Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Dihydromotuporamine C increases Na⁺/H⁺exchange activity via a Rho-kinase-dependent pathway and induces.. Sinotte, Ryan Richard 2003

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
[if-you-see-this-DO-NOT-CLICK]
ubc_2003-0426.pdf [ 3.88MB ]
Metadata
JSON: 1.0091028.json
JSON-LD: 1.0091028+ld.json
RDF/XML (Pretty): 1.0091028.xml
RDF/JSON: 1.0091028+rdf.json
Turtle: 1.0091028+rdf-turtle.txt
N-Triples: 1.0091028+rdf-ntriples.txt
Original Record: 1.0091028 +original-record.json
Full Text
1.0091028.txt
Citation
1.0091028.ris

Full Text

DIHYDROMOTUPORAMINE C INCREASES Na+/H+ EXCHANGE ACTIVITY VIA A RHO-KINASE-DEPENDENT PATHWAY AND INDUCES DISRUPTION OF INTERCELLULAR JUNCTIONS IN MAMMARY EPITHELIA by RYAN RICHARD SINOTTE B.Sc. (Cell Biology & Genetics), The University of British Columbia, 2000 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF ANATOMY & CELL BIOLOGY) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 2003 © Ryan Richard Sinotte, 2003 UBC Rare Books and Special Collections - Thesis Authorisation Form Page 1 of 1 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. 1/ The University of British Columbia Vancouver, Canada http://www.library.ubc.ca/spcoll/thesauth.html 17/07/2003 ABSTRACT Invasion and angiogenesis are two major steps in the progression towards metastatic cancer. Dihydromotuporamine C (dh-MotC) inhibits both of these processes. To better characterize the mechanism of dh-MotC action, I examined the compound's effects on Na+/H+ exchange (NHE) activity and normal mammary epithelial cell viability and junctional architecture. In contrast to squalamine, an anti-angiogenic compound that inhibits Na+/H+ exchanger isoform 3 (NHE3), dh-MotC (5 or 10u.M) increased the rate of recovery from an imposed acid load in nominally bicarbonate free conditions. This increased rate of recovery was inhibited by HOE694, a selective NHE inhibitor, and the Rho-kinase (ROCK) inhibitor Y-27632. Dh-MotC also induced an upward shift in steady state intracellular pH (pHi) of 0.14 +/- 0.02 pH units (n=6), which was not sensitive to ROCK inhibition. Therefore, dh-MotC activates Na+/H+ exchange via a ROCK-mediated cascade, and increases steady-state pHj in a ROCK-independent manner. Dh-MotC has been described as a Rho activator and is known to induce stress fibre and focal adhesion formation in serum-starved Swiss 3T3 cells. Inhibition of Na+/H+ exchange by HOE694 did not prevent dh-MotC-induced formation of stress fibres or focal adhesions. Similarly, the dh-MotC-mediated inhibition of tumour cell migration was not prevented by co-treatment with HOE694. These data suggest that increased Na+/H+ exchange activity is not required for dh-MotC ii induced stress fibres/focal adhesions and that dh-MotC inhibits tumour cell migration via a pathway that is independent of Na+/H+ exchange. Concentrations of dh-MotC that are effective at preventing tumour cell migration were toxic to normal mammary epithelial cells cultured on three dimensional basement membrane gels as evidenced by increased levels of active caspase-3 staining in dh-MotC treated mammary spheroids. Adherens and tight junction architecture was also disrupted under these conditions as evidenced by the loss of junctional E-cadherin and apically polarized ZO-1, components of adherens and tight junctions, respectively. In two-dimensional monolayer mammary epithelial cell cultures, dh-MotC did not affect cell morphology or localization of E-cadherin and ZO-1. Thus, there appears to be a differential response to dh-MotC between monolayer and spheroid cell culture. iii TABLE OF CONTENTS ABSTRACT II LIST OF TABLES VILIST OF FIGURES VIII LIST OF ABBREVIATIONS XACKNOWLEDGEMENTS XIII CHAPTER I. INTRODUCTION Motuporamin.es 1 Squalamine 4 Na+/H+ Exchange (NHE) 4 Pharmacological Inhibition of Na+/H+ Exchange 6 Dual Functions of NHEs 6 Signal Transduction and Regulation of NHE1 12 Signal Transduction and Regulation of NHE3 17 The Role of Na+/H+ Exchange in Cellular Migration 21 Rho GTPases 22 Rho GTPase Effectors 24 Signalling to Rho GTPases 5 Pharmacological Inhibition of Rho and Rho-Kinase (ROCK) 26 Actin Stress Fibres and Focal Adhesions 27 iv Rho and Cellular Migration The Role of Rho in Intercellular Junctions Objectives and Hypotheses 29 30 32 CHAPTER II. MATERIALS AND METHODS Cell Lines 33 Dihydromotuporamine C (dh-MotC) 3Cell Culture 34 Maintenance of a Pure Mammary Epithelial Cell Population 35 Matrigel™ Basement Membrane Matrix (EHS) 35 Preparation of Matrigel Coated Dishes and Coverslips 36 Differentiation Assay 3Western Blot Analysis - For P-casein 37 Determination of Intracellular pH via Fluorescence Ratio Imaging Loading Cultured Cells with BCECF 39 Fluorescence Ratio Imaging 3Acid Loading 41 Calculation of Intracellular pH 42 Analysis of Steady-State pHi and 3 Rates of Recovery from Acid Loads Wound Healing Assay 44 Stress Fibre/Focal Adhesion Assay 4V Immunofluorescence Staining and Microscopy 45 E-cadherin staining 4ZO-1 staining 6 Active caspase-3 staining 4Vinculin staining 47 Actin stainingCHAPTER III. RESULTS Dihydromotuporamine C activates Na+/H+ exchange 48 in Swiss 3T3 cells. Dihydromotuporamine C alters normal mammary epithelial 82 cell morphology. CHAPTER IV. DISCUSSION 94 V. REFERENCES 109 vi LIST OF TABLES Table # Table 1. Page # Dihydromotuporamine C increases steady-state intracellular pH (pHj) in Swiss 3T3 cells. 57 Table 2. Dihydromotuporamine C increases steady-state 79 intracellular pH (pH|) in Swiss 3T3 cells in the presence of Y-27632. vii LIST OF FIGURES Figure # Page # Fig. 1 Chemical structures of motuporamine C, 3 dihydromotuporamine C (dh-MotC) and squalamine. Fig. 2 Chemical structures of HOE694, a selective inhibitor of Na+/H+ exchange, and Y-27632, a Rho-kinase (ROCK) inhibitor. Fig. 3 Dual functions of the Na+/H+ exchanger. 10 Fig. 4 Summary of molecular pathways leading 14 to NHE1 activation. Fig. 5 Summary of molecular pathways that 19 either activate or inhibit NHE3. Fig. 6 Swiss 3T3 cells recover from an imposed acid to 50 steady-state pHj. Fig. 7 Dihydromotuporamine C does not inhibit Na+/H+ 53 exchange in Swiss 3T3 cells. Fig. 8 Dihydromotuporamine C increases steady-state 55 pHj in Swiss 3T3 cells. Fig. 9 Dihydromotuporamine C increases the rate of 60 intracellular pH (pHj) recovery (c/pHj/dt) following an imposed acid load at a test pHj of 6.6. viii Fig. 10 Dihydromotuporamine C increases the rate of intracellular pH (pHj) recovery (cfpHj/dt) across a range of physiological pHj values following an imposed acid load. Fig. 11 Acid load recovery in Swiss 3T3 cells can be inhibited, in a reversible manner, by a known inhibitor of Na+/H+ exchange, HOE694. Fig. 12 Dh-MotC-induced increases in the rates of recovery from acid loads can be inhibited, in a reversible manner, by HOE694 Fig. 13 Dh-MotC and HOE694 inhibit migration of MDA-MB-231 breast carcinoma cells. Fig. 14 HOE694 treatment does not prevent stress fibre . formation in Swiss 3T3 cells. Fig. 15 HOE694 does not inhibit focal adhesion formation in Swiss 3T3 cells. Fig. 16 Swiss 3T3 cells co-treated with dh-MotC and Y-27632, a ROCK inhibitor, do not exhibit an increase in the rate of recovery from an acid load. Fig. 17 ROCK inhibition prevents dh-MotC-induced increases in rates of recovery from an acid load At a test pHi of 6.45. Fig. 18 Dihydromotuporamine C treatment of mammary epithelial cells results in cell rounding in a dose-dependent manner. ix Fig. 19 Differentiated mammary epithelial cells treated 87 with dihydromotuporamine C do not express the milk protein [3-casein. Fig. 20 Undifferentiated mammary epithelial cells treated 90 with dihydromotuporamine C contain E-cadherin and ZO-1 at sites of cell-cell contact and are negative for active caspase-3. Fig. 21 Dihydromotuporamine C treated differentiated 92 mammary epithelial cells do not polarize and have elevated levels of active caspase-3. Fig. 22 Summary of the actions of Rho on cell migration 100 and focal adhesion formation. Fig. 23 Potential mechanism of dh-MotC-mediated 105 disruption of intercellular junctions. x LIST OF ABBREVIATIONS 7TM Seven-transmembrane AE Anion exchanger BCECF-AM 2\7'-bis-(2-carboxymethyl)-5-(and-6-)-carboxyfluorescein- acetoxymethyl ester BSA Bovine serum albumin cAMP Cyclic adenosine 3',5'-mono-phosphate CNF-1 Cytotoxic necrotizing factor-1 CPT Camptothecin Dh-MotC Dihydromotuporamine C DMA Dimethyl amiloride DMEM Dulbecco's modified Eagle's medium DMSO Dimethyl sulfoxide ECL Enhanced chemiluminescence ECM Extracellular Matrix EGF Epidermal growth factor EHS Engelbreth-Holm-Swarm El PA ethylisopropyl amiloride ERK Extracellular regulated kinase ERM Ezrin, radixin, moesin FAK Focal adhesion kinase FBS Fetal bovine serum FGF Fibroblast growth factor GAP GTPase activating protein GDI GDP-dissociation inhibitor GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GTP Guanosine triphosphate HBSS Hank's balanced salt solution HEPES N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) ILK Integrin-linked kinase LPA Lysophosphatidic acid MDCK Madin-Darby-canine-kidney MLC Myosin light chain MPA 5-N-(methylpropyl)amiloride NBC Sodium-bicarbonate co-transporter NDAE Sodium-driven anion exchanger NGS Normal goat serum NHE Sodium-proton exchange NHERF NHE regulatory factor PAGE Polyacrylamide gel electrophoresis PBS Phosphate-buffered saline PDGF Platelet-derived growth factor PI3K Phosphatidyl-inositol-3-kinase xi PKA Protein kinase A PKC Protein kinase C Ras Rat sarcoma Rho Ras homology ROCK Rho-kinase RTK Receptor tyrosine kinase TER Trans-epithelial resistance TM Transmembrane WASP Wiskott-Aldrich syndrome protein ZO-1 Zonula-occludens 1 xii ACKNOWLEDGEMENTS I am pleased to have this opportunity to thank those who have aided me and provided guidance during my time as a graduate student at UBC. I would first like to thank Dr. Calvin Roskelley, my supervisor and co-worker, for his guidance in these last three years and for proofing numerous drafts of this thesis. I would like to thank Dr. John Church for his guidance and for devoting his time and his lab's resources to this project. Thanks to Dr. Michel Roberge, whose lab motuporamine was discovered in, for his input into the direction of this project. Thanks to Dr. Timothy O'Connor who graciously agreed to be my external examiner on short notice. Special thanks to my fellow Roskelley lab members Marcia McCoy, Aruna Somasiri, Shona Penhale, Jane Cipollone, Adrian Lenahan, Colleen Wu and Lianne McHardy for your support and friendship over the years. Thanks to Claire Sheldon for all of your help on this project, I could not have completed it without your training and advice. I would like to thank the rest of the students, faculty and staff in the Department of Anatomy & Cell Biology for making my stay here a great experience. This is a terrific department to be a part of, as there is a great sense of community and many departmental social and recreational activities to take part in. Finally, I would like to thank my family and friends, especially Erin Neall, my future wife, for all your support during my studies. This work was supported by grants from the National Cancer Institute of Canada (C. Roskelley) and the BC & Yukon Heart and Stroke Foundation (J. Church). xiii I. Introduction Motuporamines Motuporamines are alkaloids derived from the marine sponge Xestospongia exigua (Kirkpatrick) that have been shown to inhibit tumour cell invasion and angiogenesis (Roskelley et al., 2001). Marine sponges are a rich source of structurally novel secondary metabolites that have potential as lead compounds for the development of new anticancer drugs. Extracts from X. exigua were found to be selectively cytotoxic against a panel of human cancer cell lines and bioassay-guided fractionation of these extracts led to the isolation of motuporamines A, B and C (Williams et al., 1998). Further fractionation led to the isolation of motuporamines D, E, F, G, H and I (Williams et al., 2002). In a subsequent assay, motuporamines A, B, C, G, H and I were shown to inhibit invasion. However, only motuporamine C has demonstrated anti-angiogenic properties (Williams et al., 2002). Motuporamines consist of a macrocyclic alkaloid ring with a spermidine-like tail. Acetylation of the central amine group in the spermidine-like tail of motuporamine C abolishes the anti-invasive properties of the drug, whereas the terminal amine group does not appear to be important (Roskelley et al., 2001). Motuporamines show some similarity in structure to squalamine, another inhibitor of invasion and angiogenesis (Figure 1). 1 Figure 1. Chemical structures of motuporamine C, dihydromotuporamine C (dh-MotC) and squalamine. Motuporamine consists of a macrocyclic ring attached to a polyamine tail. Dihydromotuporamine C is a water-soluble analog of the natural motuporamine C compound, which does not contain a double bond in the macrocyclic ring. Squalamine is a cholestane steroid attached to spermidine, a structure that fundamentally resembles motuporamine C. 2 Motuporamine C .NH; Di-hydro Motuporamine C NH-\/\/\ /\/\ <Mr^>" N N H OH H H Squalamine 3 Squalamine Squalamine is an aminosterol, which is isolated from the shark Gl tract. Squalamine is a 7,24-dihydroxlated 24-sulfated cholestane steroid conjugated to spermidine at C-3 (Li et al., 2002). Like squalamine, Dihydromotuporamine C (dh-MotC) also consists of a macrocyclic ring attached to a spermidine-like tail. Squalamine has been shown to inhibit invasion and angiogenesis and is currently in Phase II clinical trials for treatment of small-cell lung cancer (Li et al., 2002). It is also known that squalamine inhibits the Na+/H+ exchanger isoform NHE3, the Na+/H+ exchanger isoform found in intestinal and renal brush borders (Akhter et al., 1999). Therefore, we wanted to determine if Dihydromotuporamine C had any effects on Na+/H+ exchange, given the parallels in structure and effects on tumour cells that exist between dh-MotC and squalamine. Na*/H* Exchange Proton fluxes across membranes are involved in a number of physiological processes such as proliferation rate, cell volume changes, migration and intracellular communication (Schrode et al., 1997). Maintenance of intracellular pH (pHj) close to neutrality is a crucial element in cellular homeostasis, hence cells have evolved several mechanisms to regulate intracellular [H+]. Intracellular pH modulation at the plasma membrane is regulated by several families of ion exchangers. These include Na+/H+ exchangers (NHEs) and HCO3"transporters, such as the CI/HCO3"exchangers (AEs), the Na+ driven CI7HC03"exchanger (NDAE) and the Na+/HC03" co-transporters (NBCs) (Putney et al., 2002). 4 Na+/H+ exchangers are electroneutral and posses a 1:1 stoichiometry for exchange of extracellular Na+ with intracellular H+ (Wakabayashi et al., 1997). To date, eight NHE family members have been identified. All share structural homology with each other and with HCO3" transporters. Members of these families are predicted to have between 10 and 13 transmembrane regions with cytoplasmic C and N terminal domains (Kyte and Doolittle, 1982). NHE1 is ubiquitously expressed and plays a central housekeeping role in maintaining pH and cell volume homeostasis (Wakabayashi et al., 1997). NHEs 2 and 4 are predominantly expressed in the kidney and Gl tract; NHE3 is apically localized in renal proximal tubule epithelia, and intestinal brush border epithelia (Tse et al., 1992). NHE5 is found primarily in the brain (Baird et al., 1999). NHE6 is distributed in recycling compartments and transiently appears on the plasma membrane (Brett et al., 2002); NHE7 is found primarily in the trans-Golgi network (Numata & Orlowski, 2001). A partial sequence has been obtained for NHE8, which has been found in the renal proximal tubule (Goyal et al., 2003). NHEs have been described in many species including Escherichia coli, Arabidopsis thaliana, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, rat, mouse, pig, trout and human (reviewed by Putney et al., 2002). 