Medical Cell Biology Commons^™

Tetraspanin Kai1/Cd82 Inhibits Cell Migration-Related Cellular Events Via Reorganizing Actin Network, Wei Liu

Theses and Dissertations (ETD)

To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which motility-related cellular events are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-overexpressing cancer cells exhibit various morphologies but typically display elongated cellular extensions and a lack of lamellipodia. Live imaging demonstrated that the formation of lamellipodia and retraction of extensions were deficient upon KAI1/CD82 overexpression.

The deficiency in developing motility-related cellular events was accompanied by defects in actin cortical network and stress fiber formations. Notably, actin polymerization was attenuated by KAI1/CD82. Although Rac1 activity was diminished upon KAI1/CD82 expression, Rac1 …

Dietary Canola Oil Suppressed Growth Of Implanted Mda-Mb 231 Human Breast Tumors In Nude Mice, W. Elaine Hardman

Biochemistry and Microbiology

Long chain omega 3 (n-3) fatty acids, eicosapentaenoic (EPA) and/or docosahexaenoic acid (DHA), have been shown to suppress growth of most cancer cells. In vivo, alpha linolenic acid (ALA, 18:3n-3) can be converted to EPA or DHA. We hypothesized that substituting canola oil (10% ALA) for the corn oil (1% ALA) in the diet of cancer bearing mice would slow tumor growth by increasing n-3 fatty acids in the diet. Sixty nude mice received MDA-MB 231 human breast cancer cells and were fed a diet containing 8% w/w corn oil until the mean tumor volume was 60 mm ³ . …

A Novel Inhibitory Mechanism Of Mitochondrion-Dependent Apoptosis By A Herpesviral Protein, Pinghui Feng, Chengyu Liang, Young C. Shin, Xiaofei E, Weijun Zhang, Robyn Gravel, Ting-Ting Wu, Ren Sun, Edward Usherwood, Jae U. Jung

Dartmouth Scholarship

Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP) of murine gamma-herpesvirus 68 (gammaHV-68) interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1) in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. …

A Dipeptidyl Aminopeptidase-Like Protein Remodels Gating Charge Dynamics In Kv4.2 Channels, Kevin Dougherty, Manuel Covarrubias

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Dipeptidyl aminopeptidase–like proteins (DPLPs) interact with Kv4 channels and thereby induce a profound remodeling of activation and inactivation gating. DPLPs are constitutive components of the neuronal Kv4 channel complex, and recent observations have suggested the critical functional role of the single transmembrane segment of these proteins (Zagha, E., A. Ozaita, S.Y. Chang, M.S. Nadal, U. Lin, M.J. Saganich, T. McCormack, K.O. Akinsanya, S.Y. Qi, and B. Rudy. 2005. J. Biol. Chem. 280:18853–18861). However, the underlying mechanism of action is unknown. We hypothesized that a unique interaction between the Kv4.2 channel and a DPLP found in brain (DPPX-S) may remodel the …

Anti-Tumor Effect Of Doxycycline On Glioblastoma Cells, Andrea Wang-Gillam, Eric Siegel, Debra A. Mayes, Laura F. Hutchins, Yi-Hong Zhou

Neuroscience, Cell Biology & Physiology Faculty Publications

AIM: Glioblastoma multiforme (GBM) is the most common primary brain tumor in humans, and it is highly invasive. Doxycycline, first identified as an antimicrobial agent, is a nonspecific inhibitor of matrix metalloproteinases (MMPs). Our objective was to investigate the anti-MMP effect of doxycycline at therapeutically acceptable levels on glioma cells in vitro.

METHODS: The MTT assay was used to determine the anti-proliferative effects of doxycycline. MMP2 activity and expression were determined by gelatinase zymography and real-time quantitative RT-PCR, respectively. Cell invasion was assessed by Matrigel invasion assay.

