Medical Cell Biology Commons^™

Investigation Of Oncogenic Ras And Endoplasmic Reticulum-Mitochondria Calcium Flux And Their Relationship In The Context Of Tumorigenesis, Emma Anderson

Senior Honors Theses

Intracellular calcium as a signaling molecule is a pervasive feature of cellular pathways, especially those that manage internal homeostasis and transitions through the cell cycle, so much so that regulated, responsive calcium flux between the endoplasmic reticulum (ER) and the mitochondria has been suggested to play a major role in cancer development. Another factor commonly implicated in tumorigenesis is RAS, an oncogene that controls signaling for many pathways that are also regulated by calcium. While both calcium and oncogenic RAS signaling are implicated in cancer development, possible links between them have yet to be determined. The identification of these links …

Investigating The Role The Basolateral Amygdala Plays In The Incubation Of Cue-Induced Cocaine Seeking Behavior, Claire M. Corbett

Graduate School of Biomedical Sciences Theses and Dissertations

Cocaine use disorder is a chronic, relapsing brain disease. Sex and ovarian hormones are known to influence cocaine addiction liability and relapse vulnerability. However, little is known regarding the cellular and synaptic mechanisms contributing to sex differences in relapse vulnerability, including how these measures are influenced by hormonal fluctuations. To investigate sex differences in relapse vulnerability we use a rodent model of cocaine craving and relapse called the incubation model in which cue-induced seeking progressively increases or “incubates” during the first month of withdrawal from extended-access cocaine self-administration. Using this model, we have recently shown that females in the estrus …

The Pro-Fibrotic Response Of Mesenchymal Leader Cells To Lens Wounding Involves Hyaluronic Acid, Its Receptor Rhamm, And Vimentin, A. Sue Menko, Alison Romisher, Janice L Walker

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Hyaluronic Acid/Hyaluronan (HA) is a major component of the provisional matrix deposited by cells post-wounding with roles both in regulating cell migration to repair a wound and in promoting a fibrotic outcome to wounding. Both are mediated through its receptors CD44 and RHAMM. We now showed that HA is present in the provisional matrix assembled on the substrate surface in a lens post-cataract surgery explant wound model in which mesenchymal leader cells populate the wound edges to direct migration of the lens epithelium across the adjacent culture substrate onto which this matrix is assembled. Inhibiting HA expression with 4-MU blocked …

68-Year Old Man With Progressive Weakness And Ventilator Dependent Respiratory Failure: A Case Report Of Sporadic Late Onset Nemaline Myopathy, P. Kirupaharan, Daniel Kramer, Alan Gandler, Lawrence C. Kenyon, Ross Summer

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background: Neuromuscular pathologies must be considered when caring for patients with persistent or progressive respiratory failure. Pertinent disease states may involve skeletal muscles of respiration or associated neurologic structures including motor neurons, peripheral neurons and the neuromuscular junction. Diagnosis may require pulmonary function testing, neurophysiologic studies, imaging, and/or muscle biopsy.

Case presentation: A 68-year-old male was transferred to our intensive care unit (ICU) for management of ventilator dependent respiratory failure. Upon further historical review, he described gradually worsening gait instability and muscle weakness, which was previously attributed to vascular Parkinsonism in the setting of known cerebrovascular disease. Upon arrival to …

Fluorescence Imaging Detection Of Nanodomain Redox Signaling Events At Organellar Contacts, David M. Booth, Péter Várnai, Suresh K Joseph, György Hajnóczky

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

This protocol describes how to visualize, detect, and analyze redox signals (oxidative bursts) at the ER-mitochondrial interface. It uses drug-inducible crosslinking to target the genetically encoded glutathione redox sensor Grx1roGFP2 to organellar contact sites to measure local redox changes associated with transient depolarizations of the mitochondrial membrane potential (flickers). The strategy allows imaging of the oxidized to reduced glutathione ratio (GSSG:GSH) in subcellular regions below the diffraction limit with good temporal resolution and minimum phototoxicity. Moreover, the strategy also applies to diverse parameters including pH, H2O2, and Ca2+.