5 Pharmacological Inhibition ofNa*/H* Exchange There are two major classes of pharmacological agents that are used to inhibit NHEs. One class includes amiloride and its derivatives, ethylisopropyl amiloride (EIPA), dimethylamiloride (DMA) and 5-N-(methylpropyl)amiloride (MPA) (Orlowski, 1993). Amiloride and its derivatives have also been shown to inhibit ion translocation in a non-competitive manner (Warnock et al., 1988). The second class of inhibitors are the benzoylguanidines and their derivatives such as HOE694 (Figure 2) and HOE642 (cariporide) (Counillon et al., 1993). The benzoylguanidine derivatives (HOE694/642) are known to be approximately 4000 times more efficient in inhibiting NHE1 compared to NHE3 (Counillion and Pouyssegur, 2000). NHE inhibitors are thought to act by competitively inhibiting Na+ binding at the extracellular cation binding site (Counillon etal., 1993). Dual Functions of NHEs While, the transmembrane portion of the NHEs functions in the exchange of intracellular H+ for extracellular Na+, it has been shown that NHE1 directly associates with the actin-binding ERM proteins (ezrin, radixin and moesin) at the cytoplasmic C-terminus of the exchanger (Figure 3) (Denker et al., 2000). In addition, NHE3 associates with NHE regulatory factors 1 and 2 (NHERFs), which in turn bind to ezrin (Kurashima et al., 1999). These interactions could impact on a number of cellular functions. For example, regulated attachment of membrane proteins to filamentous actin (F-actin) is essential for many processes including cell adhesion and motility, determination of cell surface shape, cytokinesis, 6 Figure 2. Chemical structures of HOE694, a selective inhibitor of Na+/H+ exchange, and Y-27632, a Rho-kinase (ROCK) inhibitor. The benzoylguanidine derivatives (HOE694/642) are known to be approximately 4000 times more efficient in inhibiting NHE1 compared to NHE3 (Counillion and Pouyssegur, 2000). NHE inhibitors are thought to act by competitively inhibiting Na+ binding at the extracellular cation binding site (Counillon et al., 1993). Y-27632, a pyridine derived, highly-potent cell permeable selective inhibitor of Rho-Kinase (ROCK) acts by competing with ATP binding to the catalytic site. 7 Figure 3. Dual functions of the Na+/H+ exchanger. NHEs are 10-13TM proteins that exchange intracellular H+ for extracellular Na+ in an electroneutral 1:1 stoichiometric ratio. They also link to F-actin at the cytoplasmic C-terminus through the ERM family of proteins. NHE1 directly associates with ERM proteins (pictured), while NHE3 associates with ERMs through NHE regulatory factors (NHERFs). 9 10 phagocytosis and the integration of membrane transport with signalling pathways (reviewed by Bretscher et al., 2002). Mutations in NHE1 that disrupt ERM binding, but not ion translocation, cause impaired organization of focal adhesions and actin stress fibres (Denker et al., 2000). NHE1 and ERM proteins also co-localize in lamellipodia (Denker et al., 2000). However, EIPA treatment results in impaired cell adhesion and spreading in hamster lung fibroblasts, which suggests the ion translocation function and the cytoskeletal anchoring functions may be interrelated (Tominaga and Barber, 1998). Indeed, migration in a wounding assay is impaired in fibroblasts expressing NHE1 with mutations that independently disrupt ion translocation and ERM binding (Denker and Barber, 2002). NHE3 may also possess a similar dual-function as disruption of the actin cytoskeleton with cytochalasins B, D and latrunculin B results in inhibition of NHE3 activity and causes subcellular redistribution of NHE3 to sites of actin aggregation (Kurashima et al., 1999). Squalamine, an NHE3 inhibitor, prevents tumour cell invasion and angiogenesis by preventing directed cell migration (Sills et al., 1998). Taken in concert, the data supports a role for NHE1 and 3 in cell motility, actin stress fibre formation, focal adhesion formation and cell shape changes through interaction with the actin cytoskeleton. 11 Signal Transduction and Regulation ofNHEl Na+/H+ exchange can be activated via a variety of mechanisms, including growth factor signalling (Wakabayashi era/., 1992), hormones (Winkel et al., 1993) and osmotic stress (Grinstein et al., 1992). Various pathways leading to NHE1 activation are summarized in Figure 4. Activation of Na7H+ exchange is mediated by the C-terminal cytoplasmic domain, which acts to alter the affinity of the exchanger for intracellular H+ (Wakabayashi et al., 1992). The mechanism for altering H+ affinity has not been characterized at the present time, however there is evidence that in the chloride-bicarbonate anion exchanger, AE1 (which shares a similar topology with NHE1), the juxtamembrane region of the cytoplasmic domain may act as a flexible hinge that facilitates conformational changes that initiate interactions between the cytoplasmic and transmembrane domains (Wang, 1994). NHE1 is thought to remain constitutively at the plasma membrane, and is not regulated by recruitment of internal NHE1 stores (Schrode et al., 1998). This is in contrast to NHE3, which is primarily regulated by the number of exchangers present at the plasma membrane that in turn mediate changes in Vmax (the maximal velocity of Na7H+ exchange; Janecki et al., 1998). Phosphorylation of NHE1 is also important for regulation of the exchanger by receptor-mediated signalling mechanisms. Specifically, LPA, thrombin, endothelin and integrin-mediated activation of NHE1 require phosphorylation of the exchanger (Putney et al., 2002). In contrast, activation of NHE1 in response to osmotic stress (Grinstein et al., 1992) or ATP depletion (Goss et al., 1994) can 12 Figure 4. Summary of molecular pathways leading to NHE1 activation. NHE1 is regulated by a variety of intracellular second messengers. This type of regulation is also dependent on the cell type in which the exchanger is expressed. 13 14 occur without phosphorylation of the exchanger. In certain situations a combination seems optimal; activation of NHE1 in response to intracellular acidification can occur without phosphorylation of the exchanger, but it is enhanced by pre-treatment with growth factors and the resultant phosphorylation of NHE1 (Takahashi etal., 1999). There are several well-known intracellular signalling cascades which are capable of activating NHE1 (Figure 4). NHE1 activity can be increased through the actions of the Ras-mediated ERK (extracellular regulated kinase) cascade, however ERK does not directly activate or phosphorylate NHE1 (Bianchini et al., 1997). P90 ribosomal S6 kinase (p90RSK), a serine/threonine kinase that acts downstream of ERK, has been shown to directly phosphorylate NHE1 in a MEK-ERK dependent pathway (Moor and Fliegel, 1999). Receptor tyrosine kinase (RTK) activation of NHE1 can occur via a protein kinase C (PKC)-mediated cascade through a mechanism that is both dependent (Ma et al., 1994) and independent (Di Sario et al., 1999) of phosphatidyl-inositol-3 (PI3)- kinase. Other mechanisms of NHE1 activation through RTK signalling also exist, for example, PDGF stimulated activation of NHE1 is mediated via a Nek cascade. Nek is a Grb-2-like protein that binds to the cytoplasmic domains of RTKs. This activation is ultimately triggered by Nek interacting kinase (NIK), a downstream effector of Nek that directly phosphorylates NHE1 (Yan et al., 2001). 15 NHE1 contains two calmodulin binding sites (high & low affinity) located on the cytoplasmic C-terminus (Bertrand et al., 1994). The high affinity calmodulin binding domain regulates NHE activity in response to Ca2+ mediated signalling (Wakabayashi et al., 1994). When calmodulin is not bound, the domain may function as an autoinhibitory domain by interacting with the transmembrane domain and inhibiting ion translocation (Wakabayashi et al., 1994). G-protein coupled receptors are also known to affect Na7H+ exchange. In most cell types, signal transduction cascades mediated by Gaq and Gai3 are capable of activating NHE1 (Kitamura et al., 1995). Gaq activation of NHE1 is mediated through a PKC dependent mechanism (Dhanasekaran et al., 1994). G0C13 activation of NHE1 can also occur via a RhoA mediated pathway or a Cdc42-MEKK pathway (Voyno-Yasenetskaya, 1998). Thus, expression of constitutively active RhoA in fibroblasts causes an increase in NHE1 activity, while expression of an inactive RhoA protein inhibits activation of NHE1 by G0C13 and by G-protein coupled receptors for lysophosphatidic acid (LPA) and thrombin (Hooley et al., 1996). Gai3 to RhoA activation of NHE1 is accomplished via p160ROCK (a Rho associated kinase), which directly phosphorylates the cytoplasmic C-terminal region of NHE1 (Tominaga and Barber, 1998). Integrin activation also stimulates NHE1 activity, which results in an increase in pHj in the presence of HC03" (Schwartz et al., 1990; Ingber era/., 1990). Integrin based signals converge with signals from 7TM receptors (membrane proteins 16 with seven transmembrane segments) coupled to G0C13 and activate NHE1 through a RhoA-p160ROCK cascade (Tominaga and Barber, 1998). As a result, treatment of hamster lung fibroblasts with EIPA results in impaired focal adhesion formation and cell spreading (Tominaga and Barber, 1998), which indicates that NHE activation may also play a role in "inside-out" signalling to integrins. Signal Transduction and Regulation of NHE3 NHE3 activity is acutely up and down-regulated in response to G-protein linked receptors, tyrosine kinase receptors and protein kinases (Donowitz et al., 2000). In most cases, regulation of NHE3 activity occurs through changes in Vmax, although cAMP (Lamprecht et al., 1998) and squalamine (Akhter et al., 1999) have been shown to inhibit Na+/H+ exchange by altering its affinity for intracellular H+ in addition to changes in Vmax. Pathways leading to activation and inhibition of NHE3 are summarized in Figure 5. NHE3 is known to bind to NHE regulatory factors (NHERFs), which are PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins that facilitate linkage of NHE3 to the actin cytoskeleton and possibly to cell junctions (Weinman et al., 2000). NHERFs are also involved in protein kinase A (PKA) mediated inhibition of NHE3 (Weinman et al., 2000). It has been known for some time that cAMP causes inhibition of Na+ and water transport in the renal tubule (Agus et al., 1971). It was later determined that 17 Figure 5. Summary of molecular pathways that either activate or inhibit NHE3. NHE3 is regulated by a variety of intracellular second messengers. This type of regulation is also dependent on the cell type in which the exchanger is expressed. 18 19 NHE3 is the Na+/H+ exchanger isoform found in renal and intestinal brush border epithelia (Tse et al., 1992). Hormones that elevate intracellular cAMP levels are potent inhibitors of NHE3, which is partially mediated through phosphorylation of the exchanger by PKA (Szaszi et al., 2001). While, NHE1 is insensitive to changes in intracellular cAMP levels (Ganz et al., 1990), mutation of the PKA phosphorylation site in NHE3 (Ser605) reduces acute cAMP-mediated inhibition of NHE3 by -50%, suggesting the existence of an indirect inhibitory cAMP/PKA-based mechanism (Kurashima etal., 1997). Expression studies in PS120 human fibroblasts (which are devoid of NHEs and NHERFs) demonstrated that cAMP inhibited Na+/H+ exchange only if NHE3 and NHERF were co-expressed (Yun et al., 1997). In fact, it has since been shown that NHERF binds to NHE3 and ezrin, which is required for PKA phosphorylation of NHE3 and subsequent inhibition of Na+/H+ exchange activity (Weinman et al., 2000). Changes in cell surface localization of NHE3 are mediated via a PI3 kinase-dependent pathway. FGF and EGF stimulate NHE3 activity via increasing the number of exchangers present at the plasma membrane (Donowitz et al., 2000; Janecki er al., 2000). Thus, both FGF and EGF stimulated increases in NHE3 activity can be attenuated by Wortmannin, an inhibitor of PI3 kinase (Donowitz et al., 2000; Janecki etal., 2000). NHE3 activation also occurs through G-protein mediated signalling. Both Gcti2 and Goci3 have been shown to activate NHE3 (Lin er al., 1996). Expression of 20 dominant negative Rac1 or Cdc42 had no effect on NHE3 activity, however a dominant negative form of RhoA caused inhibition of NHE3 (Szaszi et al., 2000). In addition, treatment with Y-27632 or expression of dominant negative p160ROCK caused inhibition of NHE3 (Szaszi et al., 2000). These results correlate well with the findings that activation of NHE1 is mediated via a RhoA-ROCK signalling cascade, suggesting that multiple NHE isoforms may be activated downstream of Rho protein activation. The Role ofNa*/hf Exchange in Cellular Migration Many researchers have established that inhibition of Na+/H+ exchange inhibits the migration of epithelial cells (Klein et al., 2000), neutrophils (Simchowitz and Cragoe, 1986) and polymorphonuclear leukocytes (Ritter et al., 1998). Additionally, NHE1 and AE1 are both localized to leading edge lamellipodia in migrating cells (Klein et al., 2000; Lagana et al., 2000), providing further evidence for the involvement of membrane bound ion exchangers in cellular migration. Given the dual functions (ion translocation and cytoskeletal anchoring) of the NHE family of ion exchangers, one might speculate that either function or a synergistic combination of the two might be required for cell migration. This question was addressed in a recent study by Denker and Barber (2002). Through the use of various NHE1 mutants (either deficient in ion translocation or cytoskeletal anchoring), they found that disruption of either function resulted in an inhibition of migration. Specifically, disruption of ion translocation resulted in a 21 loss of polarity in the migrating cells, possibly due to a lack of Akt recruitment to the membrane, while loss of cytoskeletal anchoring by NHE1 resulted in impaired lamellipodia formation and formation of multiple pseudopodia in all directions. They also noticed that the cytoskeletal anchoring-deficient NHE1 mutants displayed a uniform distribution of NHE1 around the cell periphery compared to the localization at the leading edge normally observed, which suggests that anterior/posterior polarity was lost. Squalamine, an NHE3 inhibitor, also prevents directed cell migration (Sills et al., 1998). Therefore, given the structural similarities, I wanted to determine if the inhibition of migration observed in MDA-MB-231 breast carcinoma cells treated with dihydromotuporamine C could also be due to inhibition of Na7H+ exchange. Rho GTPases Dihydromotuporamine C has been shown to activate Rho and cause the formation of focal adhesions and stress fibres in Swiss 3T3 cells (McHardy et al., 2002). The Ras homology (Rho) family of GTPases belong to the Ras superfamily of monomeric 20-30 kDa GTP binding proteins (Madaule and Axel, 1985). Rho GTPases are involved in dynamic reorganizations of the cytoskeleton and cell adhesion that regulate many fundamental processes including migration, shape changes, cytokinesis, smooth muscle contraction and neurite outgrowth (Ridley, 2001). There are ten known mammalian Rho family members, some with many isoforms, including Rho (A,B,C isoforms), Rac (1,2,3 22 isoforms), Cdc42 (Cdc42Hs, G25K isoforms), Rnd1/Rho6, Rnd2/Rho7, Rnd3/RhoE, RhoD, RhoG, TC10 and TTF (reviewed by Bishop and Hall, 2000). Rho family members cycle between an inactive, GDP-bound state and an active, GTP-bound state (Kjoller and Hall, 1999). Like other GTP-binding proteins, Rho proteins have intrinsic GTPase activity. The active GTP-bound and inactive GDP-bound forms are interconvertible by GDP/GTP exchange and GTPase reactions (Boguski and McCormick, 1993). Guanine nucleotide exchange factors (GEFs) facilitate the release of GDP from Rho GTPases, thus promoting the binding of GTP (the cytosolic concentration of GTP is more than five-fold higher than GDP) (Cerione and Zheng, 1996). Rho GTPase activating proteins (GAPs) stimulate the intrinsic GTPase activity of Rho proteins, resulting in their conversion to the inactive GDP-bound state (Takai et al., 1995). GDP dissociation inhibitors (GDIs) bind to Rho family members and act to prevent Rho GTPase binding to the plasma membrane and they also promote extraction of Rho family members from the plasma membrane (Takai et al., 1995). Therefore, RhoGDIs are thought to act as molecular chaperones that regulate the translocation of Rho GTPases between the membrane and cytosol. In resting cells, Rho family GTPases exist in the GDP-bound form and in complexes with RhoGDP dissociation inhibitors (RhoGDIs) (Kaibuchi et al., 1999). RhoGDIs, as their name implies, also counteract Rho guanine nucleotide exchange factors (RhoGEFs) (Isomura et al., 1991). It is theorized that release 23 of RhoGDI from Rho is necessary for Rho activation by RhoGEF (Takahashi et al., 1997). Members from the ezrin, radixin, moesin (ERM) family have been shown to bind RhoGDP/RhoGDI complexes and stimulate dissociation of RhoGDI, therefore they have been termed RhoGDI dissociation factors (RhoGDFs) (Takahashi et al., 1997). Hence, ERM family members are positive regulators of Rho activation. As stated previously, ERM proteins also provide a structural link between F-actin and NHEs. At this point, the means by which dh-MotC stimulates Rho activation is not known. Rho GTPase Effectors More than 30 potential effector proteins have been identified as Rho, Rac and Cdc42 targets. These targets include Rho-kinase (ROK/ROCK), mDia1,2 (scaffolding proteins involved in actin reorganization) (Watanabe etal., 1999), the myosin binding subunit (MBS) of myosin phosphatase (Kimura et al., 1996), rhotekin (a scaffolding protein) (Reid et al., 1996), citron kinase (a kinase involved in cytokinesis) (Maudale etal., 1998), and the Wiskott-Aldrich syndrome family of proteins (WASP, N-WASP and SCAR/WAVE), which are involved in actin polymerization via activation of the Arp2/3 complex (Pollard and Borisy, 2003). Effector activation is currently thought to occur via disruption of intramolecular autoinhibitory domains upon activated Rho GTPase binding (Tu and Wigler, 1999). 