RESULTS: Doxycycline exerted mild anti-proliferative effects on all three glioma cell lines …

Uniaxial Stretch-Induced Regulation Of Mitogen-Activated Protein Kinase, Akt And P70s6 Kinase In The Ageing Fischer 344 × Brown Norway Rat Aorta, Kevin M. Rice, Devashish H. Desai, Deborah L. Preston, Paulette S. Wehner, Eric R. Blough

MIIR Faculty Research

The effects of ageing on the cardiovascular system contribute to substantial alterations in cellular morphology and function. The variables regulating these changes are unknown; however, one set of signalling molecules that may be of particular importance in mediating numerous cellular responses, including control of cell growth, differentiation and adaptation, are the proteins associated with the mitogen-activated protein kinase (MAPK) signalling systems. The MAPKs, in conjunction with the p70 S6k signalling cascade, have emerged as critical components for regulating numerous mechanotransduction-related cellular responses. Here we investigate the ability of uniaxial stretch to activate the MAPK and p70 S6k pathways in adult …

Bladder Inflammatory Transcriptome In Response To Tachykinins: Neurokinin 1 Receptor-Dependent Genes And Transcription Regulatory Elements, Ricardo Saban, Cindy Simpson, Rajanikanth Vadigepalli, Sylvie Memet, Igor Dozmorov, Marcia R. Saban

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background Tachykinins (TK), such as substance P, and their neurokinin receptors which are ubiquitously expressed in the human urinary tract, represent an endogenous system regulating bladder inflammatory, immune responses, and visceral hypersensitivity. Increasing evidence correlates alterations in the TK system with urinary tract diseases such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis. However, despite promising effects in animal models, there seems to be no published clinical study showing that NK-receptor antagonists are an effective treatment of pain in general or urinary tract disorders, such as detrusor overactivity. In order to search for therapeutic targets that could …

Molecular Mechanism Of Tetraspanin Cd9 Mediated Cell Motility, Jayaprakash Kotha

Theses and Dissertations (ETD)

CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K …

Cpg Hypomethylation In A Large Domain Encompassing The Embryonic Β-Like Globin Genes In Primitive Erythrocytes, Mei Hsu, Rodwell R. Mabaera, Christopher H. Lowrey, David I. K. Martin, Steven Fiering

Dartmouth Scholarship

There is little evidence addressing the role of CpG methylation in transcriptional control of genes that do not contain CpG islands. This is reflected in the ongoing debate about whether CpG methylation merely suppresses retroelements or if it also plays a role in developmental and tissue-specific gene regulation. The genes of the β-globin locus are an important model of mammalian developmental gene regulation and do not contain CpG islands. We have analyzed the methylation status of regions in the murine β-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types. A large (∼20-kb) …

Untangling The Signalling Wires, Boris N. Kholodenko Phd, Dsci

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Mitogen-activated protein kinase (MAPK) cascades process myriads of stimuli, generating receptor-specific cellular outcomes. New work exploits emergent mathematics of network inference to reveal distinct feedback designs of the RAF/MEK/ERK cascade induced by two different growth factors. It shows that response specificity can arise from differential signal-induced wiring of overlapping protein networks.

Retroviral Vector And Cell-Based Assay For Measuring The Mutation Rate Of Retroviruses Employing Same, Dawn P. Wooley, Kelly Jo Huang

Neuroscience, Cell Biology & Physiology Faculty Publications

Lentiviral-based retrovirus vectors and an in vivo mutation rate assay employing them. More particularly, an assay for directly determining the in vivo mutation rate of HIV-1.

Is Gene Therapy A Good Therapeutic Approach For Hiv-Positive Patients?, Jai G. Marathe, Dawn P. Wooley

Neuroscience, Cell Biology & Physiology Faculty Publications

Despite advances and options available in gene therapy for HIV-1 infection, its application in the clinical setting has been challenging. Although published data from HIV-1 clinical trials show safety and proof of principle for gene therapy, positive clinical outcomes for infected patients have yet to be demonstrated. The cause for this slow progress may arise from the fact that HIV is a complex multi-organ system infection. There is uncertainty regarding the types of cells to target by gene therapy and there are issues regarding insufficient transduction of cells and long-term expression. This paper discusses state-of-the-art molecular approaches against HIV-1 and …