Uncontrolled Mitochondrial Calcium Uptake Underlies The Pathogenesis Of Neurodegeneration In Micu1-Deficient Mice And Patients, Raghavendra Singh, Adam Bartok, Melanie Paillard, Ashley L. Tyburski, Melanie B Elliott, György Hajnóczky

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Dysregulation of mitochondrial Ca2+ homeostasis has been linked to neurodegenerative diseases. Mitochondrial Ca2+ uptake is mediated via the calcium uniporter complex that is primarily regulated by MICU1, a Ca2+-sensing gatekeeper. Recently, human patients with MICU1 loss-of-function mutations were diagnosed with neuromuscular and cognitive impairments. While studies in patient-derived cells revealed altered mitochondrial calcium signaling, the neuronal pathogenesis was difficult to study. To fill this void, we created a neuron-specific MICU1-KO mouse model. These animals show progressive, abnormal motor and cognitive phenotypes likely caused by the degeneration of motor neurons in the spinal cord and the cortex. We found increased susceptibility …

Targeting Sox10-Deficient Cells To Reduce The Dormant-Invasive Phenotype State In Melanoma, Claudia Capparelli, Timothy J. Purwin, Mckenna Glasheen, Signe Caksa, Manoela Tiago, Nicole A. Wilski, Danielle Pomante, Sheera Rosenbaum, Mai Q Nguyen, Weijia Cai, Janusz Franco-Barraza, R. Zheng, Md, Gaurav Kumar, I Chervoneva, Ayako Shimada, Vito W Rebecca, Adam E. Snook, Kim Hookim, Xiaowei Xu, Edna Cukierman, Meenhard Herlyn, A E Aplin

Department of Cancer Biology Faculty Papers

Cellular plasticity contributes to intra-tumoral heterogeneity and phenotype switching, which enable adaptation to metastatic microenvironments and resistance to therapies. Mechanisms underlying tumor cell plasticity remain poorly understood. SOX10, a neural crest lineage transcription factor, is heterogeneously expressed in melanomas. Loss of SOX10 reduces proliferation, leads to invasive properties, including the expression of mesenchymal genes and extracellular matrix, and promotes tolerance to BRAF and/or MEK inhibitors. We identify the class of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) inhibitors as inducing cell death selectively in SOX10-deficient cells. Targeted therapy selects for SOX10 knockout cells underscoring their drug tolerant properties. Combining cIAP1/2 inhibitor …

Suppression Of Pi3k Signaling Is Linked To Autophagy Activation And The Spatiotemporal Induction Of The Lens Organelle Free Zone, Rifah Gheyas, Ramon Ortega-Alvarez, Daniel Chauss, Marc Kantorow, A. Menko

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The terminal steps of lens cell differentiation require elimination of all organelles to create a central Organelle Free Zone (OFZ) that is required for lens function of focusing images on the retina. Previous studies show that the spatiotemporal elimination of these organelles during development is autophagy-dependent. We now show that the inhibition of PI3K signaling in lens organ culture results in the premature induction of autophagy within 24 h, including a significant increase in LAMP1+ lysosomes, and the removal of lens organelles from the center of the lens. Specific inhibition of just the PI3K/Akt signaling axis was directly linked to …

Human Endothelial Cells Promote Arsenic-Transformed Lung Epithelial Cells To Induce Tumor Growth And Angiogenesis Through Interleukin-8 Induction, Lei Zhao, Yi-Fang Wang, Jie Liu, Bing-Hua Jiang, Ling-Zhi Liu

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Arsenic exposure is associated with lung cancer. Angiogenesis is essential for tumor development. However, the role and mechanism of human vascular endothelial cells in tumor growth and angiogenesis induced by arsenic-transformed bronchial epithelial (As-T) cells remain to be elucidated. In this study, we found that endothelial cells significantly increased As-T cell-induced tumor growth compared to those induced by As-T cells alone. To understand the molecular mechanism, we found that endothelial cells co-cultured with As-T cells or cultured in conditioned medium (CM) prepared from As-T cells showed much higher cell migration, proliferation, and tube formation compared to those co-cultured with BEAS-2B …

The Role Of Decorin Proteoglycan In Mitophagy., Thomas Neill, Renato V. Iozzo

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Proteoglycans are emerging as critical regulators of intracellular catabolism. This rise in prominence has transformed our basic understanding and alerted us to the existence of non-canonical pathways, independent of nutrient deprivation, that potently control the autophagy downstream of a cell surface receptor. As a member of the small leucine-rich proteoglycan gene family, decorin has single-handedly pioneered the connection between extracellular matrix signaling and autophagy regulation. Soluble decorin evokes protracted endothelial cell autophagy via Peg3 and breast carcinoma cell mitophagy via mitostatin by interacting with VEGFR2 or the MET receptor tyrosine kinase, respectively. In this paper, we give a mechanistic perspective …

Mir-196a Upregulation Contributes To Gefitinib Resistance Through Inhibiting Gltp Expression, Bing-Jie Liu, Fang-Fang Li, Yun-Xia Xie, Chong-Yuan Fan, Wen-Jing Liu, Jian-Ge Qiu, Bing-Hua Jiang