24 Signalling to Rho GTPases Rho GTPase activation is regulated by signals from many classes of cell-surface receptors including receptor tyrosine kinases, G-protein coupled receptors, cytokine receptors and adhesion receptors (reviewed by Kjoller and Hall, 1999). Lysophosphatidic acid (LPA) was the first Rho agonist to be described (Ridley and Hall, 1992). Treatment of serum-starved Swiss 3T3 cells with LPA results in the formation of actin stress fibres and focal adhesions, which can be blocked by pretreating the cells with C3 exoenzyme (Ridley and Hall, 1992). LPA acts primarily through Goci3 to stimulate Rho activity (Swarthout and Walling, 2000). LPA is also known to activate Goti and Gaq; Gaq-GTP can activate Rho in some cell types, but is not a sufficient regulator of the cytoskeletal effects mediated by Rho (Sah et al., 2000). Bombesin, sphingosine 1-phosphate, thrombin and endothelin are also known to act via GPCRs to activate Rho (Kjoller and Hall, 1999). The G0C12/13 family are considered to be the primary G-protein mediators of Rho activation. For example, constitutively active forms of Goc-12 and G0C13 have been shown to induce stress fibre and focal adhesion formation in a Rho-dependent manner (Needham and Rozengurt, 1998), while blocking antibodies directed against Gai2 and G0C13 attenuate the effects of thrombin and LPA on the cytoskeleton, respectively (Majumdar et al., 1999; Gohla et al., 1998). Goci3 binds to p115RhoGEF and stimulates its activity, leading to GDP-GTP exchange and Rho activation (Roscoe et al., 1998). Recall that activation of both NHE1 25 and NHE3 can occur downstream of the Goci2/13-Rho-ROCK pathway. Hence Rho activation is likely upstream of NHE activation. Tyrosine kinases also play a role in Rho activation. The EGF-specific tyrosine kinase inhibitor, tyrphostin AG1478, blocks Goci3 and LPA induced activation of Rho (Gohla et al., 1999). The exact role of the EGF receptor is not clear at the present time; the evidence suggests that it acts upstream of Rho, i.e. in the pathway by which Ga13 activation leads to Rho binding to GTP (Gohla et al., 1999) . Thus, growth-factor-mediated chemotaxis, which is affected by dh-MotC in invading tumour cells and sprouting endothelial cells, may be mediated, at least in part by changes in Rho activation. Pharmacological Inhibition of Rho and Rho-Kinase (ROCK) Many bacterial toxins have been found which inactivate Rho. Clostridial cytotoxins Clostridium difficile toxin A and toxin B inactivate all Rho family members by glycosylating the GTP/GDP binding site (Sah et al., 2000). The ADP-ribosyltransferases, which include the Clostridium botulinum C3 exoenzyme, the Clostridium limosum transferase and the Staphylococcus aureus transferase epidermal differentiation inhibitor, show a greater specificity for the Rho subfamily proteins (Rho A, B and C) than the C. difficile toxins (Sah et al., 2000) . C3 exoenzyme irreversibly ADP-ribosylates Rho at Asn41, which is located in the effector region (Sah et al., 2000). Y-27632 (a pyridine derivative; Figure 2) is an inhibitor of p160ROCK that was discovered in 1997 by Uehata et 26 al. due to its selective inhibition of smooth muscle contraction in hypertensive rats. Y-27632 also prevents the formation of actin stress fibres in Hela cells (Uehata et al., 1997). This compound binds to the ATP-binding site in p160ROCK in a reversible fashion (Ishizaki et al., 2000). Y-27632 has been used in previous studies (McHardy et al., unpublished data) to reverse dhMotC -mediated inhibition of invasion. Inhibition of either p160ROCK or Rho abolishes the anti-invasive properties of dhMotC; inhibition of Rho also prevents dhMotC-induced focal adhesion/stress fibre formation in Swiss 3T3 cells (McHardy ef al., 2002). Actin Stress Fibres and Focal Adhesions In cultured fibroblasts, actin filaments are found in at least three types of structures: the cortical actin network, actin stress fibres and cell surface protrusions, including membrane ruffles (lamellipodia) and microspikes (filopodia). Actin stress fibres emanate from sites of cell attachment to the substratum known as focal adhesions. Focal adhesions consist of aggregated extracellular matrix (ECM) receptors (integrins) that span the plasma membrane and interact with ECM components on the extracellular side and actin stress fibres on the intracellular side (Burridge and Chrzanowska-Wodnicka, 1996). There are many other proteins present at the intracellular domains of focal adhesion sites; some play a structural role (e.g. a-actinin, talin, vinculin) and others are involved in signalling (e.g. focal adhesion kinase [FAK] and integrin linked kinase [ILK]) (reviewed by Wu and Dedhar, 2001). 27 In now classical experiments, Ridley and Hall (1992) identified the factors primarily responsible for actin stress fibre and focal adhesion formation. Swiss 3T3 fibroblasts do not display any stress fibres or focal adhesions when serum-starved for 24 hours. Ridley and Hall (1992) were able to induce stress fibre and focal adhesion formation via the re-addition of serum to these cells. This process was dependent on Rho, as the effect could be mimicked via microinjection of Rho into serum starved cells and blocked with C3 exoenzyme or addition of ADP-ribosylated RhoA (Ridley and Hall, 1992). It was also determined that LPA, a factor present in serum, was able to induce stress fibre and focal adhesion formation via Rho activation (Ridley and Hall, 1992). The mechanism by which Rho acts to induce stress fibres and focal adhesions has not been completely characterized. However, it is known that activated Rho and ROCK are required (Amano et al., 1997). It is also known that activated Rho induces myosin light chain phosphorylation, and that contractility is required for stress fibre formation (Chrzanowska-Wodnicka and Burridge, 1996; Katoh et al., 2001). There is no clear answer as to the true functions of stress fibres and focal adhesions, nor is it clear whether these structures actually exist in vivo or if they are merely a phenomenon exhibited by cells in culture. Burridge and Chrzanowska-Wodnicka (1996) hypothesized that stress fibres and focal adhesions represent the response of cells in culture to an apparent wound environment. In non-muscle tissue, large contractile bundles of actin are present 28 only during cytokinesis and wound contracture (Gabbiani et al., 1973). Many of the factors present in serum that activate Rho (LPA, thrombin, endothelin) are released by platelets or other cells in response to wounding in vivo. Therefore, cells cultured in the presence of serum may behave as if they are in a wound environment, particularly at low density. Rho and Cellular Migration The ability of certain cell types to migrate plays a central role in many biological processes including embryonic development, wound healing, angiogenesis and tumour metastasis. Cell locomotion is very complex, requiring coordinated activity between cytoskeletal, membrane and adhesion systems. Locomotion can be broken down into subtypes of motility. Forward motility of the membrane at the leading edge of the cell is often referred to as protrusion. Protrusive structures include filopodia (finger-like projections composed of a tight bundle of long actin filaments) and lamellipodia (thin protrusive sheets of actin). Adhesion is then required for the leading edge protrusion to be converted into movement along the substrate. Traction or cytoskeletal force generation is required to move the cell body forward. The last step involves two processes: de-adhesion and tail retraction (reviewed by Webb et al., 2002). The Rho family of GTPases are intimately linked to cell motility. In cultured fibroblasts, it is generally accepted that Rac is required for lamellipodia formation, Cdc42 is required for filopodia formation and RhoA is required for focal 29 adhesion/stress fibre formation. The role of stress fibres in motility is less clear. Studies by Nobes and Hall (1999) have shown that inhibition of p160ROCK increases cell motility, providing support for the idea that that stress fibres and focal adhesions inhibit forward movement. They also demonstrated that disruption of stress fibres with the myosin ATPase inhibitor, BDM, inhibited cell migration (Nobes and Hall, 1999). These data suggest that there may be other myosin-based contractile elements that provide the force required for forward movement. Cell migration is also prevented when Rho is inactivated (Mukai et al., 2003; van Nieuw Amerongen et al., 2003), which suggests that some level of Rho activation is required for migration. In fact, RhoA and its downstream effector kinase, p160ROCK, have been implicated in de-adhesion and tail-end retraction. Liu et al. (2002) demonstrated that inhibition of p160ROCK with Y-27632 resulted in an inhibition of de-adhesion and an increase in spreading in migrating leukocytes. Studies by Worthylake et al. (2001) also demonstrated that inhibition of RhoA or p160ROCK resulted in impaired tail retraction in migrating monocytes. The Role of Rho in Intercellular Junctions Any potential anti-cancer drug must be assayed for its effects on non-tumourigenic cells in order to weigh the potential benefits vs. drawbacks to developing the drug for therapeutic use. Therefore, I was also interested in 30 examining the effects of dihydromotuporamine C on the intercellular junctions of non-tumourigenic mammary epithelial cells. Intercellular junctions occur between epithelial cells at sites of cell-cell contact and can be classified as desmosomes, gap junctions, adherens junctions or tight junctions (reviewed by Knust and Bossinger, 2002). Each type of junction differs in its morphology, localization and constituent proteins. I was interested in examining the effects of dihydromotuporamine C on differentiated mammary epithelial cells, which polarize when plated on basement membrane and thus form both adherens and tight junctions (Somasiri and Roskelley, 1999). I used E-cadherin (an adherens junction component) and zonula occludens-1 (ZO-1, a tight junction component) as junctional markers in these studies. Reports have demonstrated that Rho subfamily members are necessary for both adherens and tight junction formation in MDCK cells (Takaishi et al., 1997). Dominant active Rho expression did not affect E-cadherin or ZO-1 localization in these studies (Takaishi etal., 1997). Hopkins et al. (2003) used cytotoxic necrotizing factor-1 (CNF-1), a bacterial toxin that results in constitutive activation of Rho, Rac and Cdc42, to examine the effects of over-activated Rho proteins on adherens and tight junctions. They found that constitutive Rho family activation resulted in displacement of ZO-1, occludin and JAM-3 from tight junctions and a concomitant decrease in transepithelial resistance (TER) (Hopkins et al., 2003). There was no observed effect on the adherens junctions. As 31 dihydromotuporamine C is known to activate Rho, it is plausible to expect that dh-MotC may affect E-cadherin and ZO-1 localization in differentiated mammary epithelial cells. Objectives and Hypotheses At the present time, we know that dh-MotC is a Rho activator and that it causes stress fibre and focal adhesion formation in Swiss 3T3 cells. Furthermore, the anti-invasive properties of dh-MotC can be attenuated by treatment with the Rho-kinase (ROCK) inhibitor, Y-27632. My main objective in undertaking this thesis was to determine additional mechanisms of action of dh-MotC. Squalamine, another inhibitor of invasion/angiogenesis, is structurally similar to dh-MotC and it inhibits NHE3 (Akhter et al., 1999). In addition, as described above, Na+/H+ exchange can be stimulated via a Rho-ROCK signalling cascade (Tominaga and Barber, 1998). Therefore, my first hypothesis was that di-hvdro motuporamine C would affect Na7H* exchange in Swiss 3T3 cells. The secondary objective that I wanted to pursue was an examination of the effects of dh-MotC on the adherens and tight junctions of non-tumourigenic mammary epithelial cells. Given the involvement of Rho family members in the formation and disruption of intercellular junctions (Takaishi et al., 1997; Hopkins et al., 2003), my second hypothesis was that dh-MotC treatment would alter E-cadherin and ZO-1 localization in non-tumourigenic mammary epithelial cells. 32 II. MATERIALS AND METHODS Cell lines Scp2 mouse mammary epithelial cells were cloned from the CID-1 p-casein expressing population of the mouse mammary cell line, COMMA-1D (Desprez et al., 1993). They polarize and differentiate (i.e. expand and secrete milk proteins) when cultured on three-dimensional reconstituted basement membrane gels (Somasiri and Roskelley, 1999). MDA-MB-231 cells were derived from an adenocarcinoma of the breast from a 51 year-old Caucasian female (Chandrasekaran and Davidson, 1979). These cells are estrogen receptor negative and highly metastatic. Swiss 3T3 cells were obtained from ATCC (Manassas, VA). The Swiss 3T3 cell line is an embryonic fibroblast line established by G. Todaro and H. Green in 1962 from disaggregated Swiss mouse embryos. They are routinely used in Rho activation assays (Nobes and Hall, 1999). Dihydromotuporamine C (dh-MotC) Dh-MotC was obtained as a gift from Dr. Raymond Anderson (Dept. of Chemistry, UBC). Dh-MotC was reconstituted in either DMSO or ddH20, appropriate vehicle controls were used in all assays performed. There were variations from batch to batch of the compound in potency, however, 33 concentrations of 2.5 to 10uM always inhibited tumour cell invasion and mediated Rho-dependent stress fibre formation. Cell culture MDA-MB-231 and scp2 cells were routinely cultured in Dulbecco's modified Eagle's medium:F12 Ham's (DMEM/F12) medium (1:1) Sigma, St. Louis, MO) supplemented with 5% fetal bovine serum (FBS, Hyclone, Logan, UT) and insulin (5ug/ml, Sigma) at 37°C in a 5% C02 humidified incubator. Swiss 3T3's were cultured in DMEM (Sigma, St. Louis, MO) supplemented with 10% FBS (Hyclone, Logan, UT) and insulin (5pg/ml, Sigma). Gentamycin, an antibiotic, was added at 50ug/ml final concentration to the media in order to prevent bacterial contamination. Cells were seeded at approximately 1.5 X 106 cells/100mm tissue culture plastic dishes. The culture medium was changed every 48 hrs. When the cells were approximately 90% confluent, they were trypsinized (0.5% trypsin [StemCell, Vancouver, BC]/ 0.06% EDTA in Ca2+/Mg2+ free Hank's balanced salt solution [HBSS, Sigma, St. Louis]) and removed from the culture plate. Cell stocks were maintained in DMEM/F12 or DMEM media containing 20% dimethyl sulfoxide (DMSO) and 25% FBS, and were stored in liquid N2 (-196°C). Swiss 3T3 cells were serum starved in culture with either DMEM or DMEM/F12 base media (no FBS, insulin or gentamycin present) 24 hours prior to stress fibre/focal adhesion experiments and ratio imaging experiments. 34 Maintenance of a pure mammary epithelial cell population During routine cell culture, epithelial cells can undergo phenotypic drift and acquire fibroblastic characteristics. In order to maintain a pure epithelial population, these cells were removed by using a differential plating technique, because cells with fibroblastic characteristics adhere and spread on tissue culture plastic more rapidly than do epithelial cells (Somasiri and Roskelley, 1999). To separate the two populations, cells were placed in the incubator for 20min following trypsinization, and unattached cells (epithelial) were transferred to new tissue culture dishes. In a pure epithelial culture, the great majority of the cells exhibit the classic "cobblestone" epithelial morphology, which is indicative of the presence of cell-cell junctions. Matrigel™ Basement Membrane Matrix (EHS) Matrigel™ (BD Pharmingen, Mississauga, ON) Basement Membrane is a solubilized basement membrane extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumour rich in extracellular matrix proteins (Kleinman et al., 1982). Laminin is the most abundant protein, followed by collagen IV, heparin sulfate proteoglycans and entactin. Other components present are: TGF-p\ fibroblast growth factor, tissue plasminogen activator and other naturally occurring growth factors from the EHS tumour. 