The Enigma Of Struma Ovarii, Lawrence M. Roth, Aleksander Talerman

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Since its first description in the early part of the twentieth century, struma ovarii has elicited considerable interest because of its many unique features; however, at present a number of aspects remain enigmatic. Although the typical presentation is that of a pelvic mass, unusual clinical manifestations such as hyperthyroidism, ascites, and Meigs' syndrome have been recognised. Uncommon macroscopic and especially histological patterns in struma can cause difficulties in diagnosis. Cystic strumas are challenging to diagnose both macroscopically and histologically. Proliferative changes within struma can be misdiagnosed as cancer. In regard to the occurrence of thyroid-type carcinoma in struma ovarii, precise …

The Myc Transactivation Domain Promotes Global Phosphorylation Of The Rna Polymerase Ii Carboxy-Terminal Domain Independently Of Direct Dna Binding, Victoria H. Cowling, Michael D. Cole

Dartmouth Scholarship

Myc is a transcription factor which is dependent on its DNA binding domain for transcriptional regulation of target genes. Here, we report the surprising finding that Myc mutants devoid of direct DNA binding activity and Myc target gene regulation can rescue a substantial fraction of the growth defect in myc^−/− fibroblasts. Expression of the Myc transactivation domain alone induces a transcription-independent elevation of the RNA polymerase II (Pol II) C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) and CDK9 and a global increase in CTD phosphorylation. The Myc transactivation domain binds to the transcription initiation sites of these promoters …

Mlck/Actin Interaction In The Contracting A7r5 Cell And Vascular Smooth Muscle, Sean Eric Thatcher

Theses, Dissertations and Capstones

Myosin light chain kinase (MLCK) is an enzyme that phosphorylates the serine-19 residue on myosin regulatory light chains (MLCs) which serves to activate the Mg2+-ATPase of myosin. This catalytic activity is thought to be the primary role of MLCK; however, it has recently been suggested that MLCK’s actin binding and bundling properties may also be of importance in smooth muscle contraction. In the absence of calcium and calmodulin (CaM), MLCK will bundle actin filaments with its N-terminus. During calcium influx and subsequent CaM activation, MLCK binding to actin decreases resulting in unbundling of actin filaments and allows myosin and actin …

The Expression And Function Of Ppar And Hif-1 In Human Melanoma, Caroline Mills

Theses, Dissertations and Capstones

The first part of my dissertation focuses on the expression and function of PPARs in human melanoma. I found that the A375 cells were significantly growth inhibited in response to _PGJ2 and troglitazone treatment. HEMn-LP showed significant growth inhibition in response to troglitazone. I found that PPARγ and PPARδ mRNA is present in both the SK-Mel 28 and A375 cells. The relative level of PPARα mRNA expression is highest in SK-Mel 28 cells, ~3 fold higher relative to both the normal human melanocytes and A375 cells. PPARγ protein was ~50% higher in both SK-Mel 28 and A375 cells relative to …

Intercenter Reliability And Validity Of The Rhesus Macaque Genechip, Fenghai Duan, Eliot R. Spindel, Yu-Hua Li, Robert B. Norgren

Journal Articles: Genetics, Cell Biology & Anatomy

BACKGROUND: The non-human primate (NHP) research community has been intensely interested in obtaining whole-genome expression arrays for their work. Recently, novel approaches were used to generate the DNA sequence information for a rhesus GeneChip. To test the reliability of the rhesus GeneChip across different centers, RNA was isolated from five sources: cerebral cortex, pancreas, thymus, testis, and an immortalized fibroblast cell line. Aliquots of this RNA were sent to each of three centers: Yerkes National Primate Research Center, Oregon National Primate Research Center and the University of Nebraska Medical Center. Each center labeled the samples and hybridized them with two …

Silencing Of The Pink1 Gene Expression By Conditional Rnai Does Not Induce Dopaminergic Neuron Death In Mice., Hongxia Zhou, Björn H Falkenburger, Jörg B Schulz, Kim Tieu, Zuoshang Xu, Xu Gang Xia

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson's disease. The expression of the shRNA …

