionality requires its conjugation to phosphatidylethanolamine through the activity of the Atg8 conjugation system. In this thesis, we characterized the proteins that interact with TgAtg8 and TgAtg3, a component of the Atg8 conjugation system, to further define their functions in Toxoplasma and identify opportunities for targeted inhibition of Atg8-related processes. We previously identified that TgAtg8 is acetylated at lysine 23 (K23) and assessed the role of this modification in this thesis. Using mutagenesis, we showed that K23 acetylation did not modulate the interaction with TgAtg3, but appeared to promote TgAtg8 protein stability. Additionally, endogenous mutation of K23 to the nonacetylatable amino acid arginine resulted in severe impairment of parasite replication and spontaneous differentiation into bradyzoites. To gain insight into the role of TgAtg8 in Toxoplasma biology, we next characterized TgAtg8 and TgAtg3 interacting proteins using affinity purification and mass spectrometry. We identified a novel group of interacting proteins that are unique to Toxoplasma, including the dynamin-related protein DrpC. Functional characterization of DrpC identified a potential role of TgAtg8 in trafficking of membrane from the Golgi to the nascent daughter parasites during replication. Lastly, we examined a group of small molecules recently identified as Atg3-Atg8 inhibitors in Plasmodium falciparum and assessed their activity against Toxoplasma. Although the compounds effectively inhibited Toxoplasma replication, they did so through novel mechanisms of action unrelated to the disruption of the TgAtg3-Atg8 interaction. Together, this work provides insight into the function of the Atg8 conjugation system in Toxoplasma that will help guide the future development of novel therapeutics targeting Atg8-related processes.
Cardiac sodium channel palmitoylation regulates channel function and cardiac excitability with implications for arrhythmia generation
(2016-12-09) Pei, Zifan; Cummins, Theodore R.; Oxford, Gerry S.; Hudmon, Andy; Rubart-von der Lohe, Michael; Sheets, Patrick L.
The cardiac voltage-gated sodium channels (Nav1.5) play a specific and critical role in regulating cardiac electrical activity by initiating and propagating action potentials in the heart. The association between Nav1.5 dysfunctions and generation of various types of cardiac arrhythmia disease, including long-QT3 and Brugada syndrome, is well established. Many types of post-translational modifications have been shown to regulate Nav1.5 biophysical properties, including phosphorylation, glycosylation and ubiquitination. However, our understanding about how post-translational lipid modification affects sodium channel function and cellular excitability, is still lacking. The goal of this dissertation is to characterize Nav1.5 palmitoylation, one of the most common post-translational lipid modification and its role in regulating Nav1.5 function and cardiac excitability. In our studies, three lines of biochemistry evidence were shown to confirm Nav1.5 palmitoylation in both native expression background and heterologous expression system. Moreover, palmitoylation of Nav1.5 can be bidirectionally regulated using 2-Br-palmitate and palmitic acid. Our results also demonstrated that enhanced palmitoylation in both cardiomyocytes and HEK293 cells increases sodium channel availability and late sodium current activity, leading to enhanced cardiac excitability and prolonged action potential duration. In contrast, blocking palmitoylation by 2-Br-palmitiate increases closed-state channel inactivation and reduces myocyte excitability. Our computer simulation results confirmed that the observed modification in Nav1.5 gating properties by protein palmitoylation are adequate for the alterations in cardiac excitability. Mutations of potential palmitoylation sites predicted by CSS-Palm bioinformatics tool were introduced into wild-type Nav1.5 constructs using site-directed mutagenesis. Further studies revealed four cysteines (C981, C1176, C1178, C1179) as possible Nav1.5 palmitoylation sites. In particular, a mutation of one of these sites(C981) is associated with cardiac arrhythmia disease. Cysteine to phenylalanine mutation at this site largely enhances of channel closed-state inactivation and ablates sensitivity to depalmitoylation. Therefore, C981 might be the most important site that regulates Nav1.5 palmitoylation. In summary, this dissertation research identified novel post-translational modification on Nav1.5 and revealed important details behind this process. Our data provides new insights on how post-translational lipid modification alters cardiomyocyte excitability and its potential role in arrhythmogenesis.
Cellular Mechanisms Mediating the Actions of Nerve Growth Factor in Sensory Neurons
(2007-08-08T15:24:37Z) Park, Kellie Adrienne; Vasko, Michael R.