35 Preparation of Matrigel coated dishes and coverslips Matrigel™ stocks (-80°C) were thawed overnight at 4°C, then diluted 1:1 with DMEM/F12 base media. In order to prevent premature gelling, Matrigel™ solutions were always kept on ice. 6-well tissue culture plates were chilled on ice and 150uJ of diluted Matrigel™ solution was added to the center of each dish. Matrigel™ was spread evenly over the entire surface of each well with the blunt end of a 200u1 pipette tip and incubated at 37°C for at least 30min to allow gelling to occur. For immunofluorescent staining, cells were cultured on Matrigel™ coated coverslips. 18mm glass coverslips were sterilized by immersing in 70% ethanol then placed on edge in 12-well tissue culture plates and allowed to dry for 30min in the tissue culture biosafety cabinet. The plates were then shaken to drop each coverslip to the bottom of each well. The 12 well plates were then chilled on ice and 80uJ of Matrigel™ solution was placed on each coverslip and spread evenly. The plates were incubated for at least 30min to allow gelling. Differentiation assay To induce polarization and milk production, scp2 cells were trypsinized and resuspended in DMEM/F12 media containing gentamycin (50u.g/ml) and the lactogenic hormones insulin (5u.g/ml), prolactin (3ug/ml), and hydrocortisone (1u.g/ml). Cells were seeded at 5 X 106/35mm onto Matrigel™-coated dishes or coverslips and incubated at 37°C, 5% C02. The medium was changed every 36 48hrs and cells were collected for immunostaining or western analysis following three or six days, respectively, of treatment with dh-MotC. Cells were collected and assayed for p-casein expression following 6 days of culture on Matrigel™ in the absence or presence of 5pM dh-MotC (reconstituted in DMSO). Immunostaining for junctional proteins was performed following 3 days of culture on Matrigel™ in the absence of prolactin, followed by three days of culture in the presence of prolactin +/- 2.5pM dh-MotC (reconstituted in ddH20). N-values refer to the number of times the experiment was performed. Western Blot Analysis For P-casein When differentiated, scp2 cells express the milk protein, p-casein (Roskelley et al., 1994). Cells cultured on Matrigel™ were rinsed once with DMEM/F12 base media and 1ml of Dispase (Collaborative Research, Waltham MA) was added to each well of a 6-well plate. The plate was incubated for 30min at 37°C. Dispase treatment is required to digest the ECM proteins and free the rounded mammary epithelial cell clusters. Cells were collected into 1.5ml microcentrifuge tubes and centrifuged at 4000 x g for 4min. This was repeated two times with re-suspension of the intact cell pellet in DMEM/F12 base media to rinse away the remaining Dispase. Pellets were then resuspended in 75pl of RIPA lysis buffer (150mM NaCI, 50mM Tris pH 7.4, 5mM EDTA, 5% NP-40, 1% sodium deoxycholate [DOC], and 0.1% sodium dodecyl sulphate [SDS], aprotinin, leupeptin, phenylmethyl-sulfonyl fluoride [PMSF]) and incubated on ice for 10min. 37 Following centrifugation at 13,000 x g in a 4°C microfuge, supernatants, which contain the milk proteins, were collected and stored at -80°C. Protein concentrations were determined via the BioRad protein assay kit (cat. # 500-0120, Mississauga, ON). Lysates were normalized to equal protein concentrations by adding RIPA buffer and sample buffer (20% glycerol, 0.1M Tris pH6.8, 4% SDS, 0.004% bromphenol blue, 10% 2-mercaptoethanol). 10ug of total protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 1.5mm thick 13% gels and transferred to PVDF (BioRad, Mississauga, ON) membranes. Membranes were incubated with blocking solution (4% bovine serum albumin [BSA] and 5% FBS in Tris (pH 7.5) buffered saline [TBS]-Tween 20) for 12hr at 4°C to block non-specific antibody binding sites. (3-casein was detected by probing with mouse monoclonal B-casein antibody (gift from Dr. C. Kaetzel, Institute of Pathology, Case Western University, Cleveland, OH) overnight at 4°C. Unbound primary antibodies were removed by washing 3x5 min with TBS-Tween 20. Bound primary antibody was detected by incubation with HRP-conjugated anti-mouse IgG for 1 hr, followed by visualization with enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL) reagents followed by exposure to Kodak X-OMAT film. Film was developed using a Kodak X-OMAT 2000A film processor, n-values refer to the number of times the experiment was performed. 38 Determination of intracellular pH via fluorescence ratio imaging Loading Cultured Cells with BCECF 2'>7'-bis-(2-carboxymethyl)-5-(and-6-)-carboxyfluorescein acetoxymethyl ester (BCECF-AM, Molecular Probes, Eugene, OR) was prepared as a 1mM stock in DMSO. Swiss 3T3 cells plated as monolayers on 18mm glass coverslips were grown to -80% confluency and serum-starved for 24hrs. Coverslips were then placed in DMEM/F12 base media containing 2pM of the pH-sensitive fluorescent cell permeable dye BCECF-AM for 30min at 37°C, followed by 5min in media containing no dye. Coverslips were then mounted in a temperature-controlled perfusion chamber at 37°C, and superfused at 2ml/min with the initial experimental solution for 5-10 minutes to allow de-esterification of the dye to occur. Fluorescence Ratio Imaging Intracellular pH values were determined via the dual-excitation fluorescence ratio method, which utilizes an Attofluor Digital Fluorescence System (Atto Instruments Inc., Rockville, MB) operating in conjunction with a Zeiss Axiovert 10 microscope (Carl Zeiss Canada Ltd.). BCECF was excited at two different wavelengths, 488nm and 452nm and the ratio of emitted light (measured at 520nm) was used in the determination of pH values. The excitation of BCECF at 488nm results in a fluorescence emission whose intensity is sensitive to changes in intracellular pH. BCECF was subsequently excited at 452nm, a wavelength largely insensitive to changes in pHj as this wavelength is close to the 39 isoexcitation point of the dye. The ratiometric method has been shown to significantly reduce signal artefacts caused by intracellular dye concentration, dye leakage, variations in optical path length and photobleaching (Bright et al., 1989). A 100W mercury arc burner was used a source of photons for excitation of BCECF. In order to minimize photo-induced damage to the cells, the light path of the burner was interrupted by a computer controlled high speed shutter. Neutral density filters were also placed in the light path to minimize photo-toxicity. 488 and 452 nm short band-pass filters were mounted on a computer activated filter changer which sequentially interrupted the light path during excitation. Excitation radiation was reflected by a long band-pass dichroic mirror (FT-495) and was focussed through a 40x LD Achroplan objective (numerical aperture 0.75) onto the cells in the perfusion chamber. Emitted fluorescent light passed back through the dichroic beam splitter before being filtered by a 520nm long-pass filter, the wavelength at which the emission was measured. An intensified charge-coupled device camera mounted on the microscope was used to measure the fluorescence emissions. Images were digitized to 8 bit resolution with a 512 x 480 pixel frame size. One image was captured for each of the two excitation wavelengths. Each image was displayed by a video terminal in order to select regions of interest (ROIs). The recorded values after 40 excitation at 488 and 452 nM reflected the mean emitted intensity within each ROI. Acid loading While mounted in the perfusion chamber, Swiss 3T3 cells were superfused at 2ml/min at 36-37°C with a standard HEPES-buffered, nominally HC03" free medium containing (mM): 136.5 NaCl, 3 KCI, 2 CaCI2, 1.5 NaH2P04, 1.5 MgS04, 17.5 D-glucose and 10 HEPES, titrated to pH 7.49 at room temperature with 10M NaOH. The pH of all solutions at 37°C was 7.35 as determined by the equation pH37 = 0.18 + 0.96 x pHRT (Baxter, 1995). pH of experimental solutions was measured using a Coming 240 pH meter, which was calibrated daily. Cytoplasmic H+ loading was induced via the NH4+ prepulse technique (Boron and De Weer, 1976), during which the cells were exposed to 40mM NH4+CI (substituted into standard HEPES medium for equimolar amounts of NaCl) for 2-3 minutes. This produces an intracellular alkalinization due to the passive influx of neutral NH3l the dissociated form of NH4+, across the cell membrane, which becomes protonated once inside the cell thereby forming NH4+ + OH". Removal of the ammonium solution results in the rapid evacuation of the highly permeable NH3 molecules from the cell, and leaves significant concentrations of NH4+ trapped in the cytoplasm by the opposing electrical gradient generated by membrane potential. Dissociation of NH4+ into NH3 + H+ and subsequent efflux of NH3 out of the cell results in an increased concentration of protons in the cytoplasm (acid loading), which causes pHj to fall below resting levels. Analysis 41 of the recovery back to steady-state pHj is a valid method for investigation of Na+/H+ exchange and other acid extrusion mechanisms (Baxter and Church, 1996; Smith et al., 1998). HOE694 (a gift from Aventis Pharma, Germany), Y-27632 (Calbiochem, San Diego, CA) and dh-MotC were added to standard HEPES media and superfused through the chamber to investigate effects on intracellular pH and acid load recovery. The pH of each solution was tested after the addition of each compound, and no changes in solution pH were observed. Calculation of intracellular pH BCECF emission intensities at each excitation wavelength were recorded by Attograph version 6.08.02 and were exported as comma-delimited text files to Microsoft Exel after selection of data from viable cells. Cell viability was determined by the ability of the cell to retain the fluorescent indicator BCECF. Conversion of BCECF emissions to absolute pH values was accomplished by comparing the ratio of background-subtracted values of the emission intensities at each excitation wavelength (488nm/452nm) and the ratio value corresponding to pH 7.00 determined by the one-point KVnigericin calibration method (Chaillet and Boron, 1985). Nigericin is a charged electron carrier that balances intra- and extracellular pH if the concentrations of K+ are the same on both sides of the cell membrane (Chaillet and Boron, 1985). Nigericin (reconstituted in ethanol) was used at 10uM in a solution containing high K+ levels (1.5mM Na^PCu, 1.5mM MgS04, 17.5mM D-glucose, 1mM CaCI2, 10mM HEPES, 10mM Na gluconate, 10mM K gluconate and 10uM nigericin) and adjusted to pH 7:00 at 37°C with 42 10M KOH. Conversion pf 488nm/452nm ratio values into pH units was accomplished via Microsoft Exel macros written by Keith Baxter, which utilize a derivation of the Henderson-Hasselbalch equation of the form: pH = log[(Rn -Rn(min))/(Rn(max) - Rn)] + pKa to determine intracellular pH (Baxter, 1995). Rn(min) and Rn(max), the theoretical minimum and maximum ratio values possible, and pKa were obtained from routine calibration experiments performed by John Church and Claire Sheldon (UBC Depts. of Anatomy & Cell Biology and Physiology); Rn refers to normalized experimental ratio values. Analysis of steady-state pH, and rates of recovery from acid loads Changes in steady-state pHj were measured by subtracting the resting pHj before treatment from that following an experimental treatment. These results were pooled and the average and standard error were calculated (Tables 1/2). Analysis of acid load recovery was performed by fitting the recovery portion of an acid load to a single exponential function, pHj = a + b(1-10("ct)) (where a, b and c are the exponential parameters and t represents time), in Sigmaplot v8.0. The first derivatives of this equation, dpHJdt = -bc10("ct) represent the change in absolute pHj as a function of time and so were used as the values for rates of recovery from acid loads at absolute values of pHj. Recovery rates were calculated from the peak of intracellular acidification to full recovery to steady-state pHj. Statistical significance was assumed at the 5% level; analysis was carried out using the two-tailed Student's t-test. In all cases, error bars represent 43 the standard error of the mean; n values have been reported where possible and represent individual coverslips unless otherwise noted. Wound healing assay Confluent MDA-MB-231 cell monolayers on 18mm coverslips in supplemented DMEM/F12 media (containing 10% FBS, 5u,g/ml insulin, 50u.g/ml gentamycin) were wounded in a cross-hatched pattern with a sterile toothpick, rinsed three times and allowed to recover for 60min at 37°C prior to treatment with dh-MotC (2.5u.M) or HOE694 (100uM). Experiments were either carried out in supplemented DMEM/F12 or standard HEPES buffered media, which is nominally bicarbonate free. Live, phase-contrast pictures were taken using a Nikon F camera attached to a Nikon TMS microscope (10x objective), at eight hour intervals, until the wound was no longer visible. N-values refer to the number of times the experiment was performed. Stress Fibre/Focal Adhesion Assay Swiss 3T3 cells cultured in supplemented DMEM (containing 10% FBS, 5u.g/ml insulin, 50u.g/ml gentamycin) were grown on 18mm glass coverslips until ~80% confluent and then rinsed three times with DMEM base media to remove fetal bovine serum. Cells were then serum starved in DMEM base media for 24 hours to remove stress fibres and focal adhesions (Ridley and Hall, 1992). LPA (1uJV1; reconstituted in DMEM/F12 base media with 1% bovine serum albumin), 10% fetal bovine serum, 10uM dh-MotC (reconstituted in DMSO) or DMSO, all in the 44 presence or absence of 100uM HOE694, were then applied to cells for 30min at 37°C in nominally HCO3" free HEPES-buffered media. Cells were then collected for immunofluorescence analysis of actin (stress fibres) and vinculin (focal adhesions). N-values refer to the number of times the experiment was performed. Immunofluorescence staining and microscopy Indirect immunofluorescence staining and microscopy were used to identify junctional proteins, cytoskeletal proteins, focal adhesions and detect activated caspase-3. After treatment (detailed above), cells were fixed and stained for the proteins of interest. Non-specific staining was assessed via incubation with secondary (fluorochrome-conjugated) antibody alone. Images were obtained as a Z-series with a 60x objective using the Deltavision Spectris 3D microscopy system (Applied Precision, Issaquah WA) for two and three dimensional mammary epithelial scp2 cell cultures. Images of Swiss 3T3 cells were taken on Kodak T-Max 400 film using a Zeiss Axiophot microscope (40x objective). E-cadherin staining Cells were fixed with absolute methanol for 15min at -20°C, rinsed with PBS, incubated with blocking solution (10% NGS/1% BSA in PBS) for 20min at room temperature and rinsed again with PBS. Cells were then incubated with a mouse monoclonal primary anti-E-cadherin antibody (1:500 mouse monoclonal a-E-cadherin #C20820, Transduction Laboratories, Mississauga, ON). Primary 45 antibody binding was detected by incubating for 1hr at room temperature with either FITC or Texas Red-conjugated Affinipure goat anti-mouse IgG (1:100, Jackson ImmunoResearch, West Grove, PA). The coverslips were rinsed 3X5 min each with PBS and mounted on glass slides with anti-fade (95% glycerol, 2.5% DABCO in PBS). ZO-1 staining Cells were processed as described above, then incubated with antibodies against mouse ZO-1 (1:100 rat monoclonal oc-ZO-1 #MAB1520 Chemicon, Temecula, CA). Primary antibody binding was detected by incubating for 1 hr at room temperature with FITC-conjugated Affinipure goat anti-rat IgG (1:100, Jackson ImmunoResearch, West Grove PA). The coverslips were rinsed 3X5 min each with PBS and mounted on glass slides with anti-fade (95% glycerol, 2.5% DABCO in PBS). Active caspase-3 staining Cells were fixed with absolute methanol for 15min at -20°C then rinsed with PBS. Coverslips were then incubated with blocking solution (10% NGS/1% BSA in PBS) for 20min at room temperature and rinsed with PBS. Cells were then incubated with antibodies against the active form of caspase-3 (1:250 rabbit a-active caspase-3 monoclonal #559565, BD Pharmingen, Mississauga, ON). Primary antibody binding was detected by incubating for 1 hr at room temperature with either FITC or Texas Red-conjugated Affinipure goat anti-rabbit IgG (1:100, 46 Jackson ImmunoResearch, West Grove, PA). The coverslips were rinsed 3X5 min each with PBS and mounted on glass slides with anti-fade (95% glycerol, 2.5% DABCO in PBS). Vinculin staining Cells were processed as described above, then incubated with antibodies against mouse vinculin (1:100 mouse monoclonal cc-vinculin V-4505, Sigma, St. Louis, MO). Primary antibody binding was detected by incubating for 1 hr at room temperature with either FITC or Texas Red-conjugated Affinipure goat anti-mouse IgG (1:100, Jackson ImmunoResearch, West Grove, PA). The coverslips were rinsed 3X5 min each with PBS and mounted on glass slides with anti-fade (95% glycerol, 2.5% DABCO in PBS). Actin staining Cells on coverslips were fixed for 15min at room temperature in warm (37°C) 3.7% paraformaldehyde in PBS, further permeabilized by incubation with acetone at -20°C for 5min, and allowed to air dry. Coverslips were incubated with rhodamine-conjugated phalloidin (1:40 Molecular Probes, Eugene, OR) to visualize filamentous actin (F-actin) for 20min at room temperature, rinsed 3 x 5min, then mounted on a slide with anti-fade (95% glycerol, 2.5% DABCO in PBS). 