Assessment of Rapid Morphological Changes Associated with Elevated cAMP Levels in Human Orbital Fibroblasts

Reddy, Lavanya; Wang, Hwai Shi; Keese, Charles R.; Giæver, Ivar; Smith, Terry J.

doi:10.1006/excr.1998.4273

Cited by 42 publications

(29 citation statements)

References 27 publications

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“…[10][11][12] Specifically, cell spreading is associated with Gi activation 31,32 whereas a rounded or stellate morphology is associated with Gs activation or cAMP elevation. [33][34][35] More recently, actin polymerization has been shown to be required for the movement of GPCRs within the membrane associated with β-arrestin-mediated receptor recycling. 36,37 Thus, not surprisingly, blocking actin polymerization inhibited or eliminated detection of GPCR responses by impedance assays (Fig.…”

Section: Discussionmentioning

confidence: 99%

Evaluating Cellular Impedance Assays for Detection of GPCR Pleiotropic Signaling and Functional Selectivity

Peters¹,

Scott²

2009

SLAS Discovery

115

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G-protein-coupled receptors can couple to different signal transduction pathways in different cell types (termed cell-specific signaling) and can activate different signaling pathways depending on the receptor conformation(s) stabilized by the activating ligand (functional selectivity). These concepts offer potential for developing pathway-specific drugs that increase efficacy and reduce side effects. Despite significant interest, functional selectivity has been difficult to exploit in drug discovery, in part due to the burden of multiple assays. Cellular impedance assays use an emerging technology that can qualitatively distinguish Gs, Gi/o, and Gq signaling in a single assay and is thereby suited for studying these pharmacological concepts. Cellular impedance confirmed cell-specific Gs and Gq coupling for the melanocortin-4 receptor and dual Gi and Gs signaling with the cannabinoid-1 (CB1) receptor. The balance of Gi versus Gs signaling depended on the cell line. In CB1-HEKs, Giand Gs-like responses combined to yield a novel impedance profile demonstrating the dynamic nature of these traces. Cellspecific signaling was observed with endogenous D1 receptor in U-2 cells and SK-N-MC cells, yet the pharmacological profile of partial and full agonists was similar in both cell lines. We conclude that the dynamic impedance profile encodes valuable relative signaling information and is sufficiently robust to help evaluate cell-specific signaling and functional selectivity. (Journal of Biomolecular Screening 2009:246-255)

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Section: Discussionmentioning

confidence: 99%

Evaluating Cellular Impedance Assays for Detection of GPCR Pleiotropic Signaling and Functional Selectivity

Peters¹,

Scott²

2009

SLAS Discovery

115

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“…Micromotion detected by the ECIS technique is directly related to conventional cell motility (19). Drugs that inhibit cell migration and cell motility in cultured cells, such as cytochalasin B, also inhibit micromotion (14,20). Micromotion detected by the ECIS technique has been used to detect cell motility, cell morphology, and cell-ECM interactions in different systems (20,21).…”

Section: Pd098059 Inhibits Basal Cell Micromotion In a Concentrationdmentioning

confidence: 99%

“…Drugs that inhibit cell migration and cell motility in cultured cells, such as cytochalasin B, also inhibit micromotion (14,20). Micromotion detected by the ECIS technique has been used to detect cell motility, cell morphology, and cell-ECM interactions in different systems (20,21). More recently, ECIS has been used to establish the metastatic behavior of cells in culture (22).…”

Section: Pd098059 Inhibits Basal Cell Micromotion In a Concentrationdmentioning

confidence: 99%

Integrin α2 and Extracellular Signal-regulated Kinase Are Functionally Linked in Highly Malignant Autocrine Transforming Growth Factor-α-driven Colon Cancer Cells