Nerve growth factor (NGF) is a neurotrophin upregulated with injury and inflammation. Peripheral administration of NGF causes hyperalgesia and allodynia in animals. Blocking NGF signaling reverses these effects. At the cellular level, chronic exposure of sensory neurons to NGF enhances expression the neurotransmitter, calcitonin gene-related peptide (CGRP). Acute exposure to NGF increases capsaicin-evoked CGRP release from sensory neurons in culture. Thus, NGF increases peptide release from neurons by: (1) increasing expression of peptides, and/or (2) altering their sensitivity. The increase in peptide outflow by either mechanism could contribute to development of hyperalgesia and allodynia. The signaling cascades mediating the actions of NGF in sensory neurons are unclear. Therefore, experiments were designed to determine which pathways regulate changes in iCGRP content and evoked release from primary sensory neurons in culture. The Ras/MEK/ERK cascade was identified as a possible regulator of iCGRP expression in response to NGF. To test this pathway, it was manipulated in neurons by (1) expression of dominant negative or constitutively active isoforms of Ras, (2) farnesyltransferase inhibition, (3) manipulation of the RasGAP, synGAP, and (4) blocking MEK activity. When the pathway was blocked, the NGF-induced increase in iCGRP expression was attenuated. When the Ras pathway was activated, iCGRP expression increased. These data indicate that Ras, and downstream signaling kinases, MEK and ERK, regulate the NGF-induced increases in CGRP in sensory neurons. To determine which pathway(s) regulate the increase in capsaicin-evoked iCGRP release upon brief exposure to NGF, the Ras/MEK/ERK pathway was manipulated as described above, and pharmacological inhibitors of the PI3 kinase, PLC, and Src kinase pathways were used. There were no differences observed in NGF-sensitization when the Ras and PI3 kinase pathways were inhibited, suggesting these two pathways were not involved. However, when the Src kinase inhibitor PP2 was used, the NGF-induced increase in release was completely blocked. Furthermore, the PKC inhibitor, BIM, also inhibited the sensitization by NGF. This data indicate Src and PKC regulate of sensitivity of sensory neurons in response to brief exposure to NGF. Thus, there is differential regulation of iCGRP content and evoked release from sensory neurons in response to NGF.
Characterization of a Novel Hunk Inhibitor in HER2+ Breast Cancer
(2024-07) Dilday, Tinslee Y.; Yeh, Elizabeth; Fehrenbacher, Jill; Brustovetsy, Nickolay; Safa, Ahmad; Sankar, Uma
Human Epidermal Growth Factor Receptor 2 (HER2)-targeted agents have proven to be effective, however, the development of resistance to these agents has become an obstacle in treating HER2+ breast cancer. Prior evidence implicates Hormonally Upregulated Neu-associated Kinase (HUNK) as an anti-cancer target for primary and resistant HER2+ breast cancers. An inhibitor Staurosporine (STU) has been identified as a HUNK inhibitor in HER2+ breast cancer. While STU was determined as a promising tool for inhibiting HUNK, it is a broad-spectrum kinase inhibitor and has not moved forward clinically. Therefore, identifying a more selective inhibitor of HUNK could be critical for targeting HUNK in HER2+ breast and understanding mechanisms by which HUNK promotes resistance to HER2-inhibitors. Specifically, HUNK has been implicated in promoting autophagy as a resistance mechanism in HER2+ breast cancer. Previously, we have identified that HUNK binds and phosphorylates an autophagy inhibitory protein, Rubicon, at Serine (S) 92 in 293T cells. This phosphorylation event causes Rubicon to switch to being an autophagy promoter. However, the role that Rubicon S92 plays in HER2+ breast cancer has yet to be examined. In this study, a novel inhibitor of HUNK is characterized alongside Rubicon S92 phosphorylation. This study establishes that HUNK-mediated phosphorylation of Rubicon at S92 promotes tumorigenesis in HER2/neu+ breast cancer. HUNK inhibition prevents S92 Rubicon phosphorylation in HER2/neu+ breast cancer models and inhibits both autophagy and tumorigenesis. This study characterizes a downstream phosphorylation event as a measure of HUNK activity and identifies a novel HUNK inhibitor that has meaningful efficacy toward HER2+ breast cancer.
CONTRIBUTIONS OF TM5, ECL3 AND TM6 OF HUMAN BCRP TO ITS OLIGOMERIZATION ACTIVITIES AND TRANSPORT FUNCTIONS
(2012-03-16) Mo, Wei; Safa, Ahmad R.; Zhang, Jian-Ting; Chou, Kai-Ming; Hocevar, Barbara A.; Smith, Martin L.
Human BCRP is one of the major ATP-binding cassette transporters involved in the development of multidrug resistance in cancer chemotherapy. Overexpression of BCRP in the tumor cell plasma membrane and apical membrane of the gastrointestinal tract leads to decreased intracellular accumulation of various anticancer drugs as well as reduced drug bioavailability. BCRP has been shown to exist on the plasma membrane as higher forms of homo-oligomers. In addition, the oligomerization domain of BCRP has been mapped to the carboxyl-terminal TM5-ECL3-TM6 and this truncated domain, when co-expressed with the full-length BCRP, displays a dominant inhibitory activity on BCRP function. Thus, the oligomerization of BCRP could be a promising target in reversing multidrug resistance mediated by BCRP. To further dissect the oligomerization domains of human BCRP and test the hypothesis that TM5, ECL3, and TM6 each plays a role in BCRP oligomerization and function, we engineered a series of BCRP domain-swapping constructs with alterations at TM5-ECL3-TM6 and further generated HEK293 cells stably expressing wild-type or each domain-swapping construct of BCRP. Using co-immunoprecipitation and chemical cross-linking, we found that TM5, ECL3, and TM6 all appear to partially contribute to BCRP oligomerization, which are responsible for the formation of oligomeric BCRP. However, only TM5 appears to be a major contributor to the transport activity and drug resistance mediated by BCRP, while ECL3 or TM6 is insufficient for BCRP functions. Taken together, these findings suggest that homo-oligomeric human BCRP may be formed by the interactions among TM5, ECL3 and TM6, and TM5 is a crucial domain for BCRP functions and BCRP-mediated drug resistance. These findings may further be used to explore targets for therapeutic development to reverse BCRP-mediated drug resistance and increase the bioavailability of anti-cancer drugs for better treatment of multidrug resistant cancers.

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