47 III. Results Dihydromotuporamine C activates Na*/hf exchange in Swiss 3T3 cells Motuporamine C and squalamine are both compounds that contain a polyamine tail attached to a macrocyclic ring and have been shown to inhibit directed cell migration, thus they inhibit invasion and angiogenesis (Roskelley et al., 2001; Sills et al., 1998). As squalamine is a known inhibitor of NHE3 (Akhter et al., 1999), and because Na+/H+exchange has been implicated in cell migration (Denker and Barber, 2002), I decided to investigate the effects of dihydromotuporamine C on Na+/H+ exchange. I chose to use Swiss 3T3 cells for these experiments because it is known that dh-MotC induces stress fibre and focal adhesion formation in serum starved Swiss 3T3 cells (Roskelley et al., 2001), and they have also been used as a model cell line to investigate Na+/H+ exchange in fibroblasts (Bright etal., 1987). Swiss 3T3 cells were loaded with the pH sensitive fluorophore, BCECF-AM and were then subjected to imposed acid loads (Boron and De Weer, 1976) in the presence or absence of dihydromotuporamine C. Under this paradigm, the addition of ammonium ions to the perfusion media for approximately two minutes caused an internal alkalinization in the cells (Figure 6). Upon removal of the ammonium, NH3 passively diffuses out of the cell, leaving the charged NH4+ ions trapped in the cytosol. NfV dissociates into NH3 + H+, and the NH3 continues to 48 Figure 6. Swiss 3T3 cells recover from an imposed acid load to steady-state pHj. Recovery from an imposed acid loads in Swiss 3T3 cells was measured via fluorescence ratio imaging. All experiments were carried out under bicarbonate free conditions to minimize the action of bicarbonate-dependent mechanisms of intracellular pH regulation. 49 8 6 -I 1 1 1 1 1 1 0 2 4 6 8 10 12 Time (min) 50 diffuse out of the cells, leaving charged H+ ions in the cytosol (acid loading) (Figure 6). At this point, intrinsic pH regulatory mechanisms (such as Na+/H+ exchange and/or HC03" transport) are activated to extrude excess H+ and return the cell to steady-state pHi levels (Figure 6). All of these experiments were carried out in the standard HEPES buffered media, such that the influence of HCO3" dependent transport mechanisms on intracellular pH was extremely minimal. Steady-state pHi under these conditions was 6.83 +/- 0.04 (n=6 coverslips; 38 cells), which agrees with previous reports using Swiss 3T3 cells (Bright etal, 1982; Schuldiner and Rozengurt, 1982). Dh-MotC (5 or 10 ixM) treatment of serum-starved Swiss 3T3 cells did not result in inhibition of recovery to steady-state pHi following an imposed acid load (Figure 7). In fact, upon addition of 5u.M dh-MotC, there was an increase in steady-state pHi (Figure 8). Table 1 shows that cells treated with dh-MotC (5 or 10u,M) underwent an average rise in steady-state pHj of 0.14 +/- 0.02 pH units (n=6 coverslips; 38 cells). I also observed that cells would recover to a higher value of steady-state pH in a shorter time after acid loading in the presence of dh-MotC, which indicates that the rate of recovery from an acid load may be faster in the presence of dh-MotC. This can be seen in Figure 7, where the cells recovering in the presence of dh-MotC recover from ~pH6.6 to pH7.0 in ~5min compared to a recovery from pH6.6 to pH6.8 in ~5min observed in the control. 51 Figure 7. Dihydromotuporamine C does not inhibit Na7hT exchange in Swiss 3T3 cells. Recovery from an imposed acid load was measured in the presence or absence of 5pM dh-MotC in Swiss 3T3 cells. Addition of dh-MotC appeared to cause an increase in steady state pHj and to increase the rate of pHj recovery following an imposed acid load. 52 53 Figure 8. Dihydromotuporamine C increases steady-state pHi in Swiss 3T3 cells. Dh-MotC was added to Swiss 3T3 cells for 40min under nominally bicarbonate-free conditions, and an increase in steady-state pHi was observed. 54 Table 1. Dihydromotuporamine C increases steady-state intracellular pH (pHi) in Swiss 3T3 cells. Changes in steady-state pH from six independent experiments were analyzed. Addition of dh-MotC (5 or 10pM) resulted in an average increase in steady-state pHi of (0.14 +/- 0.02) pH units (n=6 coverslips; 38 cells). 56 Experiment Treatment Initial pHi Final pHi ApH 139 dh-MotC (10uM) 6.90 7.08 0.18 143 dh-MotC (10uM) 6.75 7.02 0.27 151 dh-MotC (10uM) 6.90 7.05 0.15 244 dh-MotC (5uM) 6.72 6.84 0.12 254-4 dh-MotC (5uM) 6.88 7.00 0.12 254-5 dh-MotC (5uM) 6.88 6.99 0.11 255-3 dh-MotC (5uM) 6.64 6.76 0.12 255-4 dh-MotC (5uM) 6.94 7.02 0.08 Average 0.14 Std. Err. 0.02 57 To further quantify the observed increases in rates of recovery induced by dh MotC, acid loads in the presence and absence of dh-MotC were fitted to a single exponential function and the first derivative of this function was used in the determination of the rates of change in intracellular pH (dpHj/dt). Figure 9 shows that the rate of recovery from an imposed acid load at a test pH, of 6.6 in the presence of dh-MotC (5 or 10 uM) was significantly faster than the control treatment (n=5 coverslips; 30 cells; p<0.05, Student's t-test). dpHJcft was consistently higher than control rates of acid load recovery across a range of physiological pHj values (Figure 10; n=6 coverslips; 38 cells). The slopes of these lines represent the maximal velocity of Na+/H+ exchange across the membrane (Vmax), therefore cells treated with dh-MotC most likely possessed a higher maximal velocity of Na+ and H+ ion translocation compared to controls. The x-intercepts (which signify that the cells have recovered to steady-state pH; i.e. dpHj/dt = 0) of each curve also showed a shift in steady-state pH in the dh MotC treated cells, which correlates well with the finding that dh-MotC caused an increase in steady-state pHj. It is unlikely that dh-MotC increases dpH/dt via altering intracellular buffering power as the increases in pHj observed during the application of NH4CI in the presence and absence of dh-MotC were 0.75 +/- 0.08 (n=17) and 0.80 +/- 0.06 (n=15), respectively (p<0.05). Therefore, we can tentatively say that, unlike squalamine, dh-MotC increases Na7H+ exchange 58 Figure 9. Dihydromotuporamine C increases the rate of intracellular pH (pHi) recovery (dpHJdt) following an imposed acid load at a test pHi of 6.6. Intracellular pH (pHj) recovery following acid loading in Swiss 3T3 cells in the presence or absence of dh-MotC (5 or 10uM) was measured and rates of recovery (dpHJdi) were analyzed. dpHJdt at pH 6.6 is ~2 times faster in the presence of dh-MotC compared to control (*p<0.05, Student's t-test; n=5 coverslips; 30 cells). 59 60 Figure 10. Dihydromotuporamine C increases the rate of intracellular pH (pHj) recovery (dpHJdt) across a range of physiological pHj values following an imposed acid load. Intracellular pH (pHi) recovery following acid loading in Swiss 3T3 cells in the presence or absence of dh-MotC (5 or 10pM) was measured and rates of recovery (dpHJdt) were analyzed. dpHJdt is increased in the presence of dh-MotC compared to control over a range of physiological pHj values and there is a shift in the x-intercepts in the dh-MotC treated cells indicating a shift in steady-state pHj (n=6 coverslips; 38 cells). 61 V 0.008 0.006 1 • control O dihydromotuporamine C 62 activity. To provide additional evidence that the effects of dh-MotC on pHj and ofpHj/dt were due to modulation of Na+/H+ exchanger activity, I used HOE694, a selective inhibitor of Na+/H+ exchange (Counillon et al., 1993). Figure 11 demonstrates that recovery from an acid load under control conditions was inhibited by 50 or 100 uM HOE694 (n=5). Upon washout of the inhibitor, the cells recovered to steady-state pHj (Figure 11). Thus I concluded that acid load recovery in Swiss 3T3 cells under nominally HC03" free conditions was indeed due to Na7H+ exchange. I next determined if acid load recovery in the presence of dh-MotC could be inhibited by HOE694. Figure 12 shows that HOE694 (100uM) inhibited acid load recovery in the presence of dh-MotC (n=5). This inhibition was also reversible; upon washout of HOE694, recovery to steady-state pH occurred (Figure 12). Thus, the rapid recovery to steady state pHi in the presence of dh-MotC is due to Na+/H+exchange mechanisms. At this point, it was clear that dh-MotC increased Na+/H+activity in Swiss 3T3 cells, and that this effect could be inhibited by HOE694. Thus, I determined if the effects of dh-MotC on cell migration and stress fibre/focal adhesion formation could also be inhibited by HOE694. Figure 13 depicts a wound healing assay in which a confluent monolayer of MDA-MB-231 cells (derived from human breast carcinoma) was "scratched" with a sterile toothpick (n=8). In the absence of dh MotC the cells migrated into the wound space and "healed" the scratch within 16 63 Figure 11. Acid load recovery in Swiss 3T3 cells can be inhibited, in a reversible manner, by a known inhibitor of Na+/H+exchange, HOE694. Recovery from an imposed acid load in Swiss 3T3 cells was measured via fluorescence ratio imaging. Addition of HOE694 (50u,M), a known inhibitor of Na7H+ exchange, inhibited intracellular pH (pH) recovery following an acid load. After washout of HOE694, pHj recovery occurred in Swiss 3T3 cells (n=5). 64 65 Figure 12. Dh-MotC-induced increases in the rates of recovery from acid loads can be inhibited, in a reversible manner, by HOE694. Acid load recovery in Swiss 3T3 cells was measured in the presence or absence of dh-MotC +/- HOE694. Addition of HOE694 (100uM) inhibited intracellular pH (pH) recovery following an acid load in the presence of dh-MotC. After washout of HOE694, pHi recovery was observed in Swiss 3T3 cells treated with dh-MotC (n=5). 66 67 Figure 13. Dh-MotC and HOE694 inhibit migration of MDA-MB-231 breast carcinoma cells. MDA-MB-231 cells were seeded on glass coverslips and grown until confluent. Monolayers were wounded with a sterile toothpick, rinsed and allowed to recover for 1 hr. dh-MotC (2.5uM) and/or HOE694 (IOOUM) were then added to experimental wells. Pictures were taken after treatment (t=0) and when control cells had migrated into the wound space (t=16hrs). Treatment with dh-MotC and/or HOE694 resulted in inhibition of cell migration into the wound space (n=8). Bar = 50um. 68 t = Ohrs t = 16hrs 69 hours in HEPES-buffered HC03" free media (Figure 13). As expected, treatment with dh-MotC (2.5uM) resulted in inhibition of migration (Figure 13). However, HOE694 (100pM) alone also inhibited cellular migration into the wound space (Figure 13). Treatment with both HOE694 (100pM) and dh-MotC (2.5p.M) resulted in inhibition of migration (Figure 13). The same results were obtained in the presence of HC03" (data not shown). It is apparent that inhibition of migration by dh-MotC cannot be reversed by co-treatment with HOE694. Studies by Lianne McHardy (Roberge and Roskelley labs, UBC) have demonstrated that dh-MotC induces stress fibre/focal adhesion formation in serum starved Swiss 3T3 cells. It is also known that dh-MotC treatment results in Rho activation in serum starved Swiss 3T3 cells (McHardy et al,. 2002). Therefore I decided to use this system to determine the effects of HOE694 on dh-MotC-mediated stress fibre/focal adhesion formation. Figure 14 depicts a typical stress fibre assay in Swiss 3T3 cells. It is well known that fetal bovine serum (FBS) and lysophosphatidic acid (LPA) activate Rho and lead to stress fibre/focal adhesion formation (Ridley and Hall, 1992); therefore I used these reagents as positive controls in this assay. Treatment with HOE694 (100pM) alone did not induce stress fibre formation. As expected, LPA (500nM), 10% FBS and dh-MotC (10uM) treatment resulted in stress fibre formation. Co-treatment with HOE694 (100u.M) did not abrogate stress fibre formation in any of the conditions listed above (n=6). Figure 15 depicts a similar assay where the 70 Figure 14. HOE694 treatment does not prevent stress fibre formation in Swiss 3T3 cells. Swiss 3T3 cells were grown on glass coverslips until -80% confluent, then serum starved for 24 hours. Cells were then treated with either DMSO, LPA, FBS or dh-MotC (10pM in DMSO) +/- HOE694 (100pM) for 30min, then fixed in 3.7% paraformaldehyde and stained for actin using rhodamine-conjugated phalloidin. Treatment with HOE694 does not inhibit stress fibre formation induced by FBS, LPAordh-MotC (n=6). Bar=20um 71 Actin Hepes +100uMHOE694 72 Figure 15. HOE694 does not inhibit focal adhesion formation in Swiss 3T3 cells. Swiss 3T3 cells were gown on glass coverslips until -80% confluent, then serum starved for 24 hours. Cells were then treated with ddH20, LPA, FBS or dh-MotC (2.5uM in ddH20) +/- HOE694 (100uM) for 30min, then fixed in absolute methanol and stained for vinculin. Treatment with HOE694 does not inhibit focal adhesion formation induced by FBS, LPA or dh-MotC (n=3). Bar=20um 73 Vinculin HEPES +100uMHOE694 DMSO 74 cells were immunostained for vinculin, a component of focal adhesion complexes that are also induced by Rho activation in Swiss 3T3 cells (reviewed by Wu and Dedhar, 2001). Clearly, HOE694 did not prevent dh-MotC-induced focal adhesion formation in Swiss 3T3 cells (n=3). Again, it appears that although HOE694 inhibited acid load recovery in the presence of dh-MotC in Swiss 3T3 cells, the other observed effects of dh-MotC (inhibition of migration, stress fibre/focal adhesion formation) are unaffected by treatment with HOE694. Taken together, the data presented above suggested that dh-MotC-mediated activation of Na7H+exchange may be either downstream of the Rho-ROCK pathway or a parallel pathway. To address this question, I attempted to determine if dh-MotC induced activation of Na+/H+exchange was dependent on Rho-kinase (ROCK) activity. Figure 16 contains a control acid load followed by an acid load conducted in the presence of dh-MotC and Y-27632 (a ROCK inhibitor; Narumiya et al., 2000). There was an average rise in steady-state pHj of 0.16 +/- 0.05 pH units in serum-starved Swiss 3T3 cells treated with dh-MotC + Y-27632 (Table 2; n=3). This rise in steady-state was not significantly different from the previously determined increase of 0.14 +/- 0.02 pH units (Table 1; n=6) observed following dh-MotC treatment alone. In contrast, the recovery rate from an acid load in the cells treated with Y-27632 and dh-MotC together did not seem to differ from the untreated controls (Figure 16). In fact, there was no significant 75 Figure 16. Swiss 3T3 cells co-treated with dh-MotC and Y-27632, a ROCK inhibitor, do not exhibit an increase in the rate of recovery from an acid load. Acid load recovery in Swiss 3T3 cells was measured in the presence or absence of dh-MotC (5uM) + Y-27632 (30uM). A rise in steady-state pH was observed upon addition of dh-MotC + Y-27632. The rate of recovery from an imposed acid load does not appear to be increased compared to control. Cells were pre-incubated with Y-27632 for 1 hr before acid loading. 76 77 Table 2. Dihydromotuporamine C increases steady-state intracellular pH (pHj) in Swiss 3T3 cells in the presence of Y-27632. Changes in steady-state pHj from three independent experiments were analyzed. Addition of dh-MotC (5uM) + Y-27632 (30ufv1) resulted in an average increase in steady-state pHi of (0.16 +/- 0.05) pH units (n=3). 