Sawhney

Sharma

Humphrey

et al. 2003

Journal of Biological Chemistry

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Recently, we have shown that autocrine transforming growth factor-␣ (TGF-␣) controls the expression of integrin ␣ 2 , cell adhesion to collagen IV and motility in highly progressed HCT116 colon cancer cells (Sawhney, R. S., Zhou, G-H. K., Humphrey, L. E., Ghosh, P., Kreisberg, J. I., and Brattain, M. G. (2002) J. Biol. Chem. 277, 75-86). We now report that expression of basal integrin ␣ 2 and its biological effects are controlled by constitutive activation of the extracellular signal-regulated/mitogen-activated protein kinase (ERK/MAPK) pathway. Treatment of cells with selective mitogenactivated protein kinase kinase (MEK) inhibitors PD098059 and U0126 showed that integrin ␣ 2 expression, cell adhesion, and activation of ERK are inhibited in a parallel concentration-dependent fashion. Moreover, autocrine TGF-␣-mediated epidermal growth factor receptor activation was shown to control the constitutive activation of the ERK/MAPK pathway, since neutralizing antibody to the epidermal growth factor receptor was able to block basal ERK activity. TGF-␣ antisensetransfected cells also showed attenuated activation of ERK. Using a real time electric cell impedance sensing technique, it was shown that ERK-dependent integrin ␣ 2 -mediated cell micromotion signaling is controlled by autocrine TGF-␣. Thus, this study implicates ERK/MAPK signaling activated by endogenous TGF-␣ as one of the mechanistic features controlling metastatic spread. Epidermal growth factor receptor (EGFr)1 activation is known to control cancer cell growth as a result of autocrine and/or paracrine stimulation. We have recently shown that constitutive endogenous activation of the EGFr by TGF-␣ provides a critical growth and survival advantage to colon cancer cells (1, 2). Similarly, it is conceivable that human colon cancer cells involved in metastasis may derive an advantage in cell motility from a strong autocrine TGF-␣ loop, because the initial number of cells contributing to metastatic behavior is small. Recently, we showed that in HCT116 cells, the DNA synthesis response is saturated by a relatively low EGFr occupation resulting from autocrine TGF-␣ (3). The expression level of integrin ␣ 2 and its functions, on the other hand, show a much wider window of response, increasing from low EGFr occupation by autocrine TGF-␣ to saturation by complete receptor occupation with exogenous EGF. The initiation of this response at low level receptor occupation was shown by its attenuation with treatment by an EGFr blocking antibody that inhibits basal EGFr activation resulting from autocrine TGF-␣ as well as by stable transfection with a TGF-␣ antisense cDNA to inhibit basal EGFr activation in HCT116 cells. We also observed that the addition of exogenous EGF results in further EGFr activation, which is associated with higher expression of integrin ␣ 2 , enhanced cell adhesion, and micromotility. Thus, there is a difference in response windows based on the extent of EGFr activation. While the activation of EGFr was demonstrated to be critical to ␣ 2 integrin-medi...

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“…The enormous sensitivity of the ECIS device was demonstrated by detecting the morphological alterations of cultured cells caused by the periodic injections of CO 2 in ordinary cell culture incubators (Lo et al, 1994). The rapid changes in cell shape that can be induced in human orbital fibroblasts by exposure to prostaglandin E have also been monitored by ECIS and shown to be mediated by cAMP-dependent signal transduction pathways (Reddy et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Bioelectrical impedance assay to monitor changes in cell shape during apoptosis

Arndt

Seebach

Psathaki

et al. 2004

Biosensors and Bioelectronics

297

197

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Apoptosis is a strictly regulated and genetically encoded cell 'suicide' that may be triggered by cytokines, depletion of growth factors or certain chemicals. It is morphologically characterized by severe alterations in cell shape like cell shrinkage and disintegration of cell-cell contacts. We applied a non-invasive electrochemical technique referred to as electric cell-substrate impedance sensing (ECIS) in order to monitor the apoptosis-induced changes in cell shape in an integral and quantitative fashion with a time resolution in the order of minutes. In ECIS the cells are grown directly on the surface of small gold-film electrodes (d = 2 mm). From readings of the electrical impedance of the cell-covered electrode, performed with non-invasive, low amplitude sensing voltages, it is possible to deduce alterations in cell-cell and cell-substrate contacts. To improve the sensitivity of this impedance assay we used endothelial cells derived from cerebral micro-vessels as cellular model systems since these are well known to express electrically tight intercellular junctions. Apoptosis was induced by cycloheximide (CHX) and verified by biochemical and cytological assays. The time course of cell shape changes was followed with unprecedented time resolution by impedance readings at 1 kHz and correlated with biochemical parameters. From impedance readings along a broad frequency range of 1-10 6 Hz we could assign the observed impedance changes to alterations on the subcellular level. We observed that disassembly of barrier-forming tight junctions precedes changes in cell-substrate contacts and correlates strongly with the time course of protease activation.

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Assessment of Rapid Morphological Changes Associated with Elevated cAMP Levels in Human Orbital Fibroblasts

Cited by 42 publications

References 27 publications

Evaluating Cellular Impedance Assays for Detection of GPCR Pleiotropic Signaling and Functional Selectivity

Evaluating Cellular Impedance Assays for Detection of GPCR Pleiotropic Signaling and Functional Selectivity

Integrin α2 and Extracellular Signal-regulated Kinase Are Functionally Linked in Highly Malignant Autocrine Transforming Growth Factor-α-driven Colon Cancer Cells

Bioelectrical impedance assay to monitor changes in cell shape during apoptosis

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