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Tyrosine kinase inhibitor (TKI) therapy has greatly improved lung cancer survival in patients with epidermal growth factor receptor (EGFR) mutations. However, the development of TKI-acquired resistance is the major problem to be overcome. In this study, we found that miR-196a expression was greatly induced in gefitinib-resistant lung cancer cells. To understand the role and mechanism of miR-196a in TKI resistance, we found that miR-196a-forced expression alone increased cell resistance to gefitinib treatment in vitro and in vivo by inducing cell proliferation and inhibiting cell apoptosis. We identified the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) bound to the …

Ras-Mediated Tumor Stress Adaptation And The Targeting Opportunities It Presents, Alexandra Redding, A E Aplin, Elda Grabocka

Department of Cancer Biology Faculty Papers

Cellular stress is known to function in synergistic cooperation with oncogenic mutations during tumorigenesis to drive cancer progression. Oncogenic RAS is a strong inducer of a variety of pro-tumorigenic cellular stresses, and also enhances the ability of cells to tolerate these stresses through multiple mechanisms. Many of these oncogenic, RAS-driven, stress-adaptive mechanisms have also been implicated in tolerance and resistance to chemotherapy and to therapies that target the RAS pathway. Understanding how oncogenic RAS shapes cellular stress adaptation and how this functions in drug resistance is of vital importance for identifying new therapeutic targets and therapeutic combinations to treat RAS-driven …

A Twist To The Kirby-Bauer Disk Diffusion Susceptibility Test: An Accessible Laboratory Experiment Comparing Haloferax Volcanii And Escherichia Coli Antibiotic Susceptibility To Highlight The Unique Cell Biology Of Archaea, Heather Schiller, Criston Young, Stefan Schulze, Manuela Tripepi, Mechthild Pohlschroder

College of Life Sciences Faculty Papers

Archaea, once thought to only live in extreme environments, are present in many ecosystems, including the human microbiome, and they play important roles ranging from nutrient cycling to bioremediation. Yet this domain is often overlooked in microbiology classes and rarely included in laboratory exercises. Excluding archaea from high school and undergraduate curricula prevents students from learning the uniqueness and importance of this domain. Here, we have modified a familiar and popular microbiology experiment-the Kirby-Bauer disk diffusion antibiotic susceptibility test-to include, together with the model bacterium Escherichia coli, the model archaeon Haloferax volcanii. Students will learn the differences and similarities between …

Reactive Oxygen Species Reprogram Macrophages To Suppress Antitumor Immune Response Through The Exosomal Mir-155-5p/Pd-L1 Pathway, Xiang Li, Shaomin Wang, Wei Mu, Jennifer Barry, Anna Han, Richard L Carpenter, Bing-Hua Jiang, Stephen C Peiper, M G Mahoney, A E Aplin, Hong Ren, Jun He

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background: Cancer cells have an imbalance in oxidation-reduction (redox) homeostasis. Understanding the precise mechanisms and the impact of the altered redox microenvironment on the immunologic reaction to tumors is limited.

Methods: We isolated exosomes from ovarian cancer cells through ultracentrifuge and characterized by Western-blots and Nanoparticle Tracking Analysis. 2D, 3D-coculture tumor model, and 3D live cell imaging were used to study the interactions between tumor cells, macrophages and CD3 T cells in vitro. The role of exosomal miR-155-5p in tumor growth was evaluated in xenograft nude mice models and immune-competent mice models. Flow cytometry and flow sorting were used to …

Novel Oncogenic Transcription Factor Cooperation In Rb-Deficient Cancer, Amy C. Mandigo, Ayesha A Shafi, Jennifer J Mccann, Wei Yuan, Talya Laufer, Denisa Bogdan, Lewis Gallagher, Emanuela Dylgjeri, Galina Semenova, Irina A Vasilevskaya, M J Schiewer, Chris M Mcnair, Johann S De Bono, Karen E Knudsen

Department of Cancer Biology Faculty Papers

The retinoblastoma tumor suppressor (RB) is a critical regulator of E2F-dependent transcription, controlling a multitude of protumorigenic networks including but not limited to cell-cycle control. Here, genome-wide assessment of E2F1 function after RB loss in isogenic models of prostate cancer revealed unexpected repositioning and cooperation with oncogenic transcription factors, including the major driver of disease progression, the androgen receptor (AR). Further investigation revealed that observed AR/E2F1 cooperation elicited novel transcriptional networks that promote cancer phenotypes, especially as related to evasion of cell death. These observations were reflected in assessment of human disease, indicating the clinical relevance of the AR/E2F1 cooperome …