78 Experiment Treatment Initial pHi Final pHi ApH 263-3 dh-MotC (5uM) + Y-27632 (30uM) 6.63 6.77 0.14 263-4 dh-MotC (5uM) + Y-27632 (30uM) 6.64 6.74 0.10 264 dh-MotC (5uM) + Y-27632 (30uM) 6.62 6.71 0.09 265 dh-MotC (5uM) + Y-27632 (30uM) 6.75 7.04 0.29 Average 0.16 Std. Err. 0.05 79 Figure 17. ROCK inhibition prevents dh-MotC-induced increases in rates of recovery from an acid load at a test pHi of 6.45. Intracellular pH (pHj) recovery following acid loading in Swiss 3T3 cells in the presence or absence of dh-MotC (5u.M) +/- Y-27632 (30pM) was measured and rates of recovery (dpHj/dt) were analyzed. dpH/dt is increased in the presence of dh-MotC compared to control at a test pHi of 6.45 (n=6). Co-treatment with Y-27632 (30uM) and dh-MotC (5pM) did not result in an increase in dpH/dt at pH 6.45 (n=3). 80 difference in the rate of recovery from an imposed acid load between control treatments (n=6) and treatment with dh-MotC + Y-27632 (n=3) at a test pH of 6.45 (Figure 17). Acid load recovery in the presence of dh-MotC alone was significantly faster compared to control (p<0.05; n=6) and dh-MotC + Y-27632 (p<0.05; n=3) treated cells (Figure 17). These findings indicated that ROCK activation was upstream of acid-load induced Na7H+exchanger activation. Dihydromotuporamine C alters normal mammary epithelial cell morphology. Any potential therapeutic agent is maximally beneficial if it specifically targets the pathology in question, with minimal side effects on non-pathogenic tissue. It has been demonstrated that motuporamine C treatment for 24hrs does not have a detrimental effect on MDA-MD-231 breast carcinoma cell viability or proliferation (Roskelley et al., 2001). I extended these initial studies by examining the effects of dh-MotC on non-tumourigenic, functional scp2 mammary epithelial cells cultured either as undifferentiated monolayers or differentiated spheroids on three dimensional reconstituted basement membrane gels. I first examined the effect of dh-MotC on the morphology of scp2 cells that had been allowed to attach and spread as monolayers on tissue culture plastic for 16 hours prior to dh-MotC treatment. Dh-MotC used at concentrations of 5 or 10 uM 82 Figure 18. Dihydromotuporamine C treatment of mammary epithelial cells results in cell rounding in a dose-dependent manner. Scp2 cells were cultured for 24 hours in the presence of either 2.5, 5 or 10 pM dh-MotC. Concentrations of 5 and 10 pM dh-MotC cause scp2 cells grown on tissue culture plastic to "round-up" and detach from the substratum (n=6). Bar = 50pm. 83 10hrs 24hrs 84 induced cell rounding at 10hrs; by 24hrs most cells had detached from the tissue culture plastic substratum. In contrast, treatment with 2.5uM dh-MotC, a concentration that still inhibits tumour cell migration, had little effect on scp2 morphology in monolayer cultures (Figure 18; n=6). In addition, given the increase in confluency at 24 hours, it is clear that proliferation occurs in monolayers treated with 2.5uM dh-MotC. Next, I examined the effects of dh-MotC on milk protein secretion in differentiated scp2 mammary epithelial cell spheroids cultured on basement membrane gels. Differentiation and p-casein expression was detected in fully differentiated control scp2 mammary epithelial cell cultures via immunoblotting (Figure 19; n=3). In contrast, scp2 cells trated with 5pM dh-MotC did not produce _-casein (Figure 19; n=3). This experiment was conducted with a different batch of motuporamine C (reconstituted in DMSO) than the one used in figures 18, 20 and 21 (reconstituted in ddH20). We have found no differences in anti-adhesive or anti-migratory activity using the two diluents (L. McHardy, R. Sinotte and C. Roskelley, unpublished data). This result suggests that dh-MotC prevents lactogenic differentiation. Adherens junction and tight junction formation are markers of epithelial cell polarity and are sensitive to Rho activation (Hopkins et al., 2003; Takaishi et al., 1997). Thus, I looked at the localization of E-cadherin and zonula occludens-1 (ZO-1) to determine the effects of dh-MotC on adherens and tight junctions, 85 Figure 19. Differentiated mammary epithelial cells treated with dihydromotuporamine C do not express the milk protein B-casein. Scp2 mammary epithelial cells were cultured on matrigel for six days in the presence of prolactin +/- 5uM dh-MotC (DMSO was used as a vehicle control for this batch of dh-MotC). Scp2 cells cultured on tissue culture plastic without prolactin were used as a negative control for B-casein expression. Polarized scp2 cells treated with dh-MotC did not express the milk protein B-casein (-32 KDa), which was expressed in the untreated and vehicle control cells (n=3). 10uig of protein was loaded in each lane, determined via the Bradford protein assay (see Materials & Methods). 86 2- DMSO ""atrix P.s.ic P-casein Degradatio WB: p-casein J Products 87 respectively, in undifferentiated scp2 monolayers and polarized scp2 cell spheroids. To decrease stringency with respect to cytoskeletal association, immunostaining for E-cadherin and ZO-1 was performed without detergent extraction (Ben-Ze'ev etal., 1979). Clearly, E-cadherin and ZO-1 were localized at sites of cell-cell contact in control and dh-MotC (2.5uM) treated scp2 monolayer cell cultures (Figure 20; n=3). The picture was very different in spheroid culture. Control, untreated scp2 mammary epithelial spheroids contained E-cadherin, which was localized at sites of cell-cell contact, and apically polarized ZO-1, also located at sites of cell-cell contact (Figure 21). In contrast, spheroids treated with 2.5u.M dh-MotC did not display any E-cadherin or ZO-1 at sites of cell-cell contact (Figure 21; n=3). Therefore, it appears that dh-MotC disrupts cell junctions under lactogenic, differentiative conditions. Toxic effects resulting from dh-MotC treatment of the 2-D monolayers and 3-D spheroid cultures was assessed via immunostaining for the active form of caspase-3, a marker for apoptosis (Dai and Krantz, 1999). Camptothecin has been previously described as an apoptosis-inducing agent (Hinz et al., 2003) and so was used a positive control for active caspase-3 immunostaining. Immunofluorescence staining indicates that scp2 cells cultured as monolayers and treated with dh-MotC (2.5JJ,M) were comparable to control cells in levels of active caspase-3 and displayed lower levels of active caspase-3 compared to 88 Figure 20. Undifferentiated mammary epithelial cells treated with dihydromotuporamine C contain E-cadherin and ZO-1 at sites of cell-cell contact and are negative for active caspase-3. Scp2 cells were cultured for 3 days with prolactin, in serum-free media, in the presence or absence of 2.5uM dh-MotC or camptothecin, and were dual-labelled for E-cadherin and ZO-1 or separately single-labelled for active caspase-3. Camptothecin (6uM) was used as a positive control for apoptosis (detected via immunostaining for the active form of caspase-3). Camptothecin treated cells displayed a loss of junctional E-cadherin and ZO-1 and were positive for active caspase-3. Scp2 cells treated with 2.5u.M dh-MotC contained junctional E-cadherin and ZO-1. Dh-MotC treated cells and untreated cells were negative for the active form of caspase-3 (n=3). Bar = 10um. 89 2.5uM dh-MotC 1 r'v U \ v 90 Figure 21. Dihydromotuporamine C treated differentiated mammary epithelial cells do not polarize and display elevated levels of active caspase-3. Scp2 cells were cultured for 3 days on Matrigel without prolactin, then cultured for an additional three days with prolactin, in the presence or absence of 2.5ufv1 dh-MotC or camptothecin, and were dual-labelled for E-cadherin and ZO-1 or separately single-labelled for active caspase-3; nuclei were visualized via labelling with DAPI (blue). Camptothecin (6u.M) was used as a positive control for apoptosis (detected via immunostaining for the active form of caspase-3). Camptothecin treated cells displayed a loss of polarity and were positive for active caspase-3. Scp2 cells treated with 2.5u.M dh-MotC displayed a loss of junctional E-cadherin and ZO-1. Dh-MotC treated cells were also positive for the active form of caspase-3 (n=3). Bar = 10um. 91 E-cadherin ZO-1 Caspase-3 92 camptothecin (6uM) treated cells (Figure 20; n=3). In contrast, scp2 mammary epithelial cells cultured as spheroids on three dimensional reconstituted basement gels and treated with dh-MotC (2.5uJV1) displayed elevated levels of active caspase-3 compared to control spheroids, and possessed comparable levels of active caspase-3 to those seen in camptothecin (6u.M) treated cells (Figure 21; n=3). It should also be noted that, when used at concentrations higher than 10uM, dh-MotC also caused Swiss 3T3 cells to round up and lose membrane integrity, evidenced by the leakage of BCECF from cells. These data indicate that dh-MotC used at 2.5u.M, a concentration effective against tumour cell migration, induces apoptosis in differentiated mammary epithelial cells. 93 IV. Discussion Despite the use of defined chemical libraries and molecular targeting, the screening of novel compounds derived from diverse biological sources to discover new anti-cancer drugs continues to be an exciting theme in research today (Mann, 2002). Dh-MotC is an example of such a compound. It was identified in a screen designed to identify novel agents from natural sources that prevent tumour cell invasion (Roskelley et al., 2001). Dh-MotC, a water-soluble, easily synthesized analog of motuporamine C (Williams er al., 2002), is currently being tested in animal models in order to evaluate the anti-tumour, anti-angiogenesis and anti-metastasis activity of this compound. The goals of my project were: 1) To determine if dh-MotC acts in a similar fashion as squalamine by measuring the effects of the compound on Na+/H+ exchange activity and 2) To determine the effects of dh-MotC on non-tumourigenic mammary epithelial cells. Na+/H+ exchangers possess dual functions, namely ion translocation and cytoskeletal anchoring (reviewed by Slepkov and Fliegel, 2002), both of which are required for migration (Denker and Barber, 2002). Squalamine inhibits the renal and intestinal brush border Na+/H+ exchanger, NHE3 (Akhter et al., 1999), prevents endothelial cell migration, induces disorganization of actin stress fibres and causes reductions in the amount of vascular E-cadherin (VE-cadherin) in endothelial cells (Williams et al., 2001). Williams et al. speculated that these actions of squalamine are responsible for the anti-angiogenic properties of the 94 drug. In contrast, dh-MotC treatment clearly increased both steady state intracellular pH and the rate of recovery (dpHj/dt) from an imposed acid load and induced stress fibre formation in Swiss 3T3 cells. Thus, I conclude that dh-MotC does not share a common mechanism of action with squalamine. Primary published accounts have demonstrated that NHE activity is necessary for cellular migration (Denker and Barber, 2002). Inhibition of NHE activity with HOE694 prevented migration of MDA-MB-231 breast carcinoma cells; the same result is observed in dh-MotC treated cells. Even though HOE694 prevented acid load recovery in the presence of dh-MotC, migrating MDA-MB 231 cells co-treated with HOE694 and dh-MotC were also unable to migrate. Therefore, I conclude that dh-MotC does not prevent directed cell migration via stimulation of Na+/H+ exchanger activity. Dh-MotC is known to activate Rho subfamily members, which in turn have been shown to activate Na+/H+ exchange in a ROCK dependent manner (Tominaga and Barber, 1998). The observed increases in Na+/H+ exchange may be a secondary effect due to the presence of activated Rho in dh-MotC treated cells. Use of the ROCK inhibitor Y-27632 did not prevent the increase in steady-state pHi elicited by dh-MotC in Swiss 3T3 cells. These findings indicate that dh-MotC may be acting through a ROCK-independent pathway to elicit an increase in steady-state pHj. This is not surprising, considering that steady-state pHj is regulated by many mechanisms, even under bicarbonate-free conditions (Kaplan 95 and Boron, 1994). However, Y-27632 treatment resulted in a decrease in the rates of acid load recovery in the presence of dh-MotC, indicating that activation of Na+/H+ exchange during acid load recovery is downstream of ROCK. Furthermore, HOE694 treatment did not prevent the formation of dh-MotC-induced stress fibres or focal adhesions. This suggests that stimulation of Na7H+ exchange by dh-MotC does not contribute to the formation of these structures. The combination of these data leads me to conclude that dh-MotC activates Na+/H+ exchange during acid load recovery via a Rho-ROCK dependent pathway. How does dh-MotC inhibit cellular migration and consequently inhibit invasion and angiogenesis? The answer to this question may lie in the complex actions of activated Rho. To date, we know that dh-MotC activates Rho, and we also see a number of Rho associated phenotypes in Swiss 3T3 cells including stress fibre formation, induction of focal adhesions and increased NHE activity, all of which can be abolished by inhibiting Rho or downstream ROCK activity. Inhibition of this pathway also abolishes the effects of dh-MotC on invasion (McHardy et al., 2002), suggesting a functional linkage between these dh-MotC-induced phenotypes and inhibition of invasion. Rho is both required for and can inhibit invasion and migration. Activated Rho signalling leads to the formation of membrane ruffles and is also necessary for de-adhesion and tail retraction at the distal end of a migrating cell (reviewed by Oxford and Theodorescu, 2003). In contrast, high levels of activated Rho (achieved via use of a dominant active 96 Rho) prevent lamellipodia formation and inhibit cell migration (Sugimoto et al., 2003). Dh-MotC is a Rho activator, therefore it most likely inhibits tumour cell invasion and migration via the latter mechanism described above. How does Rho accomplish these seemingly antagonistic effects? Several studies have demonstrated that Rho is required for Rac activation (Rac is required for lamellipodia formation during cell migration; Nobes and Hall, 1995). However, high levels of Rho activity lead to inhibition of Rac and cellular migration (Sugimoto era/., 2003). Tsuji et al. (2002) demonstrated that two effectors downstream of Rho, ROCK and mDial (the murine homolog of human Dial), antagonize in Rho-dependent Rac activation. Treatment of LPA stimulated Swiss 3T3 fibroblasts with the ROCK inhibitor Y-27632 results in increased levels of GTP-Rac and the formation of lamellipodia and transient focal complexes at the leading edge of migrating cells (Tsuji et al., 2002). This phenotype could be reversed by expression of dominant negative Rac (Tsuji et al., 2002). Other studies have also demonstrated that ROCK signalling can lead to reversion of malignant phenotypes. For example, overexpression of LIM-kinase (a downstream target of ROCK) induces stress fibre formation and inhibits Ras-transformed fibroblast motility (Sahai et al., 2001). Cox et al. (2001) found that high levels of activated Rho, which increased in an adhesion-dependent manner with increasing concentrations of fibronectin substrate, downregulates Rac1 and Cdc42 and prevents cell migration. Rac1 and Cdc42 initiate membrane protrusion in migrating cells via induction of SCAR/WAVE or N-97 WASP-mediated activation of the Arp2/3 complex, which results in actin polymerization (Eden et al., 2002). Therefore inhibition of migration via a Rho-ROCK-dependent pathway may occur via downregulation of Rac activation and subsequent inhibition of membrane protrusion (Figure 22). Rho-ROCK signalling also results in the formation of focal adhesions between migrating cells and the culture substrate (Sawada et al., 2002; Tsuji et al., 2002), hence hyper-activated Rho-ROCK signalling may prevent migration via the induction of extremely stable focal adhesions (reviewed by Riento and Ridley, 2003) (Figure 22). It is clear that Rho activation in migrating cells must be tightly regulated both spatially and temporally in order for migration to proceed. Perturbations in the levels of activated Rho in a cell by sustained treatment with dh-MotC could cause inhibition of Rac (possibly through ROCK activation) and subsequently inhibit migration. It is possible to test these ideas by examining the levels of activated Rac in dh-MotC treated cells vs. control, both with and without Y-27632. If this hypothesis is true, one would expect to see the level of activated Rac decrease in the dh-MotC treated cells, and remain the same or increase in the Y-27632 treated cells (with or without dh-MotC). Dh-MotC was originally designed as an agent to treat metastatic breast cancer, and it is currently being evaluated for efficacy in in vivo models. I examined the effects of this compound on non-tumourigenic mammary epithelial cells. 