Department Of Pathology, Thomas Jefferson University, Identification Of Conserved Gene Expression Features Between Murine Mammary Carcinoma Models And Human Breast Tumors., Jason I Herschkowitz, Karl Simin, Victor J Weigman, Igor Mikaelian, Jerry Usary, Zhiyuan Hu, Karen E Rasmussen, Laundette P Jones, Shahin Assefnia, Subhashini Chandrasekharan, Michael G Backlund, Yuzhi Yin, Andrey I Khramtsov, Roy Bastein, John Quackenbush, Robert I Glazer, Powel H Brown, Jeffrey E Green, Levy Kopelovich, Priscilla A Furth, Juan P Palazzo, Olufunmilayo I Olopade, Philip S Bernard, Gary A Churchill, Terry Van Dyke, Charles M Perou

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: Although numerous mouse models of breast carcinomas have been developed, we do not know the extent to which any faithfully represent clinically significant human phenotypes. To address this need, we characterized mammary tumor gene expression profiles from 13 different murine models using DNA microarrays and compared the resulting data to those from human breast tumors. RESULTS: Unsupervised hierarchical clustering analysis showed that six models (TgWAP-Myc, TgMMTV-Neu, TgMMTV-PyMT, TgWAP-Int3, TgWAP-Tag, and TgC3(1)-Tag) yielded tumors with distinctive and homogeneous expression patterns within each strain. However, in each of four other models (TgWAP-T121, TgMMTV-Wnt1, Brca1Co/Co;TgMMTV-Cre;p53+/- and DMBA-induced), tumors with a variety of …

Single Nucleotide Polymorphisms (Snps) Distinguish Indian-Origin And Chinese-Origin Rhesus Macaques (Macaca Mulatta), Betsy Ferguson, Summer L. Street, Hollis Wright, Carlo Pearson, Yibing Jia, Shaun L. Thompson, Patrick Allibone, Christopher J. Dubay, Eliot Spindel, Robert B. Norgren

Journal Articles: Genetics, Cell Biology & Anatomy

BACKGROUND: Rhesus macaques serve a critical role in the study of human biomedical research. While both Indian and Chinese rhesus macaques are commonly used, genetic differences between these two subspecies affect aspects of their behavior and physiology, including response to simian immunodeficiency virus (SIV) infection. Single nucleotide polymorphisms (SNPs) can play an important role in both establishing ancestry and in identifying genes involved in complex diseases. We sequenced the 3' end of rhesus macaque genes in an effort to identify gene-based SNPs that could distinguish between Indian and Chinese rhesus macaques and aid in association analysis.

RESULTS: We surveyed the …

Modulation Of Ischaemic Contracture In Mouse Hearts: A 'Supraphysiological' Response To Adenosine, Melissa Reichelt, Laura Willems, Jason Peart, Kevin Ashton, G Matherne, Michael Blackburn, John Headrick

Kevin Ashton

While inhibition of ischaemic contracture was one of the first documented cardioprotective actions of exogenously applied adenosine, it is not known whether this is a normal function of endogenous adenosine generated during ischaemic stress. Additionally, the relevance of delayed contracture to postischaemic outcome is unclear. We tested the ability of endogenous versus exogenous adenosine to modify contracture (and postischaemic outcomes) in C57/Bl6 mouse hearts. During ischaemia, untreated hearts developed peak contracture (PC) of 85 ± 5 mmHg at 8.9 ± 0.8 min, with time to reach 20 mmHg (time to onset of contracture; TOC) of 4.4 ± 0.3 min. Adenosine …

Quantitative (Real-Time) Rt-Pcr In Cardiovascular Research, Kevin Ashton, John Headrick

Kevin Ashton

Quantitative (real-time) PCR (qPCR) represents a highly sensitive, sequence-specific, and reproducible technique for the gel-free detection and quantitation of nucleic acids. Owing to its large dynamic range and throughput, this approach has become the chosen method for rapid quantification of mRNA levels in biological samples. The sensitivity of this method permits the reliable detection of low concentrations of initial template, while delivering a linear range of up to 10 orders of magnitude in copy number. This chapter details the basic methodology behind key components of a qPCR experiment, including sample preparation, fluorescent chemistries, primer/probe design, and data analysis applicable to …