Microbial Cell Factory Of Baccatin Iii Preparation In Escherichia Coli By Increasing Dbat Thermostability And In Vivo Acetyl-Coa Supply, Jia-Jun Huang, Tao Wei, Zhi-Wei Ye, Qian-Wang Zheng, Bing-Hua Jiang, Wen-Feng Han, An-Qi Ye, Pei-Yun Han, Li-Qiong Guo, Jun-Fang Lin

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Given the rapid development of genome mining in this decade, the substrate channel of paclitaxel might be identified in the near future. A robust microbial cell factory with gene dbat, encoding a key rate-limiting enzyme 10-deacetylbaccatin III-10-O-transferase (DBAT) in paclitaxel biosynthesis to synthesize the precursor baccatin III, will lay out a promising foundation for paclitaxel de novo synthesis. Here, we integrated gene dbat into the wild-type Escherichia coli BW25113 to construct strain BWD01. Yet, it was relatively unstable in baccatin III synthesis. Mutant gene dbat S189V with improved thermostability was screened out from a semi-rational mutation library …

Opa1 Modulates Mitochondrial Ca 2+ Uptake Through Er-Mitochondria Coupling, Benjamín Cartes-Saavedra, Josefa Macuada, Daniel Lagos, Duxan Arancibia, María E Andrés, Patrick Yu-Wai-Man, György Hajnóczky, Verónica Eisner

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Autosomal Dominant Optic Atrophy (ADOA), a disease that causes blindness and other neurological disorders, is linked to OPA1 mutations. OPA1, dependent on its GTPase and GED domains, governs inner mitochondrial membrane (IMM) fusion and cristae organization, which are central to oxidative metabolism. Mitochondrial dynamics and IMM organization have also been implicated in Ca2+ homeostasis and signaling but the specific involvements of OPA1 in Ca2+ dynamics remain to be established. Here we studied the possible outcomes of OPA1 and its ADOA-linked mutations in Ca2+ homeostasis using rescue and overexpression strategies in Opa1-deficient and wild-type murine embryonic fibroblasts (MEFs), respectively and in …

Phytochemical Constituents Of Aquilaria Malaccensis Leaf Extract And Their Anti-Inflammatory Activity Against Lps/Ifn-Γ-Stimulated Raw 264.7 Cell Line, Eman Ramadan, Manar A. Eissa, Yumi Z. H-Y Hashim, Dina M. El-Kersh

Pharmacy

This study aims to identify the major phytochemical constituents in Aquilaria malaccensis (Thymelaeaceae) ethanolic leaf extract (ALEX-M) and elucidate their ability to suppress nitric oxide (NO) production from a murine macrophage-like cell line (RAW 264.7) stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Dichloromethane (DCM) and ethyl acetate (EtOAc) fractions of ALEX-M were subjected to column chromatography. Eight known compounds were isolated for the first time from this species. Compounds were identified using spectroscopic techniques (IR, UV, HRESIMS, and 1D and 2D NMR). Anti-inflammatory activity of both extract and isolated compounds were investigated in vitro. The fractions offered the isolation of …

The Effect Of Creatine On Immortalized Schwann Cell Proliferation, Peyton Kimmel, Caitlyn Henry, Angela Asirvatham

Student Research Poster Presentations 2022

Creatine is an important component of the high-energy phosphate transfer to regulate cellular levels of ATP. The functions of creatine supplementation are well studied in the muscular and skeletal system, however the impact of creatine in the nervous system, specifically Schwann cells, is relatively unknown. Schwann cell growth in vitro is facilitated by heregulin, a neuron secreted growth factor, and forskolin, a pharmacological agent that activates cAMP. In peripheral nervous system injuries, recovery time is based around Schwann cell’s ability to proliferate and differentiate, and it is unknown what role creatine plays in this process. It was hypothesized that increasing …

Uveitis-Mediated Immune Cell Invasion Through The Extracellular Matrix Of The Lens Capsule, Jodirae Dedreu, Sonali Pal-Ghosh, Mary J Mattapallil, Rachel R Caspi, Mary Ann Stepp, A Sue Menko

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

While the eye is considered an immune privileged site, its privilege is abrogated when immune cells are recruited from the surrounding vasculature in response to trauma, infection, aging, and autoimmune diseases like uveitis. Here, we investigate whether in uveitis immune cells become associated with the lens capsule and compromise its privilege in studies of C57BL/6J mice with experimental autoimmune uveitis. These studies show that at D14, the peak of uveitis in these mice, T cells, macrophages, and Ly6G/Ly6C+ immune cells associate with the lens basement membrane capsule, burrow into the capsule matrix, and remain integrated with the capsule as immune …