98 Figure 22. Summary of the actions of Rho on cell migration and focal adhesion formation. Rho can both stimulate and inhibit cellular migration via activation of opposing effector proteins ROCK and mDial. Dh-MotC is known to activate Rho, though the mechanism by which this occurs is not clear at the moment. 99 100 To carry out these experiments, I chose the mouse mammary cell line scp2, which can be cultured as either undifferentiated monolayers or differentiated, polarized spheres that resemble the alveoli of a lactating mammary gland (Somasiri and Roskelley, 1999). Interestingly, dh-MotC appeared to differentially affect morphology, viability and cell junctions in the two differentiation states. Dh-MotC treatment of scp2 cell monolayers altered their morphology in a dose-dependent manner. Concentrations of 5 or 10 u.M caused the cells to round up and detach from the tissue culture plastic substratum. In contrast, 2.5u1V1 dh MotC, a concentration that is still capable of inhibiting migration in breast carcinoma cells, did not alter monolayer morphology. Therefore, concentrations of dh-MotC that have anti-tumour activity may be non-toxic to normal cells. Presumably, the level of Rho activation increased with increasing concentrations of dh-MotC, leading to the cell rounding or prevention of spreading observed at higher concentrations of the drug. As activated Rho and ROCK are required for de-adhesion from substrate during migration (Liu et al., 2002; Worthylake er al., 2001), hyper-activation of these proteins by high concentrations of dh-MotC may accentuate this process and lead to cell detachment. To further explore this idea, it would be interesting to look at the levels and kinetics of activated Rho in dh-MotC treated scp2 cells, with the concentrations used in these studies. 101 Scp2 cell spheroids treated with 2.5u.M dh-MotC for three days displayed a loss of junctional E-cadherin and loss of apically localized ZO-1. The spheroids were allowed to polarize for three days in culture prior to addition of the drug, indicating that dh-MotC acted to dismantle pre-existing junctional structures under these conditions. Importantly, dh-MotC did not dismantle cell junctions in unpolarized monolayers. One trigger for mammary epithelial cell polarity is integrin signalling (Weaver et al., 2002). Therefore, the effects of dh-MotC on integrin-mediated focal adhesion formation may have played a role in the dismantling of polarized spheroids. Dh-MotC also prevented differentiative P-casein induction and it induced apoptosis in scp2 spheroids. Interestingly, inappropriate laminin-integrin interactions can initiate both of these responses when mammary epithelial cells are cultured on basement membrane gels (Streuli et al., 1995; Boudreau et al., 1995; Muschler et al., 1999). Therefore, dh-MotC may initiate detrimental integrin "switching" (Roskelley and Bissell, 2002), which could explain the differential response of cell monolayers and spheroids to the compound. Microinjection of the Rho inhibitor C3 exoenzyme into MDCK cells (after switching from low to high extracellular Ca2+ to induce junction formation) prevents the formation of both adherens and tight junctions (Takaishi et al., 1997). Furthermore, Rac and the Rho effector Dial are both required for maintenance of adherens junctions (Takaishi et al., 1997; Sahai and Marshall, 102 2002). In contrast, constitutive activation of Rho, Rac and Cdc42 by cytotoxic necrotizing factor-1 (CNF-1) causes displacement of tight junction proteins ZO-1, occludin and junction adhesion molecule-1 (JAM-1) (Hopkins era/., 2003). In addition, Rho dependent activation of ROCK and subsequent acto-myosin contraction disrupts adherens junctions (Sahai and Marshall, 2002). These data suggest that the effects of Rho on intercellular junctions may parallel the biphasic effects of activated Rho on cellular migration. Therefore, it is possible that the dh-MotC-mediated disruption of adherens and tight junctions observed in mammary epithelial cells cultured on basement membrane may be due to Rho-ROCK activation, subsequent acto-myosin contraction, and ROCK-mediated antagonism of Dial signalling (Figure 23). Activation of Rho by dh-MotC, and subsequent inhibition of Rac could result in loss of polarity and disruption of adherens and tight junctions. However, this does not explain why 2.5u.M dh-MotC has a toxic effect on the morphology of differentiated scp2 cells, but not on undifferentiated scp2 cells. The key difference between scp2 cells in these two states is the substratum on which they are cultured. In order to polarize and secrete milk proteins, scp2 cells must be cultured on reconstituted basement membrane (Roskelley et al., 1994). Integrin clustering and activation is promoted by culture oh basement membrane, which results in "outside-in" signalling from the clustered integrins at focal contacts and 103 Figure 23. Potential mechanism of dh-MotC-mediated disruption of intercellular junctions. Rho-ROCK signalling can result in cell rounding and dismantling of intercellular junctions. Dh-MotC may act via a Rho-ROCK pathway to induce cell rounding and junction disruption in mammary epithelial cells. 104 105 is mediated through focal contact associated kinases such as focal adhesion kinase (FAK) and integrin linked kinase (ILK) (reviewed by Wu and Dedhar, 2001). Inhibition of integrin clustering results in anoikic (anchorage-independent) apoptosis (Boudreau et al., 1995), while ILK activation allows for anchorage-independent growth via suppression of anoikis (Atwell et al., 2000). Hence, the intracellular signalling network is different in the differentiated mammary epithelial cells compared to the undifferentiated state. It has been reported that blocking both |31 integrin and MAPK or PI3K in cells cultured on 3D substratum can induce apoptosis (Wang et al., 2002). A more precise definition of the specific factors present in the differentiated scp2 cells that render them more susceptible to dh-MotC is desirable because the three-dimensional culture system is a better model for an in vivo environment. In order to further test these ideas, one could examine the effects of dh-MotC treatment on the levels and phosphorylation status of FAK and ILK, as well as the levels and kinetics of Rho, Rac and Cdc42 activation in differentiated and undifferentiated mammary epithelial cells. Such experiments should yield the kind of information necessary to begin function related studies, utilizing specific inhibitors to possibly protect against the dh-MotC-induced apoptosis in normal mammary epithelia. Where has this investigation into the properties and in vitro mechanisms of dh-MotC led us? Reports from Lianne McHardy (C. Roskelley and M. Roberge labs, UBC), Armelle Troussard (S. Dedhar lab, Jack Bell Research Centre) and myself have demonstrated that dh-MotC activates a Rho-ROCK cascade, which induces 106 formation of stress fibres and focal adhesions, inhibits invasion and migration, and increases Na+/H+ exchange activity. Dh-MotC also induces loss of polarity, prevents milk protein secretion and induces apoptosis in differentiated mammary epithelial cells. These data demonstrate that considerable caution should be exercised prior to the further development of this compound for clinical use. As seen in this study, there was a negative effect on three dimensional mammary epithelial cell cultures, which mimics the in vivo microenvironment. There were also harmful effects on non-differentiated, non-tumourigenic mammary epithelial monolayer cultures at higher doses of dh-MotC. Therefore, the therapeutic window may be limited. Dh-MotC may have applications to disease states other than metastatic cancer due to its ability to stimulate NHE activity. Most of the research on Na+/H+ exchange-related disorders focuses on determining methods to inhibit NHE activity. Inhibition of Na7H+ exchange activity is protective in ischaemic myocardial tissue and it also minimizes reperfusion injury to the heart (reviewed by Karmazyn et al., 2001). However, NHE1 knockout mice have a neurological syndrome that includes ataxia and epilepsy and it is associated with selective neuronal death in the cerebellum and brainstem. There were no detected abnormalities in serum concentrations of HCO3", Na+, K+, Cl", Ca2+, or blood urea nitrogen in these mice, arguing against gross abnormalities of kidney function (Cox et al., 1997). Similarly, NHE1 deficient mice generated by gene targeting were found to have ataxia and epilepsy as well as growth retardation; no gross 107 abnormalities of acid-base balance were detected by measurements of blood pH and HC03"(Bell et al., 1999). Thus, It is conceivable that dh-MotC may have effects on neuronal excitability that could offset epileptic seizures via activation of Na+/H+ exchange. Intrauterine growth restriction (IUGR) is a common diagnosis in obstetrics and carries an increased risk of perinatal mortality and morbidity. Reduced activity and expression of NHE in preterm intrauterine growth restricted placentas may compromise placental function and may contribute to the development of fetal acidosis in IUGR preterm fetuses (Johansson et al., 2002). Therefore, agents that activate Na+/H+ exchange could be beneficial in these types of cases. While the future clinical applications of dh-MotC are not clear at the moment, further studies characterizing the compound's precise in vitro mechanisms of action may contribute to the identification and development of new and improved molecules that specifically and non-toxically inhibit invasion, angiogenesis and metastasis. 108 References Agus ZS, Puschett JB, Senesky D, Goldberg M. 1971. Mode of action of parathyroid hormone and cyclic adenosine 3',5'-monophosphate on renal tubular phosphate reabsorption in the dog. J Clin Invest. 50(3):617-26. Akhter S, Nath SK, Tse CM, Williams J, Zasloff M, Donowitz M. 1999. Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na7H+ exchanger isoform NHE3. Am J Physiol. 276(1 Pt 1):C136-44. Aronson PS, Nee J, Suhm MA. 1982. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature. 299 (5879): 161-3. Attwell S, Roskelley C, Dedhar S. 2000. The integrin-linked kinase (ILK) suppresses anoikis. Oncogene. 19(33):3811-5. Baird NR, Orlowski J, Szabo EZ, Zaun HC, Schultheis PJ, Menon AG, Shull GE. 1999. Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain. J Biol Chem. 274(7):4377-82. Baxter K. 1995. Regulation of intracellular pH in cultured foetal rat hippocampal pyramidal neurons. University of British Columbia Dept. of Anatomy M.Sc. Thesis Vancouver, BC Baxter KA, Church J. 1996. Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones. J Physiol. 493 ( Pt 2):457-70. Bell SM, Schreiner CM, Schultheis PJ, Miller ML, Evans RL, Vorhees CV, Shull GE, Scott WJ. 1999. Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures. Am J Physiol. 276(4 Pt 1):C788-95. Bertrand B, Wakabayashi S, Ikeda T, Pouyssegur J, Shigekawa M. 1994.. The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites. J Biol Chem. 269(18):13703-9. Bianchini L, L'Allemain G, Pouyssegur J. 1997. The p42/p44 mitogen-activated protein kinase cascade is determinant in mediating activation of the Na+/H+ exchanger (NHE1 isoform) in response to growth factors. J Biol Chem. 272(1):271-9. 109 Bishop AL, Hall A. 2000. Rho GTPases and their effector proteins. Biochem J. 348 Pt 2:241-55. Boguski MS, McCormick F. 1993. Proteins regulating Ras and its relatives. Nature. 366(6456):643-54. Boudreau N, Sympson CJ, Werb Z, Bissell MJ. 1995. Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science. 267(5199):891-3. Bretscher A, Edwards K, Fehon RG. 2002. ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol. (8):586-99. Brett CL, Wei Y, Donowitz M, Rab R. 2002. Human Na(+)/H(+) exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria. Am J Physiol Cell Physiol. 2002 May;282(5):C1031-41. Bright GR, Fisher GW, Rogowska J, Taylor DL. 1987. Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH. J Cell Biol. 104(4):1019-33. Bright GR, Fisher GW, Rogowska J, Taylor DL. 1989. Fluorescence ratio imaging microscopy. Methods Cell Biol. 30:157-92. Burridge K, Chrzanowska-Wodnicka M. 1996. Focal adhesions, contractility, and signaling. Annu Rev Cell Dev Biol. 12:463-518. Chaillet JR, Boron WF. 1985. Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules. J Gen Physiol. 86(6):765-94. Cerione RA, Zheng Y. 1996. The Dbl family of oncogenes. Curr Opin Cell Biol. 8(2):216-22. Chrzanowska-Wodnicka M, Burridge K. 1996. Rho-stimulated contractility drives the formation of stress fibres and focal adhesions. J Cell Biol. 133(6):1403-15. Counillon L, Pouyssegur J. 2000. The expanding family of eucaryotic Na(+)/H(+) exchangers. J Biol Chem. 275(1):1-4. Counillon L, Scholz W, Lang HJ, Pouyssegur J. 1993. Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties. Mol Pharmacol. 44(5): 1041-5. 110 Cox GA, Lutz CM, Yang CL, Biemesderfer D, Branson RT, Fu A, Aronson PS, Noebels JL, Frankel WN. 1997. Sodium/hydrogen exchanger gene defect in slow-wave epilepsy mutant mice. Cell. 91(1):139-48 Cox EA, Sastry SK, Huttenlocher A. 2001. Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases. Mol Biol Cell. 12(2):265-77. Dai C, Krantz SB. 1999. Interferon gamma induces upregulation and activation of caspases 1, 3, and 8 to produce apoptosis in human erythroid progenitor cells. Blood. 93(10):3309-16. Denker SP, Barber DL. 2002. Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1. J Cell Biol. 159(6): 1087-96. Denker SP, Huang DC, Orlowski J, Furthmayr H, Barber DL. 2000. Direct binding of the Na-H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation. Mol Cell. 6(6): 1425-36. Desprez PY, Hara E, Bissell MJ, Campisi J. 1995. Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein ld-1. Mol Cell Biol. 1995 Jun;15(6):3398-404. Desprez PY, Roskelley CD, Campisi J, Bissell MJ. 1993. Isolation of functional cell lines from a mouse mammary epithelial cell strain: the importance of basement membrane and cell-cell interaction. Mol. Cell. Diff. 1:99-110 Dhanasekaran N, Prasad MV, Wadsworth SJ, Dermott JM, van Rossum G. 1994. Protein kinase C-dependent and -independent activation of Na+/H+ exchanger by G alpha 12 class of G proteins. J Biol Chem. 269(16): 11802-6. Di Sario A, Bendia E, Svegliati Baroni G, Ridolfi F, Bolognini L, Feliciangeli G, Jezequel AM, Orlandi F, Benedetti A. 1999. Intracellular pathways mediating Na7H+ exchange activation by platelet-derived growth factor in rat hepatic stellate cells. Gastroenterology. 116(5): 1155-66. Donowitz M, Janecki A, Akhter S, Cavet ME, Sanchez F, Lamprecht G, Zizak M, Kwon WL, Khurana S, Yun CH, Tse CM. 2000. Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts. Ann N Y Acad Sci. 915:30-42. Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW. 2002. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nek. Nature. 418(6899):790-3. Ill Gabbiani G, Ryan GB, Lamelin JP, Vassalli P, Majno G, Bouvier CA, Cruchaud A, Luscher EF. 1973. Human smooth muscle autoantibody. Its identification as antiactin antibody and a study of its binding to "nonmuscular" cells. Am J Pathol. 72(3):473-88. Ganz MB, Pachter JA, Barber DL. 1990. Multiple receptors coupled to adenylate cyclase regulate Na-H exchange independent of cAMP. : J Biol Chem. 265(16):8989-92. Gohla A, Harhammer R, Schultz G. 1998. The G-protein G13 but not G12 mediates signaling from lysophosphatidic acid receptor via epidermal growth factor receptor to Rho. J Biol Chem. 273(8):4653-9. Goss GG, Woodside M, Wakabayashi S, Pouyssegur J, Waddell T, Downey GP, Grinstein S. 1994. ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter. Analysis of phosphorylation and subcellular localization. J Biol Chem. 269(12):8741-8. Goyal S, Vanden Heuvel G, Aronson PS. 2003. Renal expression of novel Na7H+ exchanger isoform NHE8. Am J Physiol Renal Physiol. 284(3):F467-73. Grinstein S, Woodside M, Sardet C, Pouyssegur J, Rotin D. 1992. Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism. J Biol Chem. 267(33):23823-8. Hinz JM, Helleday T, Meuth M. 2003. Reduced apoptotic response to camptothecin in CHO cells deficient in XRCC3. Carcinogenesis. 24(2):249-53. Hooley R, Yu CY, Symons M, Barber DL. 1996. G alpha 13 stimulates Na+-H+ exchange through distinct Cdc42-dependent and RhoA-dependent pathways. J Biol Chem. 271(11):6152-8. Hopkins AM, Walsh SV, Verkade P, Boquet P, Nusrat A. 2003. Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function. J Cell Sci. 116(Pt 4):725-42. Ingber DE, Prusty D, Frangioni JV, Cragoe EJ Jr, Lechene C, Schwartz MA. 1990. Control of intracellular pH and growth by fibronectin in capillary endothelial cells. J Cell Biol. 110(5): 1803-11. Ingber DE, Prusty D, Sun Z, Betensky H, Wang N. 1995. Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis. J Biomech.28(12):1471-84. Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, Narumiya S. 2000. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 57(5):976-83. 112 Isomura M, Kikuchi A, Ohga N, Takai Y. 1991. Regulation of binding of rhoB p20 to membranes by its specific regulatory protein, GDP dissociation inhibitor. Oncogene. 6(1): 119-24. Janecki AJ, Janecki M, Akhter S, Donowitz M. 2000. Basic fibroblast growth factor stimulates surface expression and activity of Na(+)/H(+) exchanger NHE3 via mechanism involving phosphatidylinositol 3-kinase. J Biol Chem. 275(11):8133-42. Janecki AJ, Montrose MH, Zimniak P, Zweibaum A, Tse CM, Khurana S, Donowitz M. 1998. J Biol Chem. 273(15):8790-8. Johansson M, Glazier JD, Sibley CP, Jansson T, Powell TL. 2002. Activity and protein expression of the Na7H+ exchanger is reduced in syncytiotrophoblast microvillous plasma membranes isolated from preterm intrauterine growth restriction pregnancies. J Clin Endocrinol Metab. Dec;87(12):5686-94. Kaibuchi K, Kuroda S, Fukata M, Nakagawa M. 1999. Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases. Curr Opin Cell Biol. 11(5):591-6. Karmazyn M, Sostaric JV, Gan XT. 2001. The myocardial Na7H+ exchanger: a potential therapeutic target for the prevention of myocardial ischaemic and reperfusion injury and attenuation of postinfarction heart failure. Drugs. 61(3):375-89. Katoh K, Kano Y, Amano M, Onishi H, Kaibuchi K, Fujiwara K. 2001. Rho-kinase--mediated contraction of isolated stress fibres. J Cell Biol. 153(3):569-84. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. 1996. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science. 273(5272):245-8. Kitamura K, Singer WD, Cano A, Miller RT. 1995. G alpha q and G alpha 13 regulate NHE-1 and intracellular calcium in epithelial cells. Am J Physiol. 268(1 Pt 1):C101-10. Kjoller L, Hall A. 1999. Signaling to Rho GTPases. Exp Cell Res. 253(1): 166-79. Klein M, Seeger P, Schuricht B, Alper SL, Schwab A. 2000. Polarization of Na(+)/H(+) and CI(-)/HCO (3)(-) exchangers in migrating renal epithelial cells. J Gen Physiol. 115(5):599-608. 113 Kleinman HK, McGarvey ML, Liotta LA, Robey PG, Tryggvason K, Martin GR. 1982. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 21 (24):6188-93. Knust E, Bossinger O. 2002. Composition and formation of intercellular junctions in epithelial cells. Science. 298(5600):1955-9. Kurashima K, D'Souza S, Szaszi K, Ramjeesingh R, Orlowski J, Grinstein S. 1999. The apical Na(+)/H(+) exchanger isoform NHE3 is regulated by the actin cytoskeleton. J Biol Chem. 274(42):29843-9. Kyte J, Doolittle RF. 1982. A simple method for displaying the hydropathic character of a protein. : J Mol Biol. 157(1):105-32. Lagana A, Vadnais J, Le PL), Nguyen TN, Laprade R, Nabi IR, Noel J. 2000. Regulation of the formation of tumour cell pseudopodia by the Na(+)/H(+) exchanger NHE1. J Cell Sci. 113 ( Pt 20):3649-62. Lamprecht G, Weinman EJ, Yun CH. 1998. The role of NHERF and E3KARP in the cAMP-mediated inhibition of NHE3. J Biol Chem. 273(45):29972-8. Li D, Williams Jl, Pietras RJ. 2002. Squalamine and cisplatin block angiogenesis and growth of human ovarian cancer cells with or without HER-2 gene overexpression. Oncogene. 21(18):2805-14. Lin X, Voyno-Yasenetskaya TA, Hooley R, Lin CY, Orlowski J, Barber DL. 1996. Galpha12 differentially regulates Na+-H+ exchanger isoforms. J Biol Chem. 271(37):22604-10. Liu L, Schwartz BR, Lin N, Winn RK, Harlan JM. 2002. Requirement for RhoA kinase activation in leukocyte de-adhesion. J Immunol. 169(5):2330-6. Ma YH, Reusch HP, Wilson E, Escobedo JA, Fantl WJ, Williams LT, Ives HE. 1994. Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma. J Biol Chem. 1994 Dec 2;269(48):30734-9. Machesky LM, Insall RH. 1998. Scarl and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Curr Biol. 8(25):1347-56. Madaule P, Axel R. 1985. A novel ras-related gene family. Cell. 41(1):31-40. Madaule P, Eda M, Watanabe N, Fujisawa K, Matsuoka T, Bito H, Ishizaki T, Narumiya S. 1998. Role of citron kinase as a target of the small GTPase Rho in cytokinesis. Nature. 394(6692):491-4. 114 Majumdar M, Seasholtz TM, Buckmaster C, Toksoz D, Brown JH. 1999. A rho exchange factor mediates thrombin and Galpha(12)-induced cytoskeletal responses. J Biol Chem. 274(38):26815-21. McHardy LM, Troussard A, Dedhar S, Roberge M, Roskelley C. 2002. Dihydromotuporamine C, an inhibitor of tumour cell invasion, is a Rho activator. Abstract # 261 2002 ASCB Annual Meeting Dec 14-18 San Francisco, CA Moor AN, Fliegel L. 1999. Protein kinase-mediated regulation of the Na(+)/H(+) exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways. J Biol Chem. 274(33):22985-92. Mukai M, Iwasaki T, Tatsuta M, Togawa A, Nakamura H, Murakami-Murofushi K, Kobayashi S, Imamura F, Inoue M. 2003. Cyclic phosphatidic acid inhibits RhoA-mediated autophosphorylation of FAK at Tyr-397 and subsequent tumour-cell invasion. Int J Oncol. 22(6):1247-56. Muschler J, Lochter A, Roskelley CD, Yurchenco P, Bissell MJ. 1999. Division of labor among the alpha6beta4 integrin, betal integrins, and an E3 laminin receptor to signal morphogenesis and beta-casein expression in mammary epithelial cells. Mol Biol Cell. 10(9):2817-28. Needham LK, Rozengurt E. 1998. Galpha12 and Galpha13 stimulate Rho-dependent tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130 Crk-associated substrate. J Biol Chem. 273(23): 14626-32. Nobes CD, Hall A. 1999. Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol. 144(6): 1235-44. Nobes CD, Hall A. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibres, lamellipodia, and filopodia. Cell. 81 (1 ):53-62. Numata M, Orlowski J. 2001. Molecular cloning and characterization of a novel (Na+,K+)/H+ exchanger localized to the trans-Golgi network. J Biol Chem. 276(20):17387-94. Numata M, Petrecca K, Lake N, Orlowski J. 1998. Identification of a mitochondrial Na+/H+ exchanger. J Biol Chem. 273(12):6951-9. Orlowski J. 1993. Heterologous expression and functional properties of amiloride high affinity (NHE-1) and low affinity (NHE-3) isoforms of the rat Na/H exchanger. J Biol Chem. 268(22):16369-77. Oxford G, Theodorescu D. 2003. Ras superfamily monomeric G proteins in carcinoma cell motility. Cancer Lett. 189(2): 117-28. 115 Pollard TD, Borisy GG. 2003. Cellular motility driven by assembly and disassembly of actin filaments. Cell. 112(4):453-65. Putney LK, Denker SP, Barber DL. 2002. The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions. Annu Rev Pharmacol Toxicol. 42:527-52. Reid T, Furuyashiki T, Ishizaki T, Watanabe G, Watanabe N, Fujisawa K, Morii N, Madaule P, Narumiya S. 1996. Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain. J Biol Chem. 271 (23):13556-60. Ren XD, Kiosses WB, Schwartz MA. 1999. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. (3):578-85. Ridley AJ. 2001. Rho GTPases and cell migration. J Cell Sci. 114(Pt 15):2713-22. Ridley AJ, Hall A. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibres in response to growth factors. Cell. 70(3):389-99. Riento K, Ridley AJ. 2003. Rocks: multifunctional kinases in cell behaviour. 2003. Nat Rev Mol Cell Biol. 4(6):446-56. Ritter M, Schratzberger P, Rossmann H, Woll E, Seiler K, Seidler U, Reinisch N, Kahler CM, Zwierzina H, Lang HJ, Lang F, Paulmichl M, Wiedermann CJ. 1998. Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes. Br J Pharmacol. 124(4):627-38. Roskelley CD, Bissell MJ. 2002. The dominance of the microenvironment in breast and ovarian cancer. Semin Cancer Biol. 12(2):97-104. Roskelley CD, Desprez PY, Bissell MJ. 1994. Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction. Proc Natl Acad Sci USA. 91(26):12378-82. Roskelley CD, Williams DE, McHardy LM, Leong KG, Troussard A, Karsan A, Andersen RJ, Dedhar S, Roberge M. 2001. Inhibition of tumour cell invasion and angiogenesis by motuporamines. Cancer Res. 61(18):6788-94. Sah VP, Seasholtz TM, Sagi SA, Brown JH. 2000. The role of Rho in G protein-coupled receptor signal transduction. Annu Rev Pharmacol Toxicol. 40:459-89. 116 Sahai E, Olson MF, Marshall CJ. 2001. Cross-talk between Ras and Rho signalling pathways in transformation favours proliferation and increased motility. EMBO J. 20(4):755-66. Sawada K, Morishige K, Tahara M, Ikebuchi Y, Kawagishi R, Tasaka K, Murata Y. 2002. Lysophosphatidic acid induces focal adhesion assembly through Rho/Rho-associated kinase pathway in human ovarian cancer cells. Gynecol Oncol. 87(3):252-9. Schwartz MA, Cragoe EJ Jr, Lechene CP. 1990. pH regulation in spread cells and round cells. J Biol Chem. 265(3): 1327-32. Shrode LD, Gan BS, D'Souza SJ, Orlowski J, Grinstein S. 1998. Topological analysis of NHE1, the ubiquitous Na7H+ exchanger using chymotryptic cleavage. Am J Physiol. 1998 Aug;275(2 Pt 1):C431-9. Shrode LD, Tapper H, Grinstein S. 1997. Role of intracellular pH in proliferation, transformation, and apoptosis. J Bioenerg Biomembr. (4):393-9. Sills AK Jr, Williams Jl, Tyler BM, Epstein DS, Sipos EP, Davis JD, McLane MP, Pitchford S, Cheshire K, Gannon FH, Kinney WA, Chao TL, Donowitz M, Laterra J, Zasloff M, Brem H. 1998. Squalamine inhibits angiogenesis and solid tumour growth in vivo and perturbs embryonic vasculature. Cancer Res. 58(13):2784-92. Simchowitz L, Cragoe EJ Jr. 1986. Regulation of human neutrophil chemotaxis by intracellular pH. J Biol Chem. 261 (14):6492-500. Smith GA, Brett CL, Church J. 1998. Effects of noradrenaline on intracellular pH in acutely dissociated adult rat hippocampal CA1 neurones. J Physiol. 512 ( Pt 2):487-505. Somasiri A, Roskelley CD. 1999. Cell shape and integrin signaling regulate the differentiation state of mammary epithelial cells. Methods Mol Biol. 129:271-83. Streuli CH, Schmidhauser C, Bailey N, Yurchenco P, Skubitz AP, Roskelley C, Bissell MJ. 1995. Laminin mediates tissue-specific gene expression in mammary epithelia. J Cell Biol. 129(3):591-603. Sugimoto N, Takuwa N, Okamoto H, Sakurada S, Takuwa Y. 2003. Inhibitory and stimulatory regulation of Rac and cell motility by the G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform. Mol Cell Biol. 23(5): 1534-45. Swarthout JT, Walling HW. 2000. Lysophosphatidic acid: receptors, signaling and survival. Cell Mol Life Sci. 57(13-14):1978-85. 117 Szaszi K, Kurashima K, Kaibuchi K, Grinstein S, Orlowski J. 2001. Role of the cytoskeleton in mediating cAMP-dependent protein kinase inhibition of the epithelial Na+/H+ exchanger NHE3. J Biol Chem. 276(44):40761-8. Takahashi E, Abe J, Gallis B, Aebersold R, Spring DJ, Krebs EG, Berk BC. 1999. p90(RSK) is a serum-stimulated Na7H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1. J Biol Chem. 274(29):20206-14. Takahashi K, Sasaki T, Mammoto A, Takaishi K, Kameyama T, Tsukita S, Takai Y. 1997. Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J Biol Chem. 272(37):23371-5. Takai Y, Sasaki T, Tanaka K, Nakanishi H. 1995. Rho as a regulator of the cytoskeleton. Trends Biochem Sci. 20(6):227-31. Takaishi K, Sasaki T, Kotani H, Nishioka H, Takai Y. 1997. Regulation of cell-cell adhesion by rac and rho small G proteins in MDCK cells. J Cell Biol. 139(4): 1047-59. Tominaga T, Barber DL. 1998. Na-H exchange acts downstream of RhoA to regulate integrin-induced cell adhesion and spreading. Mol Biol Cell. 9(8):2287-303. Tominaga T, Ishizaki T, Narumiya S, Barber DL. 1998. p160ROCK mediates RhoA activation of Na-H exchange. EMBO J. 17(16):4712-22. Tse CM, Brant SR, Walker MS, Pouyssegur J, Donowitz M. 1992. Cloning and sequencing of a rabbit cDNA encoding an intestinal and kidney-specific Na+/H+ exchanger isoform (NHE-3). J Biol Chem. 267(13):9340-6. Tsuji T, Ishizaki T, Okamoto M, Higashida C, Kimura K, Furuyashiki T, Arakawa Y, Birge RB, Nakamoto T, Hirai H, Narumiya S. 2002. ROCK and mDial antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts. J Cell Biol. 157(5):819-30. Tu H, Wigler M. 1999. Genetic evidence for Pak1 autoinhibition and its release by Cdc42. Mol Cell Biol. 19(1 ):602-11. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S. 1997. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 389(6654):990-4. 118 van Nieuw Amerongen GP, Koolwijk P, Versteilen A, van Hinsbergh VW. 2003. Involvement of RhoA/Rho kinase signaling in VEGF-induced endothelial cell migration and angiogenesis in vitro. Arterioscler Thromb Vase Biol. 23(2):211-7. Voyno-Yasenetskaya TA. 1998. G proteins and Na+/H+ exchange. Biol Signals Recept. 7(2): 118-24. Wakabayashi S, Bertrand B, Ikeda T, Pouyssegur J, Shigekawa M. 1994. Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective. J Biol Chem. 269(18): 13710-5. Wakabayashi S, Bertrand B, Shigekawa M, Fafournoux P, Pouyssegur J. 1994. Growth factor activation and "H(+)-sensing" of the Na7H+ exchanger isoform 1 (NHE1). Evidence for an additional mechanism not requiring direct phosphorylation. J Biol Chem. 269(8):5583-8. Wakabayashi S, Fafournoux P, Sardet C, Pouyssegur J. 1992. The Na7H+ antiporter cytoplasmic domain mediates growth factor signals and controls "H(+)-sensing". Proc Natl Acad Sci USA. 89(6):2424-8. Wakabayashi S, Shigekawa M, Pouyssegur J. 1997. Molecular physiology of vertebrate Na7H+ exchangers. Physiol Rev. 77(1):51-74. Wang F, Hansen RK, Radisky D, Yoneda T, Barcellos-Hoff MH, Petersen OW, Turley EA, Bissell MJ. 2002. Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts. J Natl Cancer Inst. 94(19):1494-503. Warnock DG, Yang WC, Huang ZQ, Cragoe EJ Jr. 1988. Interactions of chloride and amiloride with the renal Na7H/ antiporter. J Biol Chem. 263(15):7216-21. Wang DN. 1994. Band 3 protein: structure, flexibility and function. FEBS Lett. 346(1 ):26-31. Warnock DG, Yang WC, Huang ZQ, Cragoe EJ Jr. 198. Interactions of chloride and amiloride with the renal Na7H+ antiporter. J Biol Chem. 263(15):7216-21. Watanabe N, Kato T, Fujita A, Ishizaki T, Narumiya S. 1999. Cooperation between mDial and ROCK in Rho-induced actin reorganization. Nat Cell Biol. 1(3):136-43. 119 Weaver VM, Lelievre S, Lakins JN, Chrenek MA, Jones JC, Giancotti F, Werb Z, Bissell MJ. 2002. beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell. 2(3):205-16. Webb DJ, Parsons JT, Horwitz AF. 2002. Adhesion assembly, disassembly and turnover in migrating cells - over and over and over again. Nat Cell Biol. 4(4):E97-100. Weinman EJ, Minkoff C, Shenolikar S. 2000. Signal complex regulation of renal transport proteins: NHERF and regulation of NHE3 by PKA. Am J Physiol Renal Physiol. 279(3):F393-9. Williams DE, Craig KS, Patrick B, McHardy LM, van Soest R, Roberge M, Andersen RJ. 2002. Motuporamines, anti-invasion and anti-angiogenic alkaloids from the marine sponge Xestospongia exigua (Kirkpatrick): isolation, structure elucidation, analogue synthesis, and conformational analysis. J Org Chem. 67(1):245-58. Williams DE, Lassota P, Andersen RJ. 1998. Motuopramines A-C, cytotoxic alkaloids isolated from the marine sponge Xestospongia exigua (Kirkpatrick). J. Org. Chem. 63:4838-4841. Williams Jl, Weitman S, Gonzalez CM, Jundt CH, Marty J, Stringer SD, Holroyd KJ, Mclane MP, Chen Q, Zasloff M, Von Hoff DD. 2001. Squalamine treatment of human tumours in nu/nu mice enhances platinum-based chemotherapies. Clin Cancer Res. 7(3):724-33. Winkel GK, Sardet C, Pouyssegur J, Ives HE. 1993. Role of cytoplasmic domain of the Na7H+ exchanger in hormonal activation. J Biol Chem. 268(5):3396-400. Worthylake RA, Lemoine S, Watson JM, Burridge K. 2001. RhoA is required for monocyte tail retraction during transendothelial migration. J Cell Biol. 154(1 ):147-60. Wu C, Dedhar S. Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. 2001. J Cell Biol. 155(4):505-10. Yan W, Nehrke K, Choi J, Barber DL. 2001. The Nck-interacting kinase (NIK) phosphorylates the Na+-H+ exchanger NHE1 and regulates NHE1 activation by platelet-derived growth factor. J Biol Chem. 276(33):31349-56. 120 Yun CH, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman EJ, Donowitz M. 1997. cAMP-mediated inhibition of the epithelial brush border Na7H+ exchanger, NHE3, requires an associated regulatory protein. Proc Natl Acad Sci USA. 94(7):3010-5. Zhao H, Wiederkehr MR, Fan L, Collazo RL, Crowder LA, Moe OW. 1999. Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase a and NHE-3 phosphoserines 552 and 605. J Biol Chem. 274(7):3978-87. 121 

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
United States 22 1
China 8 0
Germany 5 7
Russia 3 0
Poland 3 0
Japan 3 0
Brazil 1 0
United Kingdom 1 0
City Views Downloads
Unknown 12 8
Lewes 10 0
Ashburn 7 0
Shenzhen 4 0
Tokyo 3 0
Shanghai 2 0
Washington 2 0
Beijing 2 0
Atlanta 1 0
Jacksonville 1 0
London 1 0
Saint Petersburg 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0091028/manifest

